Green Energy and Technology

Muhammad Adly Rahandi Lubis ·

Seng Hua Lee · Efri Mardawati ·
Souvia Rahimah · Petar Antov ·
Robi Andoyo · Ľuboš Krišťák ·
Bambang Nurhadi Editors

Biomass
Conversion and
Sustainable
Biorefinery
Towards Circular Bioeconomy
Green Energy and Technology
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Muhammad Adly Rahandi Lubis · Seng Hua Lee ·
Efri Mardawati · Souvia Rahimah · Petar Antov ·
Robi Andoyo · Ľuboš Krišt’ák · Bambang Nurhadi
Editors

Biomass Conversion
and Sustainable Biorefinery
Towards Circular Bioeconomy
Editors
Muhammad Adly Rahandi Lubis Seng Hua Lee
Research Center for Biomass Faculty of Applied Sciences, Department
and Bioproducts of Wood Industry
National Research and Innovation Agency Universiti Teknologi MARA (UiTM)
Cibinong, Indonesia Jengka, Pahang, Malaysia

Efri Mardawati Souvia Rahimah

Department of Agro-Industrial Technology Department of Food Industrial Technology
Padjadjaran University Padjadjaran University
Bandung, Indonesia Bandung, Indonesia

Petar Antov Robi Andoyo

Department of Mechanical Wood Faculty of Agro-industrial Technology
Technology Padjadjaran University
Faculty of Forest Industry Bandung, Indonesia
University of Forestry
Sofia, Bulgaria Bambang Nurhadi
Department of Food Technology
Ľuboš Krišt’ák Padjadjaran University
Department of Physics, Electrical Bandung, Indonesia
Engineering and Applied Mechanics
Technical University of Zvolen
Zvolen, Slovakia

ISSN 1865-3529 ISSN 1865-3537 (electronic)

Green Energy and Technology
ISBN 978-981-99-7768-0 ISBN 978-981-99-7769-7 (eBook)
https://doi.org/10.1007/978-981-99-7769-7

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Preface

The energy crisis, caused by limited natural resources, compels industry and research
society to seek alternative energy sources. Biomass, as a renewable and abundant
material on the planet, offers numerous appealing benefits. In recent decades, there
has been a growing interest in converting biomass into energy. Biomass stores solar
energy, which can then be released as heat through burning or combustion. Typi-
cally, biomass is classified according to the type of biomass found in nature, such as
wood and woody biomass, herbaceous biomass, aquatic biomass, animal and human
biomass wastes, and mixtures of those biomass. The wood and woody biomass
could include all of the woody trees on the land, as well as their trunks, branches,
and sawdust after processing. Meanwhile, herbaceous biomass is also known as non-
woody biomass, which includes bamboo (a type of grass), bagasse, straws, and so on.
Algae and lake weed, on the other hand, are examples of aquatic biomass, whereas
manures and bones are examples of animal and human biomass. These biomasses
are high in organic matter and thus have the potential to be used as energy sources.
Different conversion methods have been used due to the differences in the nature of
these biomasses. The biorefinery process is what is needed to convert these biomasses
into energy as well as other useful products in an effective manner. In a nutshell, it
can be described as an integrated system of conversion processes and equipment that
transforms biomass into useful products such as biofuels, energy, and other chem-
ical compounds. The International Energy Agency’s Bioenergy Task 42 describes
biorefining as “the sustainable processing of biomass into a spectrum of bio-based
products and bioenergy.” Biorefinery, which is an environmentally friendly method,
has the potential to make significant contributions to the circular bioeconomy through
its promotion of the recyclable use of resources as well as value-adding character-
istics. In fact, it is one of the most important strategies that enables the circular
economy, which closes loops of raw biomass materials. The creation of a cleaner
and more environmentally friendly environment is one of the potential contributions
that biorefineries could make to the development of a sustainable bio-based circular
economy. It is possible to form a circular economy as opposed to a linear economy
by adhering to the principles of the philosophy of reuse and recycling. The real-
ization of these ideologies, however, is not without limitations. Many constraints

v
vi Preface

lie ahead, and strategies to make it work are critical. The success of biorefinery
technologies is highly dependent on several factors, including the process’s tech-
nical feasibility and cost-effectiveness. It must be cost effective and, most impor-
tantly, environmentally friendly to start with, but social acceptance is also a deciding
factor to determine the effectiveness of the technologies. This book is organized
to present the Biomass Conversion and Sustainable Biorefinery: Towards Circular
Bioeconomy, consisting of 14 chapters discussing the current progress on biomass
resources, biomass pretreatment, biomass conversion, and their applications.

Cibinong, Indonesia Muhammad Adly Rahandi Lubis

Jengka, Malaysia Seng Hua Lee
Bandung, Indonesia Efri Mardawati
Bandung, Indonesia Souvia Rahimah
Sofia, Bulgaria Petar Antov
Bandung, Indonesia Robi Andoyo
Zvolen, Slovakia Ľuboš Krišt’ák
Bandung, Indonesia Bambang Nurhadi
Contents

Current Progress on Biomass Pretreatment: The Key for Its

Valorization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Roni Maryana, Eka Triwahyuni, Muryanto Muryanto, Joni Prasetyo,
Oktaviani Oktaviani, Sri Sugiwati, Teuku Beuna Bardant,
Atanu Kumar Das, and Yanni Sudiyani
Recent Updates on Biopolymers: Precursors, Process, Properties,
Challenge, and Future Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Aisyah Hanifah, Arfiathi, Melbi Mahardika, Riska Sumirat,
Rossy Choerun Nissa, and Yeyen Nurhamiyah
Potential Application of Agro-Industrial Byproduct for Bacterial
Cellulose Production; Its Challenges and Emerging Trends
for Food Packaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
Khatarina Meldawati Pasaribu, Nanang Masruchin, and Myrtha Karina
Biomass Valorization for Bioenergy Production . . . . . . . . . . . . . . . . . . . . . . 67
Sri Suhartini, Nimas Mayang S. Sunyoto, Hendrix Y. Setyawan,
Novita Ainur Rohma, Elviliana, Dinda Agustin Pratiwi, Fitria,
Efri Mardawati, Kasbawati, and Lynsey Melville
Biomass Utilization and Biorefinery By-Product from Palm Oil
and Marine Resources for Animal Feed and Feed Additive . . . . . . . . . . . . 105
Ahmad Sofyan, Hendra Herdian, and Agung Irawan
Aquatic Biomass Conversion and Biorefinery for Value-Added
Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
Novia Amalia Sholeha, Nova Rachmadona,
Fajriana Shafira Nurrusyda, Nanang Masruchin,
and Khatarina Meldawati Pasaribu

vii
viii Contents

The Production of Microalgae and Cyanobacteria Biomass

and Their Valuable Bioproducts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
Awalina Satya, Azalea Dyah Maysarah Satya, Tjandra Chrismadha,
Nofdianto Nofdianto, Gunawan Gunawan, Ika Atman Satya,
Souvia Rahimah, Efri Mardawati, Sara Kazemi Yazdi,
and Pau-Loke Show
An Overview on Managing Minute Duckweed (Lemna Perpusilla
Torr) Cultivation for Fish Feed Purpose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171
Tjandra Chrismadha, Awalina Satya, Ika Atman Satya,
Azalea Dyah Maysarah Satya, Sara Kazemi Yazdi, and Efri Mardawati
Structured Lipids Based on Palm Oil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193
Siti Nurhasanah and S. Joni Munarso
Recent Developments of the Agroindustry Byproducts Utilization
in Bacterial Cellulose Production and Its Medical Devices
Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207
Khatarina Meldawati Pasaribu, I Putu Mahendra,
Novia Amalia Sholeha, Nanang Masruchin, Efri Mardawati,
and Myrtha Karina
Extraction, Isolation, Purification, and Potential Application
of Xylose and Xylooligosaccharides from Lignocellulosic Biomass . . . . . . 229
Euis Hermiati, Hans Wijaya, and Dwi Ajias Pramasari
Carotene Production from Biomass Waste . . . . . . . . . . . . . . . . . . . . . . . . . . . 269
Made Tri Ari Penia Kresnowati and Dianika Lestari
Effect of Single Clove Black Garlic Extract on Lipid Accumulation
During Adipocyte Differentiation Using 3T3-L1 Cell Line . . . . . . . . . . . . . 279
Efri Mardawati, Erin Nur Fitriani, Desi Ayu Sundari,
Tenny Putri Wikayani, Nurul Qomarilla, Nova Rachmadona,
Eko Fuji Ariyanto, Roni Kastaman, Totok Pujianto,
and Devi Maulida Rahmah
Physical and Organoleptic Characteristic of Bread Substituted
with Spirulina Platensis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295
Souvia Rahimah, Muhammad Fadly Sonjaya, Robi Andoyo,
Awalina Satya, Siti Nurhasanah, and Tjandra Chrismadha
Current Progress on Biomass
Pretreatment: The Key for Its
Valorization

Roni Maryana, Eka Triwahyuni, Muryanto Muryanto, Joni Prasetyo,

Oktaviani Oktaviani, Sri Sugiwati, Teuku Beuna Bardant,
Atanu Kumar Das, and Yanni Sudiyani

Abstract It has become a global concern for reducing the utilization of fossil sources
for energy and chemical purposes. Not only environmental issues such as green-
houses gases increment but also depleting fossil reserves, which became the reason
to quest for new and renewable raw materials. Lignocellulosic biomass that is abun-
dantly available is being studied as a potential material to provide both fuels and
biochemicals. However, to fractionate and disintegrate its main components: cellu-
lose; hemicellulose and lignin is a very challenging step. Different pretreatment
approaches that considering energy consumption, cost effectiveness, percentage of
lignin removal, further utilization of the cellulose, hemicellulose, lignin, and environ-
mentally friendly process have been applied. The review covers the recent pretreat-
ment technology that applied physicochemical approach; chemicals pretreatment
and application of Deep Eutectic Solvent (DES); biological pretreatment; and high
energy of radiation. This study explained a deeper understanding of the pretreat-
ment technologies for developing biorefinery concepts to support the sustainable
development of utilization of lignocellulosic biomass.

Keywords Lignocellulosic · Pretreatment · Physicochemical treatment ·

Biological pretreatment

R. Maryana (B) · E. Triwahyuni · M. Muryanto · J. Prasetyo · S. Sugiwati · T. B. Bardant ·

Y. Sudiyani
Research Center for Chemistry, National Research and Innovation Agency, Building 452 Kawasan
PUSPIPTEK, Serpong Tangerang Selatan, Banten, Indonesia
e-mail: [email protected]
O. Oktaviani
Research Center for Radiation Process Technology, National Research and Innovation Agency,
Pasar Jumat, Jakarta Selatan, Indonesia
A. K. Das
Department of Forest Biomaterials and Technology, Swedish University of Agricultural Sciences,
901 83 Umeå, Sweden

© The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2024 1
M. A. R. Lubis et al. (eds.), Biomass Conversion and Sustainable Biorefinery, Green
Energy and Technology, https://doi.org/10.1007/978-981-99-7769-7_1
2 R. Maryana et al.

1 Introduction

Pretreatment of lignocellulosic biomass is the key for the separation of cellulose,

hemicellulose, and lignin. Several factors such as the energy required and the econom-
ical value of the biomass pretreatment will be the key for industrial application.
Another consideration is the end product of the fractionation. For instance, lower
molecular weight or lower density cellulose will not be suitable for dissolving pulp
application. Moreover, the enzymatic accessibility will be the key factor for the
saccharification of cellulose into the sugar on the bioethanol production process.
The main objective of the pretreatment process on lignocellulosic biomass is lignin
removal. Determination of the pretreatment methods depends on the type of ligno-
cellulosic biomass. Therefore, no universal pretreatment can be applied to all ligno-
cellulosic biomass (Costa Sousa et al. 2009). Therefore, it can be summarized that
pretreatment has to be: (a) considering energy consumption, (b) cost-effectiveness,
(c) percentage of lignin removal, (d) further utilization of the cellulose, hemicellulose,
lignin, and (e) environmentally friendly process.
Generally, the pretreatment of biomass can be divided into chemical, physical,
biological, physicochemical, and high-energy radiation treatment. However, combi-
nation of these methods is mostly applied to get desirable results. This study aims
to provide recent information and discuss the above-mentioned biomass pretreat-
ment methods as the information for choosing suitable pretreatment conditions to
get optimum fractionation for wood and non-wood biomass components.

2 Pretreatment Methods

2.1 Physicochemical Pretreatment

Physicochemical pretreatment is believed to cause more well lignin removal and

cellulose crystallinity decrement efficiency as compared with physical, chemical, and
biological pretreatment (Zhao, et al. 2021). The most widely used physicochemical
pretreatment involved liquid-hot water (LHW), steam explosion, alkali explosion or
alkali-heat pretreatment, and ammonia fiber expansion (AFEX) (Wang et al. 2015;
Elliston et al. 2015; Jacquet et al. 2015; Yu et al. 2022; Sudiyani et al. 2020; Nurani
et al. 2020; Emam 2013; Chundawat 2020; Bensah and Mensah 2018).

2.2 Liquid-Hot Water (LHW) Pretreatment

Liquid-hot water (LHW) is one of the pretreatment technologies utilizing liquid

water without other chemicals at high temperature and pressure (Yan et al. 2016;
Zhuang et al. 2016). Water serves as a solvent and also a catalyst accompanied
Current Progress on Biomass Pretreatment: The Key for Its Valorization 3

by removed organic acids from biomass to aid in disrupting the cell wall matrix
(Mosier 2013). Moreover, LHW brings increased cellulose accessibility and produces
minimal inhibitory products (Kim et al. 2009). After LHW pretreatment, the major
changes in lignocellulose involve the limited deconstruction of cellulose, dissolution
of hemicellulose, partial removal of lignin, and carbohydrate degradation. Nitsos
et al. has been demonstrated that hemicellulose is almost completely solubilized
and deconstructed from biomass in hot water pretreatment at ~200 °C for 50 min
(Nitsos et al. 2016). Different from hemicellulose, cellulose was less affected by
LHW pretreatment. Less than 22% by weight of cellulose is degraded in wood and
herbaceous biomass treated with LHW at 200–230 °C (Mok and Antal 1992).

2.2.1 Steam Explosion

Steam explosion is an effective, environmentally safe, and industrially scalable

pretreatment method. This pretreatment results in the combination of structural
disruption of biomass by high-temperature steaming and explosive decompression
as well as autohydrolysis (Singh et al. 2015; Kumari and Singh 2018). Generally,
steam explosion was carried out under high-pressure steam (0.5–5 MPa) and temper-
ature intervals between 160 and 250 °C (Paudel et al. 2017). This method uses less
chemicals and does not result in unnecessary dilution of hydrolyzates, but the steam
explosion has some disadvantages, such as energy intense, inhibitory compounds
generation, and causing the partial ruin of carbohydrate-lignin matrix (Agbor et al.
2011). To improve the effectiveness of the steam explosion method, the addition of a
catalyst or impregnating agent such as H2 SO4 , SO2 , or CO2 can decrease the produc-
tion of inhibitors and improve the enzymatic hydrolysis on the biomass (Mosier et al.
2005; Sun and Cheng 2002).
The most attractive catalyst is CO2 due to its cheap cost of CO2 , low corrosion,
low toxicity, and the possibility of biomass having high solids content (Kucharska
et al. 2018). In most cases, CO2 explosion applies supercritical CO2 or high-pressure
CO2 to help the biomass digestibility (Zheng et al. 1998; Morais et al. 2015; Hendriks
and Zeeman 2009). Moreover, adding CO2 in mild condition before steam explosion
pretreatment also has been studied can increase the surface area of the material
which can make the enzymes more easily to enter the material during the hydrolysis
process. Pretreatment of oil palm empty fruit bunch (EFB) using steam explosion
with CO2 as an impregnating agent gives more slightly lower crystallinity index,
more disrupt biomass, and increasing enzymatic hydrolysis of EFB as compared to
pretreatment EFB using conventional steam explosion (Triwahyuni et al. 2021). The
changes in the surface morphology of EFB using SEM results in the untreated EFB
fiber had a rigid surface, with a layer of matrix material, meanwhile, the surface
of EFB after CO2 -added steam explosion, appeared uniform pores in the surface of
EFB. It indicated that some silica was removed from EFB (Triwahyuni et al. 2021).
4 R. Maryana et al.

2.2.2 Alkali Explosion

Alkali explosion is one of chemical pretreatment which can provide a high yield
of delignification. Alkali-heat pretreatment uses heat and the alkali such as sodium
hydroxide, sodium carbonate, and alkaline peroxide to dissolve lignin and reduce the
crystallinity of lignocellulose by swelling, and also enlarge the specific surface area
of cellulose (Rodrigues et al. 2016). For example, the delignification of EFB using
NaOH explosion reached 58.36% and the percentage of cellulose could increase from
30.16% (untreated EFB) to 63.82% (Muryanto et al. 2015). Pretreatment of corn straw
with NaOH-heat pretreatment obtained lignin and hemicellulose removal efficiency
of 54.09% and 67.67%, meanwhile, the relative content of cellulose enhanced to
51.65% (Zhao, et al. 2021; Lopez et al. 2019; Shahabazuddin et al. 2018). The ability
of NaOH to dissolve lignin is due to the opening of aromatic rings of lignin caused
by the use of high temperatures and pressure, then, the resulting explosive effects
can dissolve these components (Rezania et al. 2020). However, this method requires
high temperature and pressure and takes a long time as well as high treatment cost
(Muryanto et al. 2015; Kumar et al. 2020).

2.2.3 Ammonia Fiber Explosion

Ammonia fiber explosion is one of physicochemical pretreatment under high pressure

with ammonia and oxyhydrogen ions released from liquid ammonia, resulting in a
prompt rise in temperature and breaking the ester bond and ether bond between
hemicellulose lignin in lignocellulose (Zhao et al. 2020) as well as reducing the
cellulose crystal. AFEX has been demonstrated to be effective in low lignin-contained
lignocellulose pretreatment. The application of AFEX shows lignin degradability of
barley straw (Beauchemin et al. 2019) and corn stover (Mankar et al. 2021) achieved
24–1.3%.

2.2.4 Chemi-Extrusion Method

Application of the combination of alkaline and thermochemical can reduce energy

consumption and remove lignin (Chen et al. 2019). However, these combinations
still cannot meet the requirements for industrial scale that need efficient operational
cost. The extrusion method then be considered as one of the solutions because it
applies both chemical and physical methods. Moreover, in the biomass processing
temperature can be applied together with compression forces.
Extruder screws are used in the extrusion method and the single or twin screws are
usually used. The screws rotate to generate shear forces, an unaligned force acting
on one part of a reactor in a particular direction, among the biomass, screw, and
barrel. These forces generally increase the temperature and the pressure inside the
reactor which will change the biomass’s physical properties. It has been studied that
shear force will strengthen the rotation of biomass, the screw, and the barrel. Pressure
Current Progress on Biomass Pretreatment: The Key for Its Valorization 5

Fig. 1 Screw continuous reactor model that has been installed at research center for chemistry—
BRIN

and temperature increased during the process will lead to the breakage of biomass
lignocellulosic fibers (Khan et al. 2021).
It has been reported that screw rotation speed, residence time in the screw reactor,
and temperature influenced the lignin removal percentage by using 10% NaOH
(Maryana et al. 2022) (Fig. 1).
Moreover, screw speed is responsible for the reduction of the length of fiber and
increasing of the surface area of biomass that improve sugar yield after the sacchari-
fication process (Karunanithy and Muthukumarappan 2010). The screw extrusion
method is usually combined with chemical and thermo-chemical methods. The
second generation of bioethanol production has used the extrusion method. For
instance, the application of a single screw extruder that used a corn stover by varying
different screw speeds and temperatures (Karunanithy and Muthukumarappan 2010;
Jorge et al. 2006).
6 R. Maryana et al.

2.3 Chemicals Pretreatment and Application of Deep

Eutectic Solvent (DES)

2.3.1 Chemicals Pretreatment

Chemical pretreatment is generally divided into alkaline and acid pretreatment. Alka-
line pretreatment can use NaOH, Ca(OH)2 , KOH, or alkaline peroxide, a combina-
tion of NaOH and H2 O2 (Alvarez-vasco and Zhang 2013). The mechanism of alka-
line pretreatment is the saponification of intermolecular ester bonds that cross-link
silane and other components (Sun and Cheng 2002). However, alkaline pretreat-
ment has some disadvantages, such as longer time pretreatment, more water for
biomass washing, and more expensive waste recovery costs (Rezania et al. 2017).
Acid pretreatment uses low concentrations of mineral acids such as sulfuric acid
(H2 SO4 ) or hydrochloric acid (HCl). Sometimes concentrated acid can be used to
conduct pretreatment at low temperatures. Acid pretreatment commonly hydrolyzes
cellulose and hemicellulose to get monomeric sugar that can be converted to some
biobased chemical. Produce inhibitor products, hard to separate, and corrosive are
the disadvantages of acid pretreatment. Therefore, many studies have been conducted
to develop green solvents in the delignification process. The ionic liquid is widely
developed because it is environmentally friendly compared to conventional solvents
(Nguyen et al. Oct. 2010; Pena-Pereira and Namieśnik 2014). However, the ionic
liquid is still as expensive as solvent pretreatment.

2.3.2 Deep Eutectic Solvent (DES)

Deep eutectic solvent (DES) has been introduced as the new green solvent (Abbott
et al. 2001). DES is a solvent consisting of a mixture of a high melting point salt (i.e.,
hydrogen bond acceptor or HBA) with a molecular hydrogen bond donor (HBD) in
a specific ratio to form a liquid. DES has properties like an ionic liquid, although the
DES ion content is lower. DES began to be used in the pretreatment process because
of several advantages such as cheaper material costs, non-toxicity, ease of manu-
facturing process, and more biodegradable and biocompatible (Haldar and Purkait
2021). Besides that, most DES have high solubility for lignin and poor solubility
for cellulose and hemicellulose (Francisco et al. 2012). DES can be explicitly made
according to the required properties that are needed in the process. Table 1 shows
the DES from a variation of HBD and HBA.
DES in the pretreatment process is divided into several types depending on the
HBD used. Several types of DES include carbohydrate-based DES, such as CHCl-
glucose and CHCl fructose. Polyalcohol-based DES consisted of CHCl-glycerol,
CHCl-ethylene glycol, and CHCl-Propanediol. Acidic DES is commonly used in
biomass processing, containing HBD such as acetic acid, lactic acid, glycolic acid,
oxalic acid, and citric acid. Basic-based DES contains amide, amine, and imidazole
Current Progress on Biomass Pretreatment: The Key for Its Valorization 7

Table 1 Deep Eutectic solvent in the variation of HBA and HBD

HBA HBD Ratio HBA:HBD Molar masses T melting point
(kg/mol) (°C)
Choline chloride Urea 1:2 0.2597 12
Malonic acid 1:1 0.24368 10
Lactic acid 1:2 0.31978 −78
Oxalic acid 1:1 0.26569 −40
dehydrate
Ethylene 1:2 0.26376 −66
glycol
Glucose 2:1 0.43237 15
Choline acetate Urea 1:2 0.27168 <−20
Choline fluoride Urea 1:2 0.24329 1
Acetylcholine+ Cl− Urea 1:2 0.26627 −14
Glucose 1:1 0.36382 −7
Proline Oxalic acid 1:1 – −14.5
Lactic acid 2:1 – −36.7
Betaine Urea 1:2 – −42.5
Oxalic acid 1:1 – −17.2
Lactic acid 2:1 – −46.9
Glucose Citric acid 1:1 – 9.8
Tartaric acid 1:1 – −18.3
AlCl3 Urea 1:1 – <25
K2 CO3 Ethylene 1:10 – −122
glycol
Source Marcus and Solvents (2019)

compounds. CHCl-Urea is a basic DES commonly used in biomass processing (Tan

et al. 2019).
The pretreatment performance may be impacted by pretreatment parameters,
including temperature, duration, or the solid-to-liquid ratio, and DES type also affects
DES pretreatment. For example, DES Pretreatment conducted by Majová et al. (2017)
used hardwood kraft pulps as raw materials and DES solution from a mixture of ChCl-
Oxalic Acid with a 1:1 molar ratio (Majová et al. 2017). The pretreatment process
was carried out for 1 h at a temperature of 60 °C. The results obtained from DES
pretreatment were able to degrade lignin in hardwood kappa pulp by 38.7%. Another
study conducted by Okur and Koyuncu (2020) regarding the delignification of rice
husks using a DES solution of ethylene glycol-citric acid obtained optimal results at
a temperature of 120 °C and a processing time of 4 h showed that 57.33% of lignin
was degraded (Okur and Koyuncu 2020). DES pretreatment consisting of K2 CO3 and
glycerol was conducted on wheat straw and corn stalks at a temperature of 80–100 C
for 8–24 h. This pretreatment can reduce the lignin content in wheat straw and corn
8 R. Maryana et al.

Table 2 DES pretreatment

DES Molar Biomass Process Delignification Sources
ratio condition (%)
CHCl:ethylene 1:2 Switchgrass 130 °C, 30 24 Chen and Wan
glycol min, S/L 10 (2018)
CHCl:glycerol 1:2 Oil palm trunk 100 °C, S/L 49 Zulkefli et al.
5 (2017)
CHCh:lactic acid Poplar wood 145 °C, 9 h, 79 Alvarez-Vasco
S/L 10 et al. (2016)
CHCl:lactic acid 1:5 OPEFB 120 °C, 8 h, 88 Tan et al.
S/L 10 (2018)
CHCl:formic acid 1:2 Akebia herbal 120 °C, 8 h, 41 Yu et al. (2018)
residue S/L 10
CHCl:urea 1:2 OPEFB 120 °C, 8 h, 34 Tan et al.
S/L 10 (2018)
CHCl: 1:6 Wheat straw 90 °C, 5 h, 81 Zhao et al.
monoethanolamine S/L 5 (2018)
CHCl:4-catechol 1:1 Switchgrass 160 °C, 3 h, 49 Kim et al.
S/L 5 (2018)

stalks from 22% to 12.1% and 24% to 16.3%, respectively (Suopajärvi et al. 2020).
DES pretreatment in variation DES type and operation condition is shown in Table 2.
DES is used in the pretreatment process in various delignifications. Some types of
DES can reduce the lignin content above 60%. However, some are still low. Like the
chemical pretreatment process, DES pretreatment can also be combined with other
technologies to improve its performance. Some integrations can include combining
microwave, ultrasonic, and sequential pretreatment with hydrothermal, biological,
or another chemical (Tan et al. 2019).

2.3.3 Organosolv Pretreatment

Organic solvents such as methanol, ethanol, acetone, ethylene glycol, and tetrahy-
drofurfuryl alcohol are commonly used as pretreatment chemicals in the organosolv
method. Moreover, catalysts like salicylic acid and organic acid as well as sodium
hydroxide are generally used in this method (Cheah et al. 2020). It was reported
by Mirmohamadsadeghi et al., that pretreated Elmwood, pinewood, and rice straw
resulted in lignin removal of 27, 21, and 37.7% respectively, after pretreated by
using 1% (w/w) sulfuric acid at 150–180 °C for 30–60 min in 75% aqueous ethanol
(Mirmohamadsadeghi et al. 2014).
Ethanol is the most common solvent due to its economical price, not harmful
properties, ease of mixing with water, and good soluble solvent for lignin. In the
case of softwood delignification, ethanol can enhance the impregnation and support
lignin fragments in the biomass to the liquor solution (Rinaldi et al. 2016). It was
Current Progress on Biomass Pretreatment: The Key for Its Valorization 9

studied that lignin removal of 16 for 50% water–ethanol system at 175 °C for southern
yellow pine that was pretreated for 80 min (McGEE and APRIL 1982).

2.4 Biological Pretreatment

The effectiveness of a biological pretreatment is determined by several factors

including composition of biomass, moisture content, incubation time, temperature,
pH, and type of microorganism (Sindhu et al. 2016). Based on the composition of
biomass, a suitable microbial consortium must be used for the effective removal of
lignin and hemicellulose from the biomass.

2.4.1 Microorganism Used in Biological Pretreatment

Many researcher-published articles revealed that common microorganisms used in

biological pretreatment are bacteria including Bacillus, Actinomycetes and some
known fungi are able to degrade organic matter (Poszytek et al. 2016). Microbial
consortia consisting of cellulolytic bacteria of the genus Bacillus, Streptomyces,
Candida, and Aspergillus exhibit broad-spectrum biodegradation. It was also that
many potent brown rot fungus, white rot fungus, and softening fungi. Among the
three fungi, the most effective for upstream treatment of lignocellulosic materials is
white rot fungi. The white rot fungus helps in delignification which in turn improves
the enzymatic saccharification rate, especially, Phanerochaete chrysosporium, Ceri-
poriopsis subvermispora, Pleurotus ostreaus, Ceriporia lanceolata and Cyanthus
stercoreus, which selectively degrading lignin by producing a wide variety of enzyme,
i.e., laccase, lignin peroxidase (LiP), and manganese peroxidase (MnP) (Maurya et al.
2015).
Currently, there is a need for unique consortia for biological pretreatment. Effec-
tive biodegradation of lignocellulosic biomass takes place by biodegradation by
synergistic action of microbial consortiums including various bacteria and fungi.
There are several advantages of using microbial consortium which include increased
adaptability, improved productivity, improved enzymatic saccharification efficiency,
control of pH during sugar utilization, and increased substrate utilization (Kalyani
et al. 2013).
The development of an eco-friendly simultaneous pretreatment and saccharifi-
cation (SPS) methodology using a cocktail of hydrolytic and oxidizing enzymes
from a fungal consortium was reported by Dhiman et al., 2015 (Dhiman et al.
2015). The novel laccase effectively functioned as a detoxifying agent. This process
completely eliminates the use of hazardous chemicals. Pretreatment and saccharifi-
cation conducted in the same vessel make the process economically viable, reduce
energy consumption, and generate a simple process for the removal of residual
biomass.
10 R. Maryana et al.

Table 3 Microorganism used in the biological pretreatment

Microorganism Biomass Major effects
Punctualaria sp. Bamboo culms for bioethanol 50% of lignin removal
TUFC20056 production
Irpex lacteus Corn stalks 82% of hydrolysis yield,
28 days pretreatment
Fungal consortium Straw Seven-fold increase in
hydrolysis
Pleurotus ostreatus/P. Eucalyptus grandis saw dust Twenty-fold increase in
pulmonarius hydrolysis
Phanerochete Rice husk Cellulase, xylanase, lignin
chrysosporium peroxidase, glyoxidase, and aryl
alcohol oxidase were produced
during 18 days pretreament
Fungal consortium Corn stover 43.8% lignin removal/
seven-fold increase in
hydrolysis
Ceriporiopsis Wheat straw Minimal cellulose loss and
subvermispora highest sugar yield up to 44%
after 10 weeks
Ceriporiopsis Corn stover 2–threefold increase in reducing
subvermispora sugar yield
Fungal consortium Plant biomass Complete elimination of the use
of hazardous chemicals

Different biological pretreatment strategies are involved in the pretreatment of

lignocellulosic biomass and its advantages (Sindhu et al. 2016) (Table 3).
Biological pretreatment is considered as an inexpensive process when compared
to other pretreatment processes such as AFEX and organo solvent. Large-scale oper-
ation leads to high operational costs since pretreatment is to be carried out in sterile
conditions and this increases the cost of the process. One approach to reducing
the pretreatment time is by applying a combination of biological processes with
physical and chemical methods. Employing more potent microbial consortia as well
as the development of specific reactors for biological pretreatment can reduce the
pretreatment time as well as the loss of carbohydrate significantly.

2.5 Pretreatment of Biomass with High Energy of Radiation

In order to produce a digestible raw material and prevent the target material from
degrading and the production of harmful by-products, an efficient pretreatment
should break down the lignocellulose structure. To enhance the available surface
area and the size of pores in lignocellulosic materials, cellulose is physically sepa-
rated by removing lignin and hemicellulose as well as breaking down the crystallinity
Current Progress on Biomass Pretreatment: The Key for Its Valorization 11

of the substance. Any pretreatment must be economically advantageous (Maryana

et al. 2014, 2017, 2020).
Among other physical methods, high-energy radiation is regarded as an attractive
method for the pretreatment process of biomass (Duarte et al. 2012; Khan et al. 2006;
Mohd Asyraf Kassim et al. 2016). In general, radiation processing technology onto
lignocellulosic material is defined as a radiolysis reaction that induces lignocellulosic
material breakdown using radioisotopes like cobalt-60 (Co-60) or cesium-137 (Cs-
137), or an electron beam generated by an electron accelerator. Generally, biomass
materials degrade similarly under gamma and electron beam irradiation. As electro-
magnetic radiation, an electron beam has a significantly lower penetration but with
a higher dose rate compared to gamma rays. The wide application of electron beams
to the degradation of biomass is due to a number of its inherent benefits. Since
electron particle energy is immediately transferred to molecules of biomass, thus
no need for heating and catalyst during the irradiation. Because the electron beam
can be precisely controlled by adjusting the accelerator’s operating condition, the
irradiation process by using EBM is highly reproducible, controllable, and precise.
High energy radiation from electron beams, X-rays, or gamma rays can create ions
and/or radicals that initiate chemical processes that primarily cause chemical bond
cleavages and decrease the molecular weight of the biomass by increasing the irra-
diation dose. The high-energy radiation produced during this process has the poten-
tial to change the properties of cellulosic biomass, due to 1,4-glycosidic linkages
being ruptured by gamma radiation and electron beams. Subsequently increasing
the lignocellulosic biomass’ specific surface area, decreasing the polymerization
and crystallinity of cellulose, hydrolyzing hemicellulose, and partially depolymer-
izing lignin (Al-Masri and Zarkawi 1994). The irradiation of biomass with an elec-
tron beam causes the complex carbohydrates to become soluble, which facilitates
processing, such as enzymatic hydrolysis for the production of bioethanol (Danu
et al. 2012).
The effectiveness of high-energy radiation as a pretreatment technique on ligno-
cellulosic material has been studied intensively (Hyun Hong, et al. 2014). Irradiation
of Jute fibers by gamma irradiation with the doses up to 100 kGy and by an electron
beam with a dose up to 400 kGy for oil palm empty fruit bunch (OPEFB) resulted in a
decrease in the weight fraction of large particles and an increase in the weight fraction
of small and medium particles both of Jute fibers and OPEFB (Khan et al. 2006; Danu
et al. 2012). It was revealed that the chemical stability of irradiated fibers gradually
decreased with an increase in irradiation dose. Studies using thermogravimetry and
differential scanning calorimetry revealed a considerable loss in thermal stability.
The wide-angle X-ray diffraction analysis revealed that lignocellulose’s chemical
response to radiation caused structural alterations in cellulose. The solubility of irra-
diated OPEFB in water and 1% sodium hydroxide solution also increases. Based on
the bands shown in the fingerprint area and the degradation found using FTIR anal-
ysis, cellulose, hemicelluloses, and lignin were identified as the primary constituents
of OPEFB and were subject to C–O stretching and C–H deformation (Danu et al.
2012).
12 R. Maryana et al.

Combining irradiation with additional pretreatment techniques, both physical and

chemical, can improve the effectiveness of gamma pretreatment. Pretreatment is more
effective in relatively milder conditions when multiple treatments are used because
they increase each other’s effects (Duarte et al. 2012; Wang et al. 2012; Abo-State
et al. 2014; Chung et al. 2012; Saini et al. 2015). The yield of total reducing sugar
following saccharification and pretreatment of rice straw during the production of
ethanol was enhanced by combining milling, autoclaving, and gamma irradiation
(700 kGy) (Abo-State et al. 2014). When sugarcane bagasse was pretreated by using
electron beam processing (EBP), after 24 and 48 h of incubation, the conversion
yield of cellulose to glucose improved from 8 to 12% becoming 15%. The yield
of the enzymatic hydrolysis of cellulose improved with EBP and thermal treatment
(60 min at 180 °C), reaching 71.55%. After EBP, hemicelluloses were completely
hydrolyzed (Kucharska et al. 2018).
The effect of gamma irradiation on the saccharification of Undaria sp. for
bioethanol production was evaluated by Yoon et al. (2012). The irradiation has been
employed as a pretreatment in conjunction with dilute acid hydrolysis. Undaria sp.
wet samples were exposed to radiation at 22 °C with irradiation dose between 10 and
500 kGy and a dose rate of 10 kGy/h. By increasing the gamma irradiation dose, the
concentration of reducing sugars increased. The reducing sugar content was 0.017
g/L for unirradiated samples, but it rose to 0.048 g/L for a sample irradiated by the
dose of 500 kGy. This reveals that the combined method of gamma irradiation and
diluted acid hydrolysis considerably enhances the saccharification process for the
generation of bioethanol from materials derived from marine algae.
To improve enzymatic hydrolysis for the manufacture of bioethanol, poplar bark
was pretreated with 3% w/w sulfuric acid and gamma radiation (0–1000 kGy). With
an increasing irradiation dose, the yields of reducing sugar were slightly increased,
going from 35.4 to 51.5%. After pretreatment by the addition of dilute acid, the yield
of reducing sugar reached 56.1%. These results clearly demonstrated that soluble
sugars were released more quickly and to a higher amount in poplar bark that had
been prepared with diluted acid as opposed to bark that had been exposed to gamma
radiation. A significant increase in reduced sugar yield (83.1%) was found after
combination pretreatment as compared to individual pretreatment, suggesting that the
convertibility of poplar bark may be improved after combined pretreatment (Chung
et al. 2012).
On the other hand, Henniges et al. (2013) found that tuning the irradiation process,
independent of the dosage, is possible to increase oxidation by introducing iron(II)
ions. They observed the same amount of oxidized cellulose functionalities at better
preservation of molar mass (less cellulose chain degradation). They found that
the addition of hydrogen peroxide speeds up the irradiation-induced degradation.
Another finding is that wet irradiation samples are more impacted than dry irradiated
samples. When wet straw was utilized instead of dry straw, the required dose of
gamma irradiation was decreased from 500 to 10 kGy.
According to some studies and reports, irradiating biomass for pretreatment uses
less energy than other methods (Mosier et al. 2005; Mohd Asyraf Kassim et al. 2016;
Henniges et al. 2013). This method is more effective and efficient for producing the
Current Progress on Biomass Pretreatment: The Key for Its Valorization 13

intended product since it is selective and easy to control. On the other hand, there
are certain points that require to be considered due to the studies conducted in recent
years only focusing on biomass irradiation at a lab scale. Meanwhile, validating the
result requires pilot-scale investigations, and the cost of the commercial deployment
must be investigated throughout technology development (Saini et al. 2015).

3 Conclusion

The Biorefinery concept is a potential approach to respond to the depletion of fossil

resources by utilizing lignocellulosic biomass. Eco-friendly and efficient biomass
pretreatment methods in order to fractionate cellulose, hemicellulose, and lignin are
continuously developed. By studying several recent pretreatment methods, it can be
concluded that the process has to overcome the recalcitrant of lignin in the chemical
bonding complex with cellulose and hemicellulose. The cellulose crystallinity and
sugars recovery are also the main concern of the pretreatment methods. Different
pretreatment approaches that considering energy consumption, cost-effectiveness,
percentage of lignin removal, further utilization of the cellulose, hemicellulose,
lignin, and environmentally friendly process can be chosen for addressing the
challenges in the biomass pretreatment method.

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Recent Updates on Biopolymers:
Precursors, Process, Properties,
Challenge, and Future Perspectives

Aisyah Hanifah, Arfiathi, Melbi Mahardika, Riska Sumirat,

Rossy Choerun Nissa, and Yeyen Nurhamiyah

Abstract Fossil sources are common raw materials in the polymer industry because
they are cost-effective and ensure a straightforward manufacturing process. However,
the insufficient supply of fossil sources failed to afford adequate feedstock for
polymer production in the future. Fossil sources are projected to reach a satura-
tion point where supply would be less than demand due to the increasing human
population. Another important concern is the fact that fossil-based polymer creates
several environmental problems, such as non-degradable products, air pollution, and
wastewater contamination (Okkerse and Bekkum 1999). These two main reasons
are the main factors why the switch of the raw materials of polymers from fossil
to renewable materials is necessary, and the research on biobased polymeric mate-
rials becomes an interesting yet urgent topic. In this chapter, we review the current
updates on the development of biopolymers. The precursors, technological processes,
and updates on the currently available biopolymers are being reviewed. Challenges
and future perspectives are also being discussed.

Keywords Biopolymers · Starch · Seaweed · Plant oil · Polymerization

A. Hanifah · M. Mahardika · R. C. Nissa · Y. Nurhamiyah (B)

Research Center for Biomass and Bioproducts, National Research and Innovation Agency
(BRIN), Jl. Raya Bogor KM. 46 Cibinong, Bogor, Indonesia
e-mail: [email protected]
Arfiathi · R. Sumirat
Department of Agricultural Industrial Technology, Universitas Padjajaran, Jl. Raya Bandung
Sumedang KM.21, Bandung, Indonesia
M. Mahardika · Y. Nurhamiyah
Research Collaboration Center for Biomass and Biorefinery between BRIN and Universitas
Padjadjaran, Bandung, Indonesia

© The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2024 19
M. A. R. Lubis et al. (eds.), Biomass Conversion and Sustainable Biorefinery, Green
Energy and Technology, https://doi.org/10.1007/978-981-99-7769-7_2
20 A. Hanifah et al.

1 Introduction

Polymer materials are the most widely used applications in everyday life. The
production of synthetic polymers continues to grow, such as the use of polycar-
bonate, polyvinyl chloride, polyethylene (PE), polyethylene terephthalate (PET),
polypropylene (PP), poly(methyl methacrylate), and polystyrene, with an estimated
1.1 billion tonnes in 2050 (Geyer 2020). Increasing awareness of the environment and
limited fossil resources have led to the importance of replacing biobased materials
(biopolymer) to reduce greenhouse gas emissions. Biopolymers are polymers derived
from natural and renewable sources obtained from chemical polymerization reactions
(Das et al. 2022; Mohanty et al. 2022). There are classifications of biopolymers:
polysaccharides, nucleic acids, polyamides (PAs), polythioesters (PTEs), polyanhy-
drides, polyphenolic biopolymers, polyisoprenoids, and polyoxoesters (Sharma and
Dhingra 2021). The most widely available biopolymers on earth are cellulose, lignin,
rubber, and protein (Bayón et al. 2018; Mikus and Galus 2022). The advantages of
biopolymer materials are low cost, easy to form, superior properties such as resis-
tance to water, biodegradable, and mechanical properties that can be improved for
various applications. However, the increased use of biopolymers is only about 1%
of the total industrial materials produced each year (Ranganathan et al. 2020).
This chapter book provides comprehensive information on biopolymers, current
research being developed, process, properties, production on a lab or commercial
scale, future, and perspective. The first is a summary of the sources of raw materials
derived from biomass for the production of biopolymers and bioprocesses. Then,
the precursors of biopolymers will be discussed in detail with a focus on current
production. The mass production of biopolymers raises concerns about economic
and environmental sustainability (Chang et al. 2016; Maraveas 2020). Consequently,
the production of biopolymers from renewable biological sources has received great
attention during the last decade. Currently, most of the precursors of biopolymers are
obtained from biomass sources such as starch, seaweed, and plant oil, which are in
direct competition with food production (de Jong and Jungmeier 2015; Popa 2018;
Gajula and Reddy 2021). To build a biobased economy, non-edible raw materials
must be used for the production of biopolymers. All petrochemical-derived chemicals
can be replaced with biopolymer materials produced from biobased materials, which
will significantly contribute to the economic progress (Isikgor and Becer 2015; Nanni
et al. 2021).
Recent Updates on Biopolymers: Precursors, Process, Properties … 21

2 Precursor of Biopolymers

2.1 Starch

Starch is a polysaccharide that plants produce to store energy. It is stored intra-

cellularly as spherical granules ranging in size from 2 to 100 μm. The majority
of market-available starches are derived from grains such as wheat, rice, and corn
or from tubers such as potato and cassava (tapioca) (Jiang et al. 2020). Starch is an
amorphous, soft white powder without any sweetness and insoluble in water, alcohol,
and ether. The starch granules are densely packed in a semicrystalline structure with
a density of about 1.5 g cm−3 . Specific shapes, sizes, and surfaces (smoothness or
roughness) of starch granules from various botanical sources play a crucial role in
functionality and digestibility (Magallanes-Cruz et al. 2017).
Amylose and amylopectin are glucans that combine to form starch; most starches
consist of 10–20% water-soluble amylose and 80–90% water insoluble amylopectin
(Pokhrel 2015). Amylose is a linear polymer polysaccharide with 1–4-D-glucose
units. Amylase’s structure contributes to the gelling properties of heated and cooled
starches. Amylopectin has a backbone of α-(1 → 4) linkages as well, but it is also
branched through α-(1 → 6) linkages to the extent of 4–5%. Amylopectin is respon-
sible for the thickening during starch preparations, but it does not make a contribution
to the formation of a gel (Pokhrel 2015).
Starch has traditionally played a significant role as a food ingredient, but it is
finally starting to be used in other applications, such as textiles, paper, pharmaceu-
ticals, and pharmaceuticals. Starch can be applied in various ways, including pure
starch as thermoplastics, starch as blends formed by combining other polymers, and
copolymers formed by combining synthetic polymers (Pokhrel 2015; Encalada et al.
2018). There have been many studies conducted (Sriroth and Sangseethong 2006;
Ibrahim et al. 2017; Area et al. 2019; Mesias and Murillo 2020) related to the devel-
opment of starch-based products, one of which is thermoplastic starch that can be
applied as a raw material for bioplastics. As a result, the use of starch in biopoly-
mers is very promising, especially given that natural starch comes from agricultural
sources, which has the advantages of abundant supply, ease of filling, low cost, and
a wide range of applications and modifications in non-food products.

2.2 Seaweed

Seaweeds are macroscopic, multicellular, and benthic algae. Seaweed has a rapid
growth rate, resulting in a fast accumulation of biomass (Carina et al. 2021; Perera
et al. 2021). Edible marine macroalgae, or seaweed, is well-known for being
extremely nutrient-rich as both food and culinary additives. Coastal populations
all across the world, including Asians (Chinese, Japanese, Indonesian), Europeans
(Irish, Icelandic), and South Americans, among others, have harvested wild seaweed
22 A. Hanifah et al.

for food and other uses. Although wild seaweeds are still gathered from the coastal
areas of several nations, farmed seaweeds make up the majority of the seaweed and
seaweed products produced today (Padam and Chye 2020). More than US$6 billion
is thought to be generated by the international trade of seaweed each year. China
continues to be the world’s largest seaweed producer, producing 35.7 million tons,
or roughly 56.75% of the total, followed by Indonesia (27.86%) and Korea (5%)
(FAO 2021).
Around the world, there are more than 10,000 different varieties of seaweed. 10
seaweed species are intensively grown globally, out of the 221 species of seaweed
that have been commercially exploited (Padam and Chye 2020). Seaweeds remain an
unexplored resource with huge potentials such as food ingredients, cosmetics, agri-
chemicals, edible foods, fishmeal, biomaterials, and bioenergy molecules, yet also
having an important aspect in the ecosystem and economic profits (Padam and Chye
2020; Carina et al. 2021; Chudasama et al. 2021). Based on their color, seaweeds
can be divided into three groups: red seaweeds (Rhodophyceae), green seaweeds
(Chlorophyceae), and brown seaweeds (Phaeophyceae) (Perera et al. 2021).
Three major polysaccharides that are present in seaweed are alginate, agar, and
carrageenan. The majority of polysaccharides can swell in water under ambient
conditions or are water soluble (at high temperatures), producing colloidal, highly
viscous solutions or dispersions with pseudoplastic flow properties. Due to their
natural functional properties, such as thickening, stabilizing suspensions and emul-
sions, water retention and binding, and gelling, polysaccharides are advantageous
in a range of applications. Terrestrial plants do not possess polysaccharides with
specific and distinct characteristics that are received from a significant oceanic source
(Chudasama et al. 2021).

2.3 Plant Oil

Plant oil is a potential source for biopolymers since it offers availability, low cost,
easy processing route, and chemical functionality. Plant oil is a promising source
for polymer building blocks as it resembles the hydrocarbon composition of petro-
chemicals. Globally, plant oil production capacity was 209.42 million metric tons in
2019/2020, and only around 20 million tonnes were used in the chemicals industry
(Stempfle et al. 2016; USDA Foreign Agricultural Services 2021).
The total carbon atoms in the chain and the way carbon atoms are joined control
the properties of fatty acids. Single carbon atoms may join each other and connect to
the hydrogen atom by a single atom. Carbon atoms are also linked by a double bond
and joined to only one hydrogen each. The double bond increases the ratio of carbon
atoms to hydrogen because of the absence of two hydrogen atoms. The disappearance
of hydrogen generates fatty acids that are less “saturated” with hydrogen. This kind of
bond is called saturated, while the double bond fatty acid is called unsaturated (Pond
1998). Unsaturated fatty acids are unstable compared with saturated fatty acids. The
presence of a double bond makes the carbon atoms in this fatty acid more reactive.
Recent Updates on Biopolymers: Precursors, Process, Properties … 23

Fig. 1 Ester linkage hydrolysis (Mallakpour and Rafiee 2012)

Double bonds adjust the crystal packs; they limit the rotation of a carbon atom and
bend the chain (Gurr et al. 2002) (Fig. 1).
Polymer made from plant oil offers material with beneficial properties such
as hydrophobicity, flexibility, low melting temperature, and biodegradability.
Polyamide (Fan et al. 1997; Hablot et al. 2010), polyester (Quinzler and Mecking
2010; Testud et al. 2017), polyurethane (Hojabri et al. 2010; More et al. 2013), and
self-healing materials (Cordier et al. 2008) are examples of polymers that have been
synthesized from fatty acids.

2.4 Others

Classification biopolymers are (1) the agro-polymers that comprise polysaccharides,

proteins, and lipids; and (2) bio-polyesters (biodegradable polyesters), such as poly-
hydroxyalkanoate (PHA), polylactic acid (PLA), and aromatic and aliphatic co-
polyesters. Biopolymers classified as agro-polymers are biomass products obtained
from agricultural materials, such as polysaccharides, proteins, and lipids. Bio-
polyester is subdivided based on its source. The polyhydroxyalkanoate (PHA) group
is obtained from activity-derived microorganisms obtained by extraction. Exam-
ples of PHAs include Poly(hydroxybutyrate) (PHB) and Poly(hydroxybutyrate co-
hydroxy valerate) (PHBV). Another group is the polyesters obtained from biotech-
nology applications, namely by the conventional synthesis of monomers obtained
biologically, called the polylactide group. An example of a polylactide is polylactic
acid. The last group is obtained from conventionally synthesized petrochemicals
made from synthetic monomers. This group consists of polycaprolactone (PCL),
polyester amides, aliphatic co-polyesters, and aromatic co-polyesters.
24 A. Hanifah et al.

3 Production Process

3.1 Plasticization

Plasticizers are substances used to improve the flexibility, extensibility, and process-
ability of polymeric materials. Plasticizers have the property of lowering the melting
temperature, melt viscosity, and glass transition temperature, as well as having high
boiling points, molecular weights ranging from 300 to 600 and elastic modulus of
polymers without changing their chemical nature (Drobny 2015; Subramanian and
Varade 2017). Plasticizers are classified into three types: polyol (glycerol, polyethy-
lene glycols, and propylene glycol), oils/glycerides (acetylated monoglycerides,
castor oil, and fragmented coconut oil), and organic ester (phthalate ester, citrate ester,
triacetin, and dibutyl sebacate) (Foroughi-dahr et al. 2017). The amount of plasticizer
added to a polymer is determined by the intended result. To improve the workability
of the polymer melt, a small amount of plasticizer can be added, contrary to the
impression that large additions can completely transform the product’s properties
(Harrison 2002).
When starch is plasticized, the plasticizers break the inter- and intra-molecular
hydrogen bonds existing in the starch, causing it to become thermoplastic. The
basic crystalline structure of the starch granules is broken by the plasticizer, which
permeates them. Thermoplastic starch (TPS) becomes extremely brittle at low mois-
ture contents while becoming more flexible and softer at high moisture amounts.
When wet, it also rapidly loses strength. The effectiveness of TPS in situations with
extremely low or high humidity is obviously greatly constrained by its sensitivity to
moisture (Janssen and Moscicki 2009).
Plasticizers work by decreasing interactions between molecules and spreading
them out. Low viscosities and temperature coefficients of viscosity are character-
istics of efficient plasticizers. A polymer with a low molar mass and a higher free
volume is frequently used as a plasticizer. A crucial need is that the plasticizer be
perfectly blended at the molecular level, either in a homogenous mixture with the
polymer or with the plasticizer itself (Janssen and Moscicki 2009). A plasticizer’s
principal function is to improve process efficiency and flexibility. The glass transition
temperature is lowered by the plasticizer (T g ). Due to different environmental issues
and rising petroleum product prices, plasticizers are becoming more and more impor-
tant. So that the process can be made both affordable and biodegradable, plasticizer
should be natural, affordable, and renewable (Khan et al. 2017).
The research about TPS and the effect of plasticizer processes obtained by melt
processing was carried out by (Carvalho et al. 2006) using 1,4-butanediol (BUT),
1,6-hexanediol, 2,5-hexanediol, glycerol, ethylene glycol (EG), diethylene oxide
glycol (DEG), trimethylene oxide glycol (TEG), ethylene glycol monomethyl ether,
D-sorbitol (SOR), propylene glycol (PG), polyethylene oxide glycol (PEG) 300 and
600, and polypropylene-oxide glycol (PPG). The results showed that compounds
containing OH had a good impact on the characteristics of TPS prepared by melt
mixing.
Recent Updates on Biopolymers: Precursors, Process, Properties … 25

3.2 Polymerization

Similar to fossil-based polymers, polymerization is a common technique to produce

biobased polymers. Polycondensation and ring-opening polymerization (ROP) are
examples of polymerization to get aliphatic biopolymers. Condensation polymeriza-
tion is a chemical reaction with the elimination of a small molecule, which results
in the repeat units in polymers having less atoms than when they are in the pres-
ence of monomers (Young and Lovell 2011). This technique was found by Wallace
Carothers who discovered the condensation polymerization technique and linear
polymers such as polyamides and polyesters which are still useful today (Carothers
1936, 1937). An example of polycondensation in biopolymers is the polycondensa-
tion of poly glycerol sebacate (PGS) which starts from glycerol and sebacic acid.
PGS is a biodegradable and bioresorbable polymer which suitable for biomedical
applications such as tissue engineering (Rai et al. 2012). Glycerol is a by-product
of the saponification process from oleochemical plants meanwhile sebacic acid is
originated from fatty acid (Tan et al. 2013). According to Wang et al. (2002), PGS
was prepared in two stages: pre-polycondensation and crosslinking. The equimolar
amount of sebacic acid and glycerol was mixed under argon at 120 °C for 24 h with
the pressure reduced over 5 h. For the crosslinking stage, the pre-polymer is kept
reacted at 40 mTorr and 120 °C for 48 h (Fig. 2).
Meanwhile, ring-opening polymerization (ROP) is a polymerization technique of
cyclic monomers that produces a polymer with identical molecular formulae to those
of the monomers (Young and Lovell 2011). For example, biobased polyamides can
be prepared from monomers with cyclic structure (lactams). An example of lactams
is ε-caprolactone as the precursor of caprolactam for PA6 and ω-laurolactam for
PA12. Evonik produces PA12 on an industrial scale with the tradename Vestamid
E Even though the monomer is mostly produced from fossil-based laurin lactam,
currently, the alternative laurin lactam is being developed by Evonik from palm
kernel through biotechnological process shows the ring-opening polymerization of
12-aminodecanoic acid (laurin lactam) to obtain PA12.
Even though ring-opening polymerization can obtain high-molecular-weight
polymers, however, due to few available cyclic monomers (Zhao 2018; Santoro et al.
2020) only limited polymers can be obtained. On the other hand, the polycondensa-
tion process brings possibilities and flexibility that are incomparable since could use
a variety of monomers.

Fig. 2 The polymerization scheme of poly glycerol sebacate (PGS) (Wang et al. 2002)
26 A. Hanifah et al.

3.3 Blending

To modify the properties of biopolymers to the intended use, various ways are
employed for their modifications, including absorption of fillers and reinforcements,
blending, plasticization, and impact modification (Rajeswari et al. 2021). Blending is
the simple mix of polymeric components without chemical reaction to generate novel
materials with appropriate qualities. From an industrial perspective, this procedure
can be conducted by employing regular machines, so a costly investment is unneeded.
Polymer blends are applied in a wide variety of industrial applications. In comparison
to alternative polymerization processes and the production of novel monomers, this
technique can obtain a wide range of characteristics, which meet the standard of the
targeted application at an efficient cost and time.
The aim of blending might be the optimization of the material performance,
reduction of the sensitivity to water, cutting down the cost, and improvement of
the application properties. The most significant motivation for mixing could differ
from other methods.

3.4 Fermentation

Biopolymers which are constructed from microorganisms need certain nutrients and
restricted environmental conditions. They are created directly by fermentation or
indirectly by chemical polymerization of the monomers produced by fermentation.
Most biopolymers are biocompatible and do not negatively affect biological systems.
Biopolymers derived from bacteria are considered to be produced due to their defense
mechanisms or as storage materials (Mohan et al. 2016). Biopolymers are synthesized
by biological organisms and produced by processive enzymes that connect building
ingredients such as sugars, amino acids, and hydroxy fatty acids to achieve a high-
molecular-weight molecule. Bacteria can produce various ranges of biopolymers,
including polysaccharides (composed of sugars and sugar acids linked by glyco-
sidic linkages), polyesters (composed of hydroxy fatty acids linked by ester bonds),
polyamides (consisting of amino acids linked by peptide bonds), and polyphosphates
(polyPs; composed of inorganic phosphates linked by anhydride bonds) (Fig. 3).
PLA and PHAs are examples of biopolymers that belong to the polyester group and
are formed when microorganisms are used in the processing step. PLA is produced
from a low molecular weight organic acid synthesized through microbial fermenta-
tion called lactic acid (LA). This acid is utilizing renewable sources such as cane
sugar, corn, and sugar beets. PHAs are an extensive family of bio-polyesters manu-
factured by diverse bacteria to store carbon and energy. Fermentation is influenced
by several parameters, including the substrate, temperature, pH, oxygen, and bacteria
used. The term “substrate” refers to the fermentable material that includes the essen-
tial nutrients for the growth and production of fermented products by bacteria. The
Recent Updates on Biopolymers: Precursors, Process, Properties … 27

Fig. 3 Bacterial polymer granules as biomaterials (Moradali and Rehm 2020)

bacterial strains used in the fermentation process can be divided into heterofermen-
tative and homofermentative methods. This homofermentative process is constantly
utilized in industry.
Lemoigne discovered PHAs in 1943 (Lemoigne and Girard 1943); they are a
family of naturally occurring polyesters generated intracellularly by many prokary-
otes as carbon and energy storage polymers. In general, PHAs can be synthe-
sized via three distinct carbon source-based steps: step I is mainly applied by
poly(hydroxybutyrate) (PHB) PHB-producing organisms such as C. necator and
Bacillus sp., whereas steps II and III exist in mcl-PHA-producing Pseudomonas sp.
Different monomers can be produced from different bacterial strains with varying
carbon substrates of the microorganisms; consequently, co-polymer proportions can
be modified based on the targeted applications of the final product (Fig. 4).
For the synthesis of PLA, raw material starch/sugar is extracted and fermented
using lactic acid bacteria (LAB), which results in the generation of lactic acid. The
fermentation of starch and other polysaccharides (sugar-containing materials), which
are readily available from corn, sugar beet, sugar cane, potatoes, and other biomasses,
accounts for 90–95% of the global lactic acid generation. Normal fermentation
provides 85–90% of L(+) lactic acid and 70–80% of D(−) lactic acid, depending
on the carbon source. During fermentation, anaerobic lactic acid bacteria (LAB)
consume pyruvic acid, the last product of the Embden–Meyerhof–Parnas (EMP)
route, via glycolysis. Prior to this, the carbon source sugars are converted to pyruvic
acid. L-lactate dehydrogenase or D-lactate dehydrogenase can convert pyruvic acid to
lactate while influencing the stereospecificity of the lactic acid generated. Lactic acid
28 A. Hanifah et al.

Fig. 4 Schematic illustration of the biosynthetic steps for producing lactate-based polymers (Nduko
and Taguchi 2021)

is chemically processed and polymerized to create the final product by polyconden-

sation reaction, ring-opening polymerization, and azeotropic dehydrative condensa-
tion. Ring-opening polymerization produces a product with a high molecular weight,
making it the most practical method for producing PLA (Fig. 5).

Fig. 5 Lactic acid production by fermentation procedure (Moradali and Rehm 2020)
Recent Updates on Biopolymers: Precursors, Process, Properties … 29

4 Biobased Polymers

In contrast to fossil fuel plastics, which are produced from petroleum, biopolymers
or organic plastics are generated from renewable biomass resources such as starch,
seaweed, and proteins. Biopolymers offer the additional advantages of conserving
fossil resources and reducing CO2 emissions, making them an essential invention for
sustainable development.

4.1 Thermoplastic Starch

Native starch exists in granular form. Pure starch is hydrophilic, brittle, has a lower
thermal decomposition temperature, poor thermal processability, a high melting
point (Khan et al. 2017), and rapidly degrades when exposed to water (Janssen and
Moscicki 2009). Starch can be processed into thermoplastics by processing under
shear of melt extrusion and high temperatures with the addition of plasticizers (Ma
et al. 2009). Low-molecular-weight plasticizers are typically used during the gela-
tinization process to help break up the starch granules (Martin and Gonz 2017).
Thermoplastic is a material that may be molded into desired shapes because of its
weak physical crosslinks, which can be melted and reshaped in a heating and cooling
cycle (Sjoo and Nillsson 2018).
Nowadays, research on the development of TPS continues to increase using
various materials due to advances in TPS production technology. The raw mate-
rials include wheat starch (Moghaddam et al. 2018), cassava bagasse (Edhirej
et al. 2017), cornstarch (Baran et al. 2022), potato starch (Niazi et al. 2015),
mango starch (Agwamba 2021), cassava starch (Chotiprayon et al. 2020), rice
starch (Prachayawarakorn et al. 2010), and pea starch (Cao et al. 2009). TPS
can be processed by extrusion, blowing, thermocompression, or injection molding
(Castillo et al. 2013). The advantages of TPS are its low cost, renewable properties,
and wide availability. However, TPS has various drawbacks, such as being hygro-
scopic, hydrophilic, having a high sensitivity to moisture, poor mechanical properties
compared with conventional polymers, low gas permeability, and inadequate water
barrier characteristics (Bangar et al. 2021). Blending TPS with a different biodegrad-
able polymer and renewable filler are examples of methods of overcoming these
drawbacks. TPS can be used for various applications like food packaging, plastic
bags, disposable cutlery, etc. (Khan et al. 2017).

4.2 Seaweed Based Polymers

Polysaccharides from seaweed can be used as a potential active agent, a raw material
that is rich in polysaccharides, or extracts. The usefulness, sensory qualities, and
30 A. Hanifah et al.

sustainability of materials can all be enhanced by seaweed-based products. Seaweed

has drawn attention because of its biodegradability, non-toxicity, antioxidant prop-
erties, and great film-forming ability. Seaweeds are applied in active packaging
(Yildirim et al. 2018), edible films (Tran et al. 2020), and edible coatings (Aayush
et al. 2022). Different seaweed polysaccharides’ capacities for biocompatibility, gel
formation, emulsification, gelation, and foaming are based on their distinctive struc-
tural features. In addition to the seaweed’s natural qualities, its nutritional value,
which includes vitamins, antioxidants, minerals, and calories, is helpful in creating
edible coatings and films. When seaweeds are mixed with other polysaccharides,
nanoparticles, essential oils, or plant extracts, their barrier, thermal, mechanical,
antioxidant, and antibacterial characteristics are improved (Perera et al. 2021).
Active packaging combining sodium alginate and lemongrass oil can inhibit E. coli
and L. monocytogenes growth (de Oliveira et al. 2019). κ-Carrageenan and mulberry
polyphenol extracts can improve antioxidant and pH-sensitive properties (Liu et al.
2019). Two seaweed polysaccharides, alginate and carrageenan, are extensively used
in the production of edible films and coatings. Film homogeneity and transparency
were favored by alginate and glycerol (Paula et al. 2015). Glycerol and k-carrageenan
enhanced the moisture barrier and tensile characteristics (Paula et al. 2015).
Food coatings serve a number of purposes, including changing the functional char-
acteristics of foods, serving as a barrier between the environment and food products,
and managing the moisture on the food’s surface (Perera et al. 2021; Aayush et al.
2022). The use of agar-based coatings has been beneficial in increasing the storage
shelf life of banana fruits by reducing fruit hardness and weight loss (Hussein Ziedan
et al. 2018). Carrageenan and chitosan, which are added to dragon fruit that is kept at
a temperature of 10 °C and a relative humidity of 90–95%, have been shown to reduce
weight loss. Bract chlorophyll concentration and freshness retention for 30 days (Thi
et al. 2021).

4.3 Polylactic Acid

Poly(lactic acid) (PLA) is a type of biodegradable thermoplastic polyester that is

projected to replace conventional petrochemical-based polymers. PLA is a desirable
biopolymer due to its processability, sustainability, and eco-friendliness; thus, it
has gained popularity in packaging, textile, automotive composites, and biomedical
applications. In terms of future industrial uses, the physical characteristics of PLA
are crucial, including glossy and translucent, stable at low temperatures, moderately
permeable to oxygen and water, and resistant to grease and oil. These characteristics
make it an excellent material for the production of film, bottles, cups, and trays
(Ranakoti et al. 2022).
PLA can be synthesized through direct lactic acid polycondensation, ring-opening
polymerization of lactide, and a lactic acid cyclic dimer. The ring-opening polymer-
ization method is a technique that combines a metal catalyst with lactide to produce
larger PLA molecules (Ebnesajjad 2012). Direct polycondensation requires extreme
Recent Updates on Biopolymers: Precursors, Process, Properties … 31

Fig. 6 Chirality structures of PLA

conditions to generate polymer with a high molecular weight at a short reaction

time, low pressure of 5 mm Hg, and temperatures as high as 180–200 °C, meanwhile
ROP can generate a high-molecular-weight PLA with a controlled molecular weight
distribution under mild reaction conditions (temperature of 130 °C at a short reaction
time) (Ashothaman et al. 2021). PLA is a kind of polyester that is often made from
hydroxy acid, one of the few polymers whose stereochemical form can be easily
modified. This is obtained by polymerizing a restricted mixture of L and D isomers,
which produces polymers with a high molecular weight that is either crystalline or
semicrystalline. Lactic Acid (LA) or 2-hydroxy propionic acid is the main monomer
component for PLA which includes an asymmetric carbon that generates two enan-
tiomeric forms, L-LA and D-LA (Fig. 6), which make PLLA and PDLA, respectively.
The combination of the two optical isomers results in PDLLA. The proportion of
D- and L-enantiomers results in various PDLLA stereo blocks. The ratio of these
isomers, which is the specific chemical composition of PLA, regardless of whether
it is PDLA or PLLA, and the structure of the PLA stereo block, may affect essential
aspects, such as crystallization extent and thermal properties. However, PLA can
be made with many qualities by modifying the polymer chains’ molecular weight,
composition, and stereoisomeric distribution (Jiménez et al. 2019).
PLA is one of the most potential biodegradable polymers due to its thermoplastic
processibility, mechanical properties, and biological properties, such as biocompat-
ibility and biodegradability. Glass transition temperature (T g ) is the most important
parameter for amorphous PLA because significant changes in polymer chain mobility
occur at the glass transition temperature. The behavior of semicrystalline PLA can
be anticipated to influence critical physical characteristics such as T g and crystalline
melting temperature (T m ) (Revati et al. 2016) (Table 1).

4.4 Polyhydroxyalkanoates

Polyhydroxyalkanoates (PHAs) are polyesters of hydroxylic acid (HA) monomers

linked by an ester group. PHAs are bacteria-based biodegradable polymers that
encourage carbon neutrality and sustainable industry. The monomer composition
determines the elastomeric/thermoplastic characteristics of these polymers. Despite
32 A. Hanifah et al.

Table 1 Mechanical
Property PLA
properties of PLA (Jiménez
et al. 2019) Density (g/cc) 1.25
Haze (%) 2.1
Tensile strength (MPa) 109.97
Tensile modulus (MPa) 3299.26
Ultimate elongation (%) 160
Tear resistance (g/mm) 0.3810

the significant potential and efforts to build cost-effective fermentative methods,

the PHAs production costs are still relatively high (5–10 $/kg), which hinders
the commercialization of these biopolymers as commodities materials (Ortelli
et al. 2019). According to ASTM standard, PHAs such as poly(3-hydroxybutyrate)
(PHB) and poly(3-hydroxybutyrate-co-3-hydroxy valerate) (PHBV) are defined as
biodegradable in all aerobic and anaerobic environments. They can be used to
make soil, marine biodegradable, and compostable products, which is an advantage
compared to synthetic non-degradable plastics (Mohapatra et al. 2021).
The main PHAs subcategories are short (3–5 carbons), medium (6–14 carbons),
and long (15+ carbons) (Meereboer et al. 2020). Unsaturated fatty acids form
short and medium-chain PHAs with double bonds. The common short-chain PHAs
are poly(3-hydroxybutyrate) (PHB) and poly(3-hydroxybutyrate-co-3-hydroxy
valerate) (PHBV). Other biodegradable PHAs include poly(3-hydroxybutyrate-
co-4-hydroxybutyrate) (PH4B), poly(3-hydroxybutyrate-co-3-hydroxyhexanoate)
(PHBHx), and poly(3-hydroxybutyrate-co-3-hydroxyoctanoate) (PHBO) (Fig. 7).
PHAs, such as poly(3-hydroxybutyrate-co-3-hydroxy valerate (PHBV) and
poly(3-hydroxybutyrate-co-3-hydroxy valerate (PHB), are brittle due to their high
crystalline degree. The chemical structure of PHAs is responsible for giving it these
characteristics. Therefore, given that these numerous varieties of PHAs each have
specific structural, physical, and chemical qualities, it is necessary to categorize them
according to those properties and then modify them so that they are simple to use for
the purposes that have been described. The general properties of the common types
of PHAs are shown in Table 2.
PHAs have various excellent properties, including a high volume-to-surface ratio,
a tiny pore size with a high chance of being recycled, biodegradability, and biocom-
patibility. Recent attention has been drawn to PHAs due to their multiple favorable
qualities, such as ease of processing, resistance to UV rays, and insolubility in water.
The biodegradable nature and other advantages of polyhydroxyalkanoates, such as
high-temperature stability, low degree of surface porosity, improved toughness, and
elasticity, result in their use in a variety of industries, including medicine, agriculture,
etc. (Chai et al. 2021).
Recent Updates on Biopolymers: Precursors, Process, Properties … 33

Fig. 7 General chemical structure and various applications of PHAs (Sharma et al. 2021)

Table 2 The general properties of the common types of PHAs (Chai et al. 2021)
Property P(3HB) P(3HB-co-4HB)
Glass transition temperature, T g (°C) 2–4 − 48–4
Melting temperature, T m (°C) 160–175 50–175
Tensile strength, σ (MPa) 15–40 17–104
Young’s modulus (GPa) 1–2 0.07–1.5
Elongation at break (%) 1–15 14–1320
Crystallinity (%) 50–80 34–60

4.5 Plant Oil-Based Polymers

Plant oil has several functional groups coming from fatty acids that are useful for
polymer synthesis. Carboxylic acid and alkyl ester are the standard functional groups
in the fatty acid. There are also additional groups such as epoxy group (vernolic acid)
and hydroxyl (ricinoleic acid). Plant oil can be directly used for polymerization due
to these functional groups. Direct polymerization usually converts plant oil to polyol
through epoxidation.
34 A. Hanifah et al.

Junming and his co-workers (Junming et al. 2012) reported the preparation of
polyester polyols from oleic acid, a common unsaturated fatty acid in plant oils. The
synthesis consists of three steps: epoxidation, ring-opening reaction, and esterifica-
tion. The polyols appear as a viscous liquid at ambient temperature with OH number
ranging from 307 to 425 mg KOH g−1 . Polyester polyol was then used to synthesize
polyurethane foams. The polyurethane synthesized from the oleic-based polyols and
isocyanate showed an acceptable result and reached the standard of rigid foam in
China as shown in Table 3.
The hazardous reaction of phosgene in isocyanates synthesis and the sustainable
demand in chemistry generated the development of the phosgene-free method. Plant
oils have double bonds and ester that can be synthesized into various new structures
including “bio” isocyanates. The major drawbacks of plant oil-based isocyanates are
that they are still not able to substitute current isocyanates on the market. Despite
that, Henkel Corporation Company and General Mills have commercialized dimer
fatty acid diisocyanates from fatty acid containing 36 carbon atoms in the chain.
Other raw materials, such as soybean oil, azelaic acid, and oleic acid, also have been
attempted to make diisocyanates and used for the synthesis of “green” thermoplastic
PU (Caylı and Kusefoglu 2008; Hojabri et al. 2010; More et al. 2013).
Rix et al. used mini-emulsion polymerization in their attempt to prepare non-
isocyanates polyurethane (NIPU). A fatty acid diamine, Priamine 1075, was reacted
with fatty acid-based bis-cyclic carbonates at 60 °C for several hours. The mini-
emulsion process was then prepared to obtain waterborne NIPU latexes with the
addition of surfactants and hydrophobic additives. The result obtained was NIPU
with a solid content reaching of 30wt%. The molar masses were low compared to bulk
NIPUs, and this is because of the existence of partial hydrolysis of the carbonates. The
NMR result of PHUs from the mini-emulsion process showed signals at 3.5–4 ppm
that are considered equal to protons of hydrolysis compounds (Rix et al. 2016).
Despite direct polymerization being an easy route to obtain plant-oil-based
polymer, only few polymers can be obtained through this route, such as bio-
polyurethane and epoxy. Other routes such as the dimerization of fatty acid, and
self-metathesis of methyl ester are needed to obtain various types of monomers with
double functional groups.

Table 3 Physical properties of polyurethane foams (Junming et al. 2012)

Properties Foam 1a Foam 2a Foam 3a China standardb
Thermal conductivity W/(m K) 0.028 0.037 0.076 0.024
Density (kg/m3 ) 31.3 40.6 58.1 30–50
Compressive strength (MPa) 150 210 230 150–300
Modulus of compression (MPa) 3.44 4.58 5.66 –
Bending strength (MPa) 0.28 0.34 0.41 –
a Foam 1 used polyols with hydroxy number 425; foam 2 used polyols with hydroxy number 361;
foam 3 used polyols with hydroxy number 307
b China standard (JC/T 998-2006): Spray polyurethane foam or thermal insulation
Recent Updates on Biopolymers: Precursors, Process, Properties … 35

Most of the fatty acids have only one functional group and act as a chain terminator
in polymer synthesis. Polymerization of fatty acid only occurs in dimer or trimer
fatty acids which have two or three functional groups. So, the fatty acid has to
be dimerized or trimerized before use (Mallakpour and Rafiee 2012). CRODA has
developed various functional dimer and trimer fatty acids for polymer synthesis such
as fatty acid, fatty diol, and fatty diamine.
Mecking and his groups (Quinzler and Mecking 2010; Stempfle et al. 2011; Trza-
skowski et al. 2011) have investigated the conversion of fatty acid into saturated
α,ω-diacid and diol as a preparation of long-chain aliphatic semicrystalline polyester.
Oleic acid, erucic acid, or 10-undecenoic acid are modified into diacid through
carbonylation and olefin metathesis. Carbonylation converts the internal double bond
in the hydrocarbon to terminal ester groups while olefin metathesis coupling of two
=CH(CH2 )nCOOR part and then the saturated product being hydrogenated. The
reduction mechanism of dicarboxylic acid is used to obtain diols. One of their results
has similar properties to low-density polyethylene (LDPE) and polyethylene. The
material has a melting temperature (T m ) of 103 °C and crystallize temperature (T c )
of 87 °C while LDPE has T m 110 °C and T c 94.09 °C. This material was prepared
by the linear incorporation of oleic acid and erucic acid. Equal amounts of dimethyl-
1,19-nonadecanoate and nonadecane-1,19-diol were prepared for polycondensation
of novel polyester catalyzed by titanium alkoxides (Quinzler and Mecking 2010;
Gaska et al. 2017).
Vilela and his groups used erucic acid to generate long-chain aliphatic polyester
via self-metathesis for monomer synthesis. Hydrogenation of erucic acid to obtain
dicarboxylic acid and reduction of the acid to get diols (Fig. 8). Polycondensation
of hexacosane-1,26-diol with α,ω,-dicarboxylic acid generates long-chain aliphatic
polyesters 26,26 with excellent properties. The polyester has the highest T m at 104 °C,
and the highest degradation temperature (T d ) at 386 °C. The DSC graph of polyester
26,26 exhibits a sharp endotherm and crystallization peak indicating a high degree
of the polyesters (Vilela et al. 2012).
A long carbon chain of fatty acids is beneficial for the synthesis of hyperbranched
polyester. Testud et al. reported the preparation of hyperbranched polyester (HPBE)
from fatty acid methyl ester (FAME) with tunable properties. They use various plant

Fig. 8 Conversion of erucic acid to 1,26 diacid and 1,26 diol and polymerization of both (Vilela
et al. 2012)
36 A. Hanifah et al.

Fig. 9 Thiol-ene scheme of polyamide monomer (Türünç et al. 2012)

oils as starting material for the synthesis of monomers of ABn type (n = 2 or 3) where
A is acid and B is diol moieties. The most efficient catalyst system that achieves high
molar masses (3000–10000 g mol−1 ) is shown by zinc acetate, sodium methoxide,
and 1,5,7-triazabicyclo [4.4.0] dec-5-ene (TBD). The glass transition of the samples
varies from −33° to 9 °C, with a degree of branching ranging from 0.07 to 0.45 and
thermal stability above 300 °C. A significant amount of hydroxyl group in HPBE
can have further modifications to reach desired properties (Testud et al. 2017).
Falkenburg et al. reported the preparation of polyamide-based fatty acid for the
first time in 1945. They synthesized polyamide from difunctional and polyfunc-
tional amines and polymeric fat acid (dimer and trimer). The polyamides result has
unique properties including strong adhesion to various surfaces, good water resis-
tance, soluble in alcohol, excellent mechanical properties, and flexible as described
in Table 2. It is suitable for resin, coating, elastomer, and hot melting adhesives
application (Falkenburg et al. 1945).
Thiol-ene addition was introduced in the synthesis of fatty acid monomers. Thiol-
ene additiona is an effective method for various transformations because it shows a
click reaction behavior. Türünç and his co-workers prepared a fatty acid-based amine
monomer through thiol-ene addition to obtain polyamide. Cysteamine hydrochloride
addition to double bonds of Methyl-10 undecenoate, methyl erucate, and methyl
oleate produced a good multifunctional monomer (Fig. 9). The products were used for
polyamides with adipic acid and 1,6-hexamethylene. 1,5,7-triazabicyclo [4.4.0] dec-
5-ene (TBD) was used as a catalysts in copolymerization. The result yields polyamide
with excellent thermal and solubility properties. The resulted polyamide gave the
highest T m at 138 °C (Türünç et al. 2012).
Nurhamiyah et al. have been synthesized a series of fully biobased polyamides
from a fatty acid biobased dicarboxylic acid and biobased diamine, PA36,36, and
PA36,9 (Nurhamiyah et al. 2021a, 2021b). PA36,36 was prepared from a facile
condensation of Pripol 1009 and Priamine 1075 at 220 °C at various times. It was
found that the optimum time to synthesize this biobased polyamide is at 24 h. PA36,36
has excellent properties, for example, zero water absorption, high toughness (14.21
± 4.58 MJ m−3 ), and large elongation at break (up to 2286%), and shows autonomous
self-healing behavior at room temperature (Nurhamiyah et al. 2021a). Meanwhile,
PA36,9 is a semicrystalline fully biobased polyamide elastomer that shows similar
Recent Updates on Biopolymers: Precursors, Process, Properties … 37

properties to the synthetic medium hardness, prospecting as an alternative substi-

tution. The example is a melting temperature (T m ) of 83.6 °C, a glass transition
temperature (T g ) of 17.6 °C, a large elongation at break (1220%), a high tensile
strength (31.8 MPa), a medium hardness (Shore A/Shore D = 90/35), and excellent
hydrophobicity.

5 Challenges and Future Perspectives

Biopolymer production is growing rapidly, and the production process of precursors

of biopolymers (starch, seaweed, and plant oil) from renewable resources continues
to increase. This is due to the high demand for biopolymer materials from various
industries ranging from packaging, the automotive industry, the agricultural sector,
bioplastics, and electronics, as well as biopolymer research and development to
increase its capacity in industrialization. In the early stages of the biopolymer
industry, biodegradable materials were developed for short-term applications to solve
the problem of synthetic polymers. Currently, biopolymer production focuses on
the application of durable materials with sustainable bioprocesses using biomass-
derived materials in the hope of reducing greenhouse gas emissions and limiting
fossil resources. Development of new applications with the advantage of even better
properties. In general, the expected development and sustainability of biopolymer
materials in the future depend on a variety of applications and the quantity of biobased
materials. Particularly, advances in bioprocessing and the utilization of lignocellu-
losic biomass as a waste product for the production of biopolymers with low produc-
tion costs, sustainability, and material properties that can be modified according to
application based on renewable energy sources are becoming more attractive.

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Potential Application of Agro-Industrial
Byproduct for Bacterial Cellulose
Production; Its Challenges and Emerging
Trends for Food Packaging

Khatarina Meldawati Pasaribu, Nanang Masruchin, and Myrtha Karina

Abstract Most packaging used today is made of plastic, which is produced from
fossil-based polymers. In terms of its ease of processing and cheapness, plastic is
non-biodegradable. Apart from being a plastic substitute, cellulose-based packaging
is bio-based and sustainable. Cellulose is commonly generated from vascular plants.
However, numerous chemicals are required for cellulose isolation and purification.
For plant cellulose replacement, bacterial cellulose is considered as the favorable
resources. Bacterial cellulose, also well known as microbial cellulose, is the cellulose
produced by the activity of non-pathogen gram-positive or gram-negative bacteria in
the substrate containing carbon and nitrogen. Possessing a three-dimensional nano-
structure, high reactive functional groups, high mechanical strength properties, and
bacterial cellulose attracts much attention for research work or commercial purposes.
However, Hestrin-Schramm, the synthetic or considered as standard medium for
bacterial cellulose production, is expensive. Recently, there has been a lot of interest
in searching for carbon and nitrogen sources as an alternative to synthetic bacterial
growth media. Agro-industrial byproducts are derived from agriculture and food
industry processing. Rich in carbohydrates and protein, these resources are suitable
for bacterial cellulose production. This chapter aims to describe the agro-industrial
residues for bacterial cellulose production and their recent possible application for
food packaging.

Keywords Agro-industrial byproduct · Bacterial cellulose · Food packaging

K. M. Pasaribu · N. Masruchin · M. Karina (B)

Research Center for Biomass and Bioproducts, National Research and Innovation Agency
(BRIN), Cibinong 16911, Indonesia
e-mail: [email protected]
Research Collaboration Center for Biomass and Biorefinery, BRIN – UNPAD, Jatinangor 45363,
Indonesia
N. Masruchin · M. Karina
Research Collaboration Center for Nanocellulose, BRIN - Andalas University, Padang 25163,
Indonesia

© The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2024 43
M. A. R. Lubis et al. (eds.), Biomass Conversion and Sustainable Biorefinery, Green
Energy and Technology, https://doi.org/10.1007/978-981-99-7769-7_3
44 K. M. Pasaribu et al.

1 Introduction

Due to its excellent properties such as high strength, hardiness, ease of processing, and
cost-effectiveness, plastic has become a popular and important material for food pack-
aging (Andrady et al. 2009). The common commercial plastics for food packaging
are generally derived from petrochemical-based polymers, specifically polypropy-
lene (PP), polystyrene (PS), polyvinyl chloride (PVC), high-density polyethylene
(HDPE), low-density polyethylene (LDPE), and polyethylene terephthalate (PET)
of which they account for almost 90% of total polymers manufacture (Napper and
Thompson 2019). These plastics are non-biodegradable, and when their lifetime is
over, the improper disposal of plastic waste would become a severe environmental
problem. Due to poor management, plastic wastes are found in rivers. It was reported
that 80% of plastic waste is distributed by more than 1,000 rivers globally (Meijer
et al. 2022) which slowly goes to the ocean, becomes a marine pollutant, and grad-
ually becomes a global oceanic serious problem (Long et al. 2022). Bulky plastics,
often known as macro-plastics, have been found in the ocean, and their accumulation
has significantly increased. As a result, marine animals were suffered and killed by
plastic entanglement (Dasgupta et al. 2022). In the ocean, plastic gradually degrades
into micro-plastic, whose accumulation is hazardous since it is easily swallowed
by crustaceans whose digestive tract is usually consumed by humans (Rainieri and
Barranco 2019). Besides causing the accumulation of plastic wastes, the manufac-
ture of petroleum-based plastic triggers the reduction of its non-renewable resources.
Nowadays, petrochemical industries are the source of more than 99% of the global
plastics raw materials (British Plastics Federation 2019). Consequently, sustainable,
renewable, and biodegradable-based biopolymers, specifically those originating from
organic resources, are important as alternative resources for not only improving food
quality but also producing environmentally-friendly food packaging.
The alternative source for petroleum-based polymer as a plastic replacement for
various materials is addressed to cellulose, the most prevalent macromolecule on
earth, a renewable and low-cost natural polymer. In general, cellulose is obtained
from vascular plants, but it can also be found in tunicin, typically from Micro-
cosmus sulcatus, Halocynthia roretzi, Ciona intestinalis, Styela plicata, and Ascidia
sp. in the form of rod-like crystals (Zhao and Li 2014). Cellulose is also synthesized
from green algae of Cladophorales (Cladophora, Chaetomorpha, and Rhizoclonium)
and Siphonocladales (Valonia, Dictyosphaeria, Siphonocladus, and Boergesenia)
(Mihranyan 2011). Additionally, cellulose is also found in the cell wall of Saprolegnia
monoica (Fèvre et al. 1990).
Cellulose has been used in various fields for a long time for chemical-based
materials, fabric supplies, and pulp paper production. For a wide range of appli-
cations, particularly when high purity and white color are considered necessary,
cellulose should be isolated from lignin, hemicellulose, and pectin as the plant cell
wall components through the pulping and bleaching process, which requires various
synthetic chemicals. In addition, cellulose isolation and purification require a large
amount of energy as well as the price of waste effluent and toxic material treatment.
Potential Application of Agro-Industrial Byproduct for Bacterial … 45

Therefore, highly pure and environmentally friendly cellulose is an important topic of

research subject. For this purpose, bacterial cellulose offers an alternative and inter-
esting role as cellulose resources. In terms of cellulose isolation, bacterial cellulose
is more beneficial than plant cellulose because it is free of lignin and hemicellu-
lose, preventing the need for a pulping and bleaching process and making it more
environmentally-friendly and energy efficient. Thus, bacterial cellulose is a source
of pure cellulose as well as indicates higher water absorptivity, higher crystallinity,
and a higher degree of polymerization (Salari et al. 2018).
Although BC possesses remarkable characteristics, it is costly to fabricate. The
synthetic medium Hestrin–Schramm (HS) medium is one of the main factors causing
to high production cost of BC. This synthetic medium is an indispensable material for
bacteria to produce cellulose (Lahiri et al. 2021). Therefore, there is a need to explore
the potential and low cost of natural resources for bacterial cellulose production. In
this chapter, the production of bacterial cellulose by using abundant and low-cost
agro-industrial byproducts and the possibility for food packaging application are
presented.

2 Agro-Industrial Wastes for Bacterial Cellulose

Production

Bacterial cellulose production depends on various basic factors such as oxygen supply
(Wu and Li 2015), temperature (Lee et al. 2014), reactor design (Islam et al. 2017),
and optimum pH (Reiniati et al. 2017). However, carbon and nitrogen play an impor-
tant role in the synthesis of bacterial cellulose (Rajwade et al. 2015). Typically,
the carbon and nitrogen sources were obtained from the Hestrin-Schramm culture
medium. This expensive medium mostly contains synthetic glucose, peptone, yeast
extracts, and various minerals. Therefore, exploring the low-cost carbon and nitrogen
resources from agro-industrial byproducts for alternative resources is the right option.
Agro-industrial byproducts are defined as various wastes from the food and agri-
culture industries (Madeira et al. 2017). It is available in a huge amount every year.
In terms of their abundance and sustainability, the agro-industrial byproducts can be
deduced from the total volume produced from the plant waste from crops, approxi-
mately 250 million tons per year (FAO 2013). It was also informed that 1/3 of total
human food production, or around 1.3 billion tons annually, is discarded worldwide
(Duque-Acevedo et al. 2020). The use of agro-industrial residues for innovative prod-
ucts not only solves waste removal issues but also reduces pollution, reduces adverse
effects on human and animal health (Zihare et al. 2018), and raises its value.
Agricultural commodities such as fruits, vegetables, legumes, and cereals and
their processing generate unclear value byproducts (Almaraz-sánchez et al. 2022).
Other agricultural primary activities such as harvesting, pruning, and collecting in
the field crops generate residues in the form of logs, straws, leaves, husks, roots,
and seeded pods from crops (Hiloidhari et al. 2020) as well as animal residues
46 K. M. Pasaribu et al.

(Forster-Carneiro et al. 2013). These underutilized products are important resources

for new materials, chemicals (Madeira et al. 2017), and energy (Vandamme 2009).
This is due to the important and valuable existing ingredients of the residues such
as carbohydrates, protein, fibers, minerals, and vitamins (Lopes and Ligabue-Braun
2021). Carbohydrate and protein will be valuable resources and mediums for bacteria
to propagate in the cellulose gel.
Depending on the material target, various pre-treatments of agro-industrial residue
are commonly carried out. Chemical, biological, enzymatic, and physical pre-
treatments such as milling, steam explosion/steam treatment, hydrothermal, irra-
diation, and chemical treatment such as alkaline hydrolysis are the most common
pre-treatments (Singh nee’ Nigam et al. 2009). These pre-treatments aim to reach
the reactive molecule followed by procedures to obtain the desired raw material (De
Corato et al. 2018). The fermentation, hydrolysis, precipitation, and filtration aim to
eliminate the poisonous and low-value chemicals (Araújo et al. 2020), so that the
existing organic substances could be changed into diverse products involving carbon
and nitrogen as the intermediate compounds. The fine and highly de-crystalized
structure was obtained by the milling process, whereas steam treatment/steam explo-
sion increased the pore size of the fiber. Biological pre-treatment aims to degrade
lignin; it generally involves the activity of white-rod fungi such as Phanerochaete
chrysosporium (Singh nee’ Nigam et al. 2009).
In agro-industrial industries, carbon and nitrogen could be found in the form of
cellulose, hemicellulose, lignin, carbohydrate, or proteins (Urbina et al. 2021). The
composition depends on the origin, type of raw material resources, and method of
analysis. Based on these descriptions, they are particularly potential for BC produc-
tion. The high carbohydrate and nitrogen contents of 56.9 and 28.5%, respectively,
are found in oil palm frond (Rhaman and Naher 2021). Paddy straw also shows a
high carbohydrate and nitrogen content of 50.2 and 84.9%, respectively.
The carbon and nitrogen generated from numerous agro-food industries is
presented in Table 1.

2.1 Agro-Wastes

Available in a huge amount worldwide annually, agro-wastes are the most promising
resource for the production of BC. Sugarcane straw is generated from sugarcane
production, which plays a role in the daily required nutrient. Recently, sugarcane
straw is utilized as bio-ethanol production, but its valorization is widely open to
realization. It contains 35–45% of cellulose, which can be used for carbon resources.
Sugarcane straw, which was previously boiled in water, was used as the medium
for Komagataeibacter xylinus ATCC 11142 and incubated statistically at 30 °C for
15 days. The dry pellicle of BC was weighed as 1.06 g/L (Dhar et al. 2019). A quite
similar amount of BC yield was obtained from the root, stalk, and leaf parts of sweet
sorghum, as of 2.28 g/L, stalk 1.82 g/L, and leaf 2.54 g/L, respectively. Commonly
considered and used as an energy crop, sweet sorghum is an important resource
Potential Application of Agro-Industrial Byproduct for Bacterial … 47

Table 1 Carbon and nitrogen content from agro-industrial byproducts

Resources Carbon content (from) Nitrogen content (from) References
Cassava wastewater Carbohydrates 58.11% Total nitrogen 1.94% Ribeiro et al. (2019)
Cheese whey Lactose, 77% Protein, 13% Lopes et al. (2013)
Coconut oil cake 48.16% 1.69% Sathish and Shetty
(2013)
Coffee husk 18% 13% Mussatto et al.
(2011)
Corn cob 53.61% 1.9% Sathish and Shetty
(2013)
Grape pulp Carbohydrate, 6.53% 1.96% Ekanah et al. (2017)
Oat straw Cellulose 40%, NA Singh nee’ Nigam
hemicellulose 27%, et al. (2009)
lignin 18%
Oil palm empty fruit 43.8–54.7% 0.25–1.21% Chang (2014)
bunch
Oil palm frond 56.9% 28.5% Rhaman and Naher
(2021)
Orange peel Cellulose (71.2 g/kg), Crude protein (57.2 g/ Ahmad et al. (2012)
hemicellulose (128 g/ kg)
kg)
Orange peel Carbohydrate 52.90% Crude protein 12.3% Gotmare and Gade
(2018)
Paddy straw 50.2% 84.9% Rhaman and Naher
(2021)
Pineapple waste 45.68% 0.61% Sathish and Shetty
(2013)
Rice bran extract Glucose 38.3% 13.2% Choi (2020)
cellulose 7.8%
Rice husk Cellulose 22%, NA Megawati et al.
hemicellulose 23%, (2011)
lignin 15%
Rice straw Cellulose 32%, NA Limayem and Ricke
hemicellulose 24%, (2012)
lignin 18%
Rice washed Carbohydrate, 90% Protein, 8% Srikandace et al.
(2022)
Sawdust 55.2% 34.3% Rhaman and Naher
(2021)
Sugarcane bagasse 48.32% 0.2% Sathish and Shetty
(2013)
Sugarcane straw Cellulose 36%, NA Saad et al. (2008)
hemicellulose 21%,
lignin 16%
(continued)
48 K. M. Pasaribu et al.

Table 1 (continued)
Resources Carbon content (from) Nitrogen content (from) References
Tofu liquid waste Carbohydrate, 25% Protein, 65% Srikandace et al.
(2022)
Tomato juice Carbohydrate, 2.52% Protein, 1% Ismail Abdullahi
et al. (2016)
Wheat straw Cellulose 27%, NA Adapa et al. (2011)
hemicellulose 21%,
lignin 23%
NA: Not available

for BC production, which will be possible for building block material application
(Wang et al. 2021). A higher BC production of 2.86 g/L was reported from corn stalk,
containing 3.87 g/L of glucose and glucan (35%), which were previously treated with
acetic acid for Acetobacter xylinum ATCC 23767 (Cheng et al. 2017). Another corn
residue can be obtained from the corn stover. Mainly contains glucose and xylose as
well as available abundantly as agricultural residues, corn stover can be used as a low-
cost feedstock in the manufacturing of BC. Enterobacter sp. FY-07 (CGMCC No.
6103) was used during the fermentation and incubated under static conditions at 30 °C
for 24 h. The productivity was 14.35 g/L/ day but with the addition of xanthan gum,
the productivity increased significantly up to 17.13 g/L/day. Interestingly, the pilot
scale for BC has been reported by using oat hulls. Previously chemically treated with
HNO3 , followed by enzymatic saccharification and the addition of sodium hydroxide,
oat hulls were performed in a 100 L fermentor. After purification, 80.5 tons of 98%-
wet BC gel per 100 tons of oat hulls were obtained with a 93% crystallinity index and
composed of 100% cellulose Iα -allomorph (Skiba et al. 2020). Another approach to
scale-up BC production has been explored. In a 30 L working volume, Acetobacter
xylinum KJ1 was used, and the BC yield was achieved at 5.6 g/L using saccharified
food wastes (Song et al. 2009). DHU-ATCC-1 strain, a mutant of Komagataeibacter
xylinus ATCC 23770, was employed in a 75 L stirred-tank reactor to scale up BC
production with the final yield of 17.3 gr/L using overripe bananas (Molina-Ramírez
et al. 2020). Other possible agro-wastes for BC production are presented in Table 2.

2.2 Fruit-Food Wastes

Various studies have been reported regarding the use of agro-waste as a source of
carbon and nitrogen for BC production. Green waste generated from fruits, vegeta-
bles, and food wastes is the potential resources due to its high glucose and fructose
content. Citrus peel and pomace enzymolysis from beverage industrial waste were
successfully used for BC by using Komagataeibacter xylinus CICC 10529 with a
yield of 5.7 ± 0.7 g/L higher than from HS medium with 50 nm for its average diam-
eter. The entire results confirmed the role of citrus peel and pomace enzymolysis
Potential Application of Agro-Industrial Byproduct for Bacterial … 49

Table 2 Agro-industrial waste for BC production

Resource Bacteria strain Yield (gr/L) References
Cacao mucilage G. xylinus 13.13 Saavedra-Sanabria
exudate et al. (2021)
Cashew tree K. rhaeticus 2.3–6.0 Pacheco et al. (2017)
Cashew tree K. rhaeticus 2.8 Silva et al. (2010)
exudate
Coffee cherry husk G. hansenii UAC09 6.24 Usha et al. (2011)
Corn stalk K. xylinum, ATCC 23767 2.86 Cheng et al. (2017)
Corn stover Enterobacter sp. FY-07 2.08 Gao et al. (2021)
Oat hulls Medusomyces gisevii 2.2 Skiba et al. (2020)
Sa-12
Pecan nutshell G. entanii 2.8 Dórame-Miranda
et al. (2019)
Prickly pear peels Lactiplantibacillus 2.94 El-Gendi et al. (2023)
plantarum strain AS.6
Sugarcane straw K. xylinus ATCC 11142 1.06 Dhar et al. (2019)
Sweet sorghum Acetobacter xylinum Root 2.28, stalk 1.82, Wang et al. (2021)
ATCC 23767 leaf 2.54
Wheat thin stillage G. sucrofermentans 6.19 Revin et al. (2018)
B-11267
Wheat straw K. xylinus ATCC 23770 8.3 Chen et al. (2013)

as potential sources for BC production with similar characteristics to HS medium,

being more environmentally-friendly and less expensive to produce (Fan et al. 2016).
Other mango peel waste was also developed as an alternative culture medium for
Komagataeibacter xylinus DSMZ 2004. The yield of BC was 6.32 g/L after 16 days of
fermentation by the static culture technique. Structural analysis showed the diameter
of BC from mango waste peel was 98.8 nm and showed a similar chemical structure to
BC synthesized from pure sugar. This resulted BC was proposed for biomedical and
pharmaceutical applications (García-Sánchez et al. 2020). Additionally, pineapple
peels as an alternative medium were used for Komagataeibacter xylinus IITR DKH20
which was incubated for 384 h resulting in 11.44 g/L dried. The resulted BC revealed
similar physicochemical properties to the BC produced using HS medium and was
proposed for biomedical application (Khan et al. 2021). When wasted rotten tomato
media was used as a substitute medium for Gluconacetobacter hansenii and culti-
vated for 7 days, the yield BC was 3.71 g/L. BC was produced after 7 days and aimed
for medical and pharmaceutical fields (Fatima et al. 2021). However, a higher dry BC
of 7.8 gr/L was obtained from the tomato juice when used as an optimization process
for the 10 L production fermentation medium for Acetobacter pasteurianus RSV-
4 after 7 days of incubation. Furthermore, Komagataeibacter xylinus DSM 6513
was successfully grown in a medium generated from red and white grape bagasse
from the wine industry. It was reported that the white grape bagasse was a better
50 K. M. Pasaribu et al.

Table 3 Fruit wastes

Resource Bacteria strain Yield (gr/L) References
Citrus peel pomace K. xylinus CICC 10529 5.7 Fan et al. (2016)
Grape bagasse G. xylinus NRRL-B42 8.0 Vazquez et al. (2013)
Kitchen waste K. rhaeticus K15 4.76 Li et al. (2021)
Litchi extract K. xylinus CH001 2.5 Yang et al. (2016)
Mango peels K. xylinus DSMZ200 6.32 Sanchez et al. (2020)
Musk melon K. persimmonis GH-2 8.08 Hungund et al. (2013)
Orange pulp A. pasteurianus RSV-4 2.8 Kumar et al. (2019)
Pineapple peels K. xylinus IITR DKH20 2.57 Khan et al. (2021)
Pineapple residue G. medellinensis 3.24 Algar et al. (2015)
Rotten fruits G. xylinus 0.06 Jozala et al. (2015)
Rotten tomato G. hansenii PJK KCTC 3.83 Fatima et al. (2021)
10505BP
Tomato juice A. pasteurianus RSV-4 7.8 Kumar et al. (2019)
Various fruit juice A. xylinum NBRC 13693 0.2–2.1 Kurosumi et al. (2009)

substitution as a low-cost medium resource than the red grape. White grape bagasse
also produced a five times higher yield, five times higher water holding capacity
as well as greater flexibility than the HS medium. Red grape bagasse-based BC is
suitable for the food industry, whereas white grape BC is appropriate for the textile
and biomedical industries (Ogrizek et al. 2021). Another study reported that dried
BC of 8.08 g/L was obtained from musk melon as a natural, cheaper carbon source
medium for Gluconeacetobacter persimmonis, which was incubated at 30 °C for
14 days (Hungund et al. 2013). Kitchen wastes could also be the promising carbon
and nitrogen resources for BC production, since it is composed of carbohydrates and
protein. A new cellulose-producing bacteria, namely, Komagataeibacter rhaeticus
K15, has been isolated from kombucha tea and shown the capability to use kitchen
wastes as a carbon source for cellulose production of as much as 4.76 g/L (Li et al.
2021). The detailed lists of fruit wastes are shown in Table 3.

2.3 Food and Beverage Industrial Wastes

Based on daily activity, food-beverage industries generate large amounts of waste.

Proper waste management can create economic benefits as well as provide free-
contamination caused by their accumulation. On the other side, the wastes are rich
in carbohydrates and protein thus promising to be a low-cost resource of media used
for BC production. Beer manufacturing is a significant economic activity. Modern
brewing is commonly a big industry that generates large quantities of byproducts
nowadays. Waste beer yeasts are the second most common byproduct of the brewing
Potential Application of Agro-Industrial Byproduct for Bacterial … 51

industry that is discarded or fed to livestock. The waste beer yeast hydrolysates with
3% sugar concentration when it was treated by ultra-sonication, resulted BC yield of
7.02 g/L, nearly 6 times compared with the untreated waste beer yeast at 1.21 g/L (Lin
et al. 2014). Cheese whey is today recognized as a source of functional and bioactive
compounds, especially proteins and peptides but a significant amount of the whey
produced globally is still not valorized whereas it contains rich nutrient components
(Pires et al. 2021). The dry BC of 6.77 g/L was synthesized from the K. xylinus
when enriched with β-galactosidase and proposed as a food packaging application.
Corn steep liquor is a byproduct of the corn wet-milling production which generally
consists mainly of water and other ingredients such as sugar and protein. Therefore,
it is potential as nutrient medium for bacteria. A strain of G. hansenii UCP1619
was incubated in corn steep liquor at 30 °C for 10 days. A dry of 7.02 gr/L BC
was resulted from this fermentation process and showed the future applications in
the textile field (Costa et al. 2017). Another promising carbon source is thin stillage
from rice wine distilleries. The strain of G. xylinus was incubated in the rice wine
distillery by static cultivation for 7 days. A dry BC of 6.26 g/l was obtained which
was reported almost 50% higher than produced in an HS-only medium with slightly
denser reticulated structures and higher crystallinity (Wu and Liu 2013). Utilization
of crude confectionery waste hydrolysates for K. sucrofermentans was reported for
BC production. The waste contained 28.3% g/g free sugars, 28.4% g/g starch, 7.1%
g/g protein, and 24.9% g/g fats was used in order to explore the lower cost alternative
medium. BC was produced with a yield of 5.7 g/L and the potential as a bio-based
packaging reinforcing agent (Efthymiou et al. 2022a, b). Other possible resources
obtained from food and beverage industrial waste are presented in Table 4.

Table 4 Food and beverage industrial wastes

Resource Bacteria strain Yield (gr/L) References
Cheese whey K. xylinus DSM 2325 6.77 Rollini et al. (2020)
Confectionery wastes K. sucrofermentans 5.7 Efthymiou et al. (2022a, b)
Corn steep liquor G. hansenii UCP1619 7.02 Costa et al. (2017)
Jujube-processing K. xylinum CGMCC 2.2 Li et al. (2015)
industry 2955
Maple syrup A. xylinum BPR 2001 1.51 Zeng et al. (2011)
Rice wine distillery G. xylinus 6.26 Wu and Liu (2013)
Sugar cane molasses K. rhaeticus 2.23–2.58 Machado et al. (2018)
Tofu liquid waste K. xylinum 3.8 Srikandace et al. (2022)
Waste beer yeast K. hansenii CGMCC 7 Lin et al. (2014)
3917
Whey G. sucrofermentans 5.45 Revin et al. (2018)
B-11267
52 K. M. Pasaribu et al.

2.4 Others

Other industrial wastes (Table 5) such as biodiesel wastes, Chinese medicinal herbs,
cotton-based waste textiles, distillery effluent, dry olive mill residues, and tobacco
extract wastes also show the potential resources for bacterial cellulose production.

3 Bacterial Cellulose and Its Properties

Bacterial cellulose, also known as bio-cellulose or microbial cellulose, is cellulose

produced by the activity of non-pathogen, either positive or negative bacteria in a
medium containing nitrogen and carbon as nutrient resources. The acetic bacte-
rial that plays a role in the cellulose formation is commonly known as Aceto-
bacter xylinum (Yamada et al. 1997), which is re-classified as Gluconeacetobacter
xylinum and recently known as Komagataeibacter xylinum. This non-phatogen-
aerobic bacteria can convert 108 glucose molecules per hour into cellulose and is
considered as the most effective strain for bacterial cellulose production commer-
cially due to its high productivity (Wang et al. 2019). Other producing bacteria are
Pseudomonas (Ude et al. 2006), Rhizobium (Robledo et al. 2012), Sarcina (Yang
et al. 2013a, b), Agrobacterium (Barnhart et al. 2013), and Lactobacillus (Khan et al.
2020). It was reported that Rhizobium and Agrobacterium produced cellulose in
Rhizobium and Agrobacterium reportedly produced cellulose in exceedingly low
yields. On the other hand, from the family of Acetobacteriaceae such as Koma-
gataeibacter, Acetobacter, Gluconacetobacter, Gluconobacter, and Asaia together
with the Bacillus, Leifsonia, Salmonella, Erwinia, Enterobacter, Pseudomonas, and
Shewanella in non Acetobacteriaceae produce high cellulose (Li et al. 2022a, b).
Generally bacterial cellulose is produced purposely, but it was hypothesized that
cellulose is formed to protect bacteria from unfavorable factors such as UV radiation,
harsh chemicals, and accessibility to oxygen (Retegi et al. 2010).
BC is illustrated by an ultrafine network structure, with emerging chains
combining to generate sub-fibrils with a width of 1.5 nm (Ross et al. 1991). The
spatial configuration of the pre-microfibril accumulation results in crystallinity of

Table 5 Other industrial wastes

Biodiesel waste G. xylinus NRRL-B42 10 Vazquez et al. (2013)
Chinese medicinal herb Taonella mepensis 0.54 Wu et al. (2021)
Cotton-based waste textiles G. xylinus ATCC 23770 10.8 Hong et al. (2012)
Distillery effluent G. oboediens 8.1 Jahan et al. (2018)
Dry olive mill residue K. sacchari sp. 0.85 Gomes et al. (2013)
Elephant grass G. xylinus CH001 6.4 Yang et al. (2013a, b)
Tobacco extract waste K. xylinum ATCC 23767 5.2 Ye et al. (2019)
Potential Application of Agro-Industrial Byproduct for Bacterial … 53

up to 84–89% (Czaja et al. 2004). The crystallinity highly relates to mechanical

strength (Nishiyama et al. 2002) such as Young’s modulus of BC, approximately in
the range of 15–35 GPa, and the tensile strength typically in the range 200–300 MPa,
respectively (Brown et al. 1976). The sub-fibrils are subsequently self-assembled to
generate microfibrils, resulting in a fibrillar ribbon then tightly aggregating each
other with a width of 50–80 nm. The resulted fibrillary ribbon is 200 times finer
than cotton fiber with an extremely large surface area (Vitta and Thiruvengadam
2002). With a high surface area to mass ratio, bacterial cellulose shows high-water
retention capability. The high-water content of bacterial celluloses is also due to the
hydrophilicity property caused by the pore structure. The relative hydrophilicity was
approximately around 40–50% (Bishop 2007). Additionally, BC shows the degree
of polymerization around 14.000––16.000 at pH 4 but its polymerization lowered
when pH increased to 5 (Tahara et al. 1997). A low degree of polymerization was
also found when bacterial cellulose was synthesized spherical-type bubble column
bioreactor (Choi et al. 2009).
BC is usually produced in a simple static method in which the container is filled
with the acidic medium containing carbon–nitrogen where the bacteria strain is inoc-
ulated at room temperature for a certain time, usually from 1 to 2 weeks. The static
method results in a thick pellicle at the top of the medium. The thickness increases
with the increase of fermentation time (Fig. 1). High crystallinity, strong tensile
strength, dense network structure, high-temperature resistance, and good flame retar-
dancy were generated from bacterial cellulose in static culture. Additionally, under
static culture conditions, bacterial cellulose was uniform in film shape as well as
showed good biocompatibility and biodegradability (Gao et al. 2020).

Fig. 1 Hestrin-Schramm-based bacterial cellulose

54 K. M. Pasaribu et al.

On the other hand, agitated cultivation is simply carried out by growing the bacteria
in a container containing growth nutrients and agitated for several days. The agitation
technique aims to increase the oxygen supply for the bacteria which finally resulted
in pellet-like aggregates. In the agitation culture, the crystallinity and cellulose Iα are
lower than from the static culture. However, the degree of polymerization is higher
than the static culture (Watanabe et al. 1998). Furthermore, through the agitation
culture, the smaller particle size of bacterial cellulose resulted in the high-water
holding capacity, compared to the static culture. Additionally, the lower Young’s
modulus and higher suspension viscosity were shown from the agitation technique
(Ougiya et al. 1997). Additionally, more porous bacterial cellulose was produced by
the agitation culture (Gao et al. 2020). So far it is considered that agitation is the
most proper method for cost-effective BC production (Hu et al. 2013). The selection
of these two methods is based on the BC application target with its various property
considerations. The pellicle-type BC was developed for a plasmonic paper sensor
(Purwidyantri et al. 2020) while the hollow-type spherical BC was proposed as a
seamless capsule for drug delivery applications (Hoshi et al. 2018). In addition to
the culture method, additional elements such as nutrients, type of bacterial strains,
oxygen availability, and the alignment of its three-dimensional network the environ-
ment of fermentation also have an impact on the bacterial cellulose properties (Kim
et al. 2019).
Generally, bacterial cellulose has far better properties than plant cellulose such as
purity higher than 99% (Klemm et al. 2005), total surface area of more than 150 m2 /g
(Ul-Islam et al. 2012), water holding capacity of more than 95% (Rebelo et al. 2018),
and tensile strength 20–300 MPa (Feng et al. 2015). Crystallinity is also an important
properties for evaluation since it relates to mechanical strength. A high crystallinity
of up to 98.4% was observed in bacterial cellulose produced from confectionery
using Komagataeibacter sucrofermentans after a 24 h HCl treatment (Efthymiou
et al. 2022a, b). Additionally, a crystallinity of 75.37% was obtained from bacte-
rial cellulose in sweet sorghum by Acetobacter xylinum ATCC 23767 (Wang et al.
2021). By using a laboratory-scale bioreactor with a 41 cm2 cross-sectional area,
overripe banana-based bacterial cellulose cultivated by Komagataeibacter medelli-
nensis showed a crystallinity of 82.93% (Molina-ramírez et al. 2020). The high
crystallinity highly corresponds with mechanical performance The tensile strength
and Young’s modulus of BC are 200–300 MPa and 15–35 GPa which are higher than
synthetic polymer (Cacicedo et al. 2016). These values usually vary depending on
the bacterial strain, cultivation method, culture nutrient as well as drying method. A
tensile strength of around 27.3–37.2 MPa was achieved when bacterial cellulose was
oven-dried (Illa et al. 2019). The tensile strength of bacterial cellulose-based stalk
and leaf of sweet sorghum when prepared in the medium of Acetobacter xylinum
ATCC 23767 was 8.24 MPa and 4.83 MPa, respectively. When bacterial cellulose
was produced in rotten guava mixed with cheese whey by using Komagataeibacter
intermedius MO, the tensile of 30 MPa was achieved (Lotfy et al. 2021). Further-
more, rotten banana-based bacterial cellulose showed tensile strength and Young’s
modulus of 280.6 MPa and 9.4 MPa, respectively, much higher than those synthesized
by Hestrin-Schramm medium (Molina-Ramírez et al. 2018). Young’s modulus of 8.7
Potential Application of Agro-Industrial Byproduct for Bacterial … 55

Fig. 2 Purification by 2% NaOH (left) (Skiba et al. 2020), purification by boiling water (right)
(Srikandace et al. 2022)

GPa was achieved from bacterial cellulose synthesized using tofu liquid water, higher
than from bacterial cellulose cultivated in the Hestrin-Schramm medium. This bacte-
rial cellulose also revealed the same irregular three-dimensional network made of
disordered dense fibrils arrangement with that produced from the synthetic medium
(Srikandace et al. 2022).
The resulted bacterial cellulose requires purification by removing the remaining
bacterial cell or nutrients in the medium. Different from plant cellulose which is
chemically linked with hemicellulose and lignin and thus makes it difficult to remove
impurities, purification of bacterial cellulose is much easier to carry out. Purification
is easily carried out by boiling dilute sodium hydroxide followed by rinsing it with
water (Revin et al. 2018). However, it has been proved that by boiling in water for
10 min with 2–3 replication after the water is decanted, pure bacterial cellulose
was obtained (Srikandace et al. 2022). Figure 2 shows the morphology of bacterial
cellulose purified by sodium hydroxide and boiling water, respectively.

3.1 Drying of Bacterial Cellulose

The drying method plays a role in the performance and properties of bacterial cellu-
lose. As bacterial cellulose is too thick and slippery, the dry state is preferable for its
wider application and it is more easily handled with stable properties. Various drying
method has been reported for bacterial cellulose treatment, such as oven drying (Illa
et al. 2019), microwave heating and air convection heating (Gao et al. 2020), and
evaporation (Zeng et al. 2014). These drying techniques provide various performance
alterations. When compared with the freeze-drying technique at −84 °C for 24 h, oven
drying of bacterial cellulose resulted in higher crystallinity, decreased fiber diameter,
narrowed size distribution, and increased mechanical properties (Illa et al. 2019).
However, the swelling ability of the bacterial cellulose gel was reduced through
freeze drying (Clasen et al. 2006). Additionally, whitish BC with higher porosity
56 K. M. Pasaribu et al.

was shown by the freeze-dried method whereas transparent and film volume reduc-
tion was deduced from bacterial cellulose by oven drying (Vasconcellos and Farinas
2018). Furthermore, a long drying time of 120 h at 100 °C provided tensile strength
of 250.7 MPa and a tensile modulus of 18.6 GPa (Abral et al. 2021). The supercritical
drying technique provided mechanically robust and extremely light films of bacterial
cellulose (Zeng et al. 2014) whereas freeze drying at −30 °C resulted in transparent
film with higher porosity (Urbina et al. 2019a, b). It was reported that the lyophilizer
technique employed at −50 to 20 °C for 36 h yielded a loose reticulated porous struc-
ture with a high-water absorption capacity (Feng et al. 2015). Microwave heating
was carried out in a short time but it provided bacterial cellulose with slightly lower
crystallinity and a higher swelling degree with the wrinkled surface (Indriyati and
Puspitasari 2019).

3.2 Bacterial Cellulose-Based Food Packaging

There have been various studies were carried out to use bacterial cellulose for food
packaging thus the evaluation of its properties for that purpose is indispensable.
Bacterial cellulose is hydrophilic due to its rich hydroxyl group content, therefore
bacterial cellulose has low barrier properties. Water vapor permeability (WVP) of
bacterial cellulose is frequently studied for food packaging applications. It appraises
the amount of water vapor that can pass through the package layer from the inner or
outer environment, which possibly leads to unfavorable alterations in the product’s
characteristics. For this purpose, bacterial cellulose produced by Gluconeacetobacter
hansenii CGMCC3917 was used as a reinforcing agent and it was incorporated with
agar for edible packaging. The concentration of 3–5% of bacterial cellulose showed
favorable WVP properties. The application of BC up to 10% decreased WVP up to
25.7% (Wang et al. 2018). The WVP ranged from 1.87 × 10−11 to 2.04 × 10−10 g /m
s Pa was obtained from a composite film containing bacterial cellulose, glycerol, and
polyvinyl alcohol. The film is the potential for food packaging to keep the quality of
food as well as increase the shelf-life (Cazón et al. 2020).
Another important characteristic consideration for food packaging is mechanical
strength which plays a role during production, storage, application, transportation,
and distribution. Mechanical properties such as ultimate tensile strength, elonga-
tion at break, tensile Young’s modulus, tensile toughness to break, ultimate puncture
strength, puncture deformation, puncture Young’s modulus, and puncture toughness
to break were evaluated for bacterial nanocomposite film incorporated with polyvinyl
alcohol, glycerol and boric acid. Due to its suitable mechanical properties, the resulted
nanocomposites are suitable for disposable packaging (Rouhi et al. 2017). Improve-
ment of mechanical properties as well as possess good antibacterial activity and
antioxidant capacity was shown from alternative edible and environment-friendly
sheets for food packaging made of bacterial cellulose, curdlan, and cinnamon essen-
tial oil (Zhou et al. 2022). Other enhancements of mechanical properties together with
Potential Application of Agro-Industrial Byproduct for Bacterial … 57

barrier and antibacterial properties were reported from a sheet prepared from bacte-
rial cellulose with the addition of konjac glucomannan and cucurmin. The resulted
film was claimed to support beef freshness (Li et al. 2022a, b). In addition, the
wrapping nanopaper generated from bacterial cellulose and Lactobacillus plantarum
was efficient against Lactobacillus monocytogenes in freshly ground beef (Shafipour
Yordshahi et al. 2020).
When bacterial cellulose was combined with cyanidin-3-glucoside, it resulted
in a smart pH-sensitive sheet that possessed an antioxidant characteristic and was
applicable for tilapia filet freshness non-destructive packaging indicator (Shi et al.
2022). Another smart film based on bacterial cellulose was developed by 2,2,6,6-
tetramethylpiperidine-1-oxyl radical (TEMPO)-oxidation containing thymol and
anthocyanin-rich purple potato extract. It was reported that the film showed improved
thermal stability, UV protection, and water vapor barrier characteristics but some-
what decreased tensile strength. With real-time assessment of freshness, these partic-
ular characteristics of composite film illustrate the prospective tool for commercial
shrimp packaging (Wen et al. 2021). Interestingly, isolates of sunflower protein and
bacterial cellulose with improved mechanical properties, water vapor permeability,
and solubility were developed for food packaging materials, specifically for fresh
fruit preservation (Efthymiou et al. 2022a, b).
Transparency as another important characteristic should be taken into account
not only for the product performance but also for consumer satisfaction. Trans-
parency and hydrophobicity improvement as well as antioxidant capacity was
obtained when bacterial cellulose-apple pomace-based nanopapers were combined
with hydrophobic medium-chain-length polyhydroxyalkanoate as a coating agent.
The film was developed for active packaging application (Urbina et al. 2019a, b).
Additionally, bacterial cellulose from sago liquid waste was developed for meat
sausage packaging. Its transparency was improved by the addition of carboxymethyl
cellulose into the bacterial cellulose. This treatment improved mechanical charac-
teristics as well as kept sausage quality for 6 days at room temperature (Yanti et al.
2021). Cheese whey permeates as a by-product of whey ultrafiltration, as a cheap
substrate for bacterial cellulose production by Komagataeibacter xylinus, and conju-
gated with Sakacin-A, produced by Lactobacillus sakei was reported as the potential
antimicrobial packaging material (Rollini et al. 2020).

4 Conclusion

As petroleum-based plastic supplies continue to decline the price rises whereas its
demand increases in line with the population growth as well as the awareness of
environmental rules have prompted an exploration for low-cost bacterial cellulose
production for environmentally-friendly food packaging. Even though the resources
are varied based on the type of activity, it has been reported that agricultural wastes
show potential as an alternative source of carbohydrates and nitrogen for bacterial
58 K. M. Pasaribu et al.

cellulose production. The studies contributed to investigating the applicable tech-

nique for food packaging application. If this biomass is used to produce bacterial
cellulose massively or on a large scale, this not only increases the value added of
the residues but also supports the waste management from the related agro-industrial
operation as well as the possibility of creating economic growth.

Acknowledgements The authors are grateful for any support provided by the National Research
and Innovation Agency, BRIN-Indonesia.

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Biomass Valorization for Bioenergy
Production

Sri Suhartini, Nimas Mayang S. Sunyoto, Hendrix Y. Setyawan,

Novita Ainur Rohma, Elviliana, Dinda Agustin Pratiwi, Fitria,
Efri Mardawati, Kasbawati, and Lynsey Melville

Abstract Biomass is currently seen as a potential to be used as bioenergy resources.

Its high availability and renewability generate extensive interest for further valoriza-
tion. In Indonesia, research and development of transforming biomass into bioenergy
via different pathways is expanding. Conversion of biomass via physical/mechanical,
biochemical, and thermochemical offers produces bioenergy in the form of liquid
(i.e., biodiesel, bioethanol, and bio-oil), gasses (i.e., biogas and syngas), and solid
(i.e., biopellets, biochars, and briquettes). These types of bioenergy are essential
for substituting fossil-based fuels, hence have positive impacts on reducing carbon
emissions and climate change. Different mechanisms of process occur during the
conversion. Specific measures to the influencing factors are crucial to ensure the
optimum performance efficacy. This chapter discusses various bioenergy routes from

S. Suhartini (B) · N. M. S. Sunyoto · H. Y. Setyawan · N. A. Rohma · Elviliana · D. A. Pratiwi

Department of Agro-Industrial Technology, Faculty of Agricultural Technology, Universitas
Brawijaya, Malang, East Java 65145, Indonesia
e-mail: [email protected]
Fitria
Research Centre for Biomass and Bioproducts, National Research and Innovation Agency
(BRIN), Cibinong, West Java, Indonesia
Department of Biological Systems Engineering, Washington State University, Richland,
Washington, USA
E. Mardawati
Department of Agro-Industrial Technology, Faculty of Agro-Industrial Technology, Universitas
Padjadjaran, Sumedang, West Java, Indonesia
Kasbawati
Applied Mathematics Laboratory, Department of Mathematics, Hasanuddin University, Makassar,
Sulawesi, Indonesia
L. Melville
Bioresource and Bioeconomy Research Group, Faculty of Computing, Engineering and Built
Environment, Birmingham City University, Birmingham, West Midlands, UK
S. Suhartini
Centre of Excellence in Bioenergy and Biorefinery, Faculty of Agricultural Technology,
Universitas Brawijaya, 65145 Malang, Indonesia

© The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2024 67
M. A. R. Lubis et al. (eds.), Biomass Conversion and Sustainable Biorefinery, Green
Energy and Technology, https://doi.org/10.1007/978-981-99-7769-7_4
68 S. Suhartini et al.

biomass substrates from the process’ mechanisms to examples, in particular anaer-

obic digestion, transesterification, fermentation, densification, and thermochemical
pathways.

Keywords Anaerobic digestion · Transesterification · Fermentation · Pelletizing ·

Pyrolysis

1 Introduction

Energy demand continues to increase in line with the increasing rate of population
growth and economic growth. For example, according to the Ministry of Energy
and Mineral Resources of the Republic of Indonesia (Kementerian ESDM 2021),
energy demand in Indonesia is expected to continue to increase until 2030 with an
average annual increase of 1.6%. Energy is used in various fields, one of which
is as fuel for industrial, transportation, and household activities. The most widely
used energy source to date comes from fossil fuels (Ulfa et al. 2021). Fossil fuels
are non-renewable fuels and their availability is limited, thus over time there will
be scarcity. In addition, fossil fuels can produce emissions (such as NOx and SO2 ),
when accumulated, will cause acid rain (Zikri et al. 2018). These problems have an
impact on climate change (Istiani et al. 2021).
Also, along with the increasing demand for energy, it is necessary to use renewable
energy as an alternative to fossil fuels. Renewable energy can be created from the
utilization of biomass including agro-industrial, plantation, agricultural, and forestry
wastes. Biomass is an organic material obtained from living things in nature (Calvo-
Serrano et al. 2019). Usually, biomass comes from products that are renewable,
urban waste, forests, and residues originating from the agricultural sector. Biomass-
derived from plants is obtained by the reaction between carbon dioxide and water, air,
and the sun through the process of photosynthesis to produce carbohydrates which
form a group of biomasses. Biomass is composed of three main components, 40–
60%(w), 10–30%(w), and 20–30%(w) (Reyes et al. 2021). Biomass-derived from
forest and agricultural plants is generally composed of components of cellulose,
hemicellulose, lignin, fat, protein, starch, and sugar. Biomass also contains water
and organic components such as nitrogen, sulfur, alkali, alkaline earth, heavy metals,
magnesium, chlorine, and potassium.
According to Kusumaningrum and Munawar (2014), biomass has the potential to
be used as an energy source as it is abundant, cheap, environmentally friendly, and
renewable. While Tursi (2019) defined biomass is organic material produced directly
or indirectly by living organisms and is available in a renewable manner. Use of
biomass as bioenergy sources can minimize the negative impacts on the environment
(Febrianti et al. 2020). Also, the benefits of using biomass-based bioenergy are that
it does not emit sulfur that causes acid rain, produces less amount of ash than coal,
is abundant in availability, renewable, and relatively fast to produce (Munawar and
Subiyanto 2014).
Biomass Valorization for Bioenergy Production 69

Sources of biomass include agricultural residues such as rice husks, corn cobs,
and corn fiber, remaining forest products such as wood, agricultural and plantation
industry residues such as palm oil waste including empty oil palm bunches (EFB),
shells, fronds, palm tree trunks, wood processing industry sawdust, cocoa shells,
sugarcane bagasse, pulp sludge, urban waste such as used paper, and dry leaves
(Febrianti et al. 2020). Agricultural industry waste is another source of biomass
(Wang et al. 2022). This waste can be detrimental to the environment, like other
industrial wastes. The form of this waste can be solids, gases, or liquids. In handling
agricultural and industrial waste, it is necessary to group it based on its form and on
its raw components, such as carbohydrates, proteins, or fats. Indonesia has potential
biomass resources of up to 50,000 MW which are dominated by plants and industrial
organic waste. Installed biomass capacity in Indonesia reaches 312 MW (Yana et al.
2022). Biomass is formed by the main lignocellulosic compounds including cellu-
lose, hemicellulose, and lignin. These three compounds function to form complex
chemical bonds to become the basic material for plant cell walls. The cellulose
content in the biomass varies and ranges from 40 to 50%. Hemicellulose in biomass
has a percentage of about 15–35%. Lignin in biomass has a percentage of around
10–25% (Tursi 2019), and is considered as a complex structure that covers the cell
walls (Hermiati et al. 2010). The lignocellulosic content contained in biomass can be
used to produce energy through the biomass conversion process. Biomass is unique
in nature and has different concentration of lignocellulosic compounds, as shown in
Table 1 (Hermiati et al. 2010).
Biomass sustainability is critically important to be created in the supply of energy
sources. The government’s role as a policymaker is vital in developing and imple-
menting renewable energy sources. The support from the Indonesian government
is by releasing a Minister of Energy and Mineral Resources Regulation regarding
the implementation of co-firing to increase biomass economies of scale and reduce
dependence on coal.

Table 1 Lignocellulose content in various biomass

Biomass Cellulose (%) Hemicellulose (%) Lignin (%)
Cocoa shell 36.47 18.90 60.67
Sugarcane bagasse 50 25 25
Corncob 41 36 16
Wheat straw 30 50 15
Empty fruit bunches of palm oil 44.21 16.68 35.51
Rice husk 34.4 24.3 19.2
Source Hermiati et al. (2010)
70 S. Suhartini et al.

2 Bioenergy from Biomass Resources: Mechanisms

and Example

Biomass as a fuel without going through the conversion process has bad properties
due to its low density. Direct use of biomass also causes respiratory problems due to
carbon monoxide (CO) and sulfur dioxide (SO2 ) gases. To reduce this, biomass can be
converted into energy such as heat, electricity, liquid, solid, and gaseous fuels (Moura
et al. 2022). Various opportunities routes for biomass valorization to bioenergy, either
to physical, biochemical, thermal, mechanical, thermal, and thermochemical path-
ways (Fig. 1). These can be done in several ways including densification, gasification,
carbonization, pyrolysis, and anaerobic digestion processes. Each of these conver-
sion principles consists of a specific process that is useful to convert biomass into
bioenergy. These routes can produce bioenergy in different forms such as liquids
(i.e., biodiesel, bioethanol), gases (biogas, syngas), and solids (biopellet, biochar,
briquettes) (Parinduri and Parinduri 2020).

Fig. 1 Biomass conversion to energy pathways

Biomass Valorization for Bioenergy Production 71

Fig. 2 Stages in the anaerobic digestion process (Hagos et al. 2017)

2.1 Anaerobic Digestion

2.1.1 Definition and Mechanisms

The anaerobic digestion (AD) process consists of four stages: hydrolysis, acido-
genesis, acetogenesis, and methanogenesis, where the dominant biogas containing
methane gas is produced in the methanogenesis process. The stages of the anaerobic
digestion process are shown in Fig. 2. Biogas produced can later be converted into
electrical and heat energy or developed into biomethane (Montingelli et al. 2016).
Besides biogas, AD also generated organic residues, known as digestate. The AD
system enhances the recovery of nutrients (N and P) in the final product. The resulting
P composition has the potential to be reused as fertilizer (Li et al. 2022).

2.1.2 Hydrolysis

Hydrolysis as the initial stage of AD is a process of breaking down large and complex
polymers such as fats, oils, starch, polysaccharides, and proteins into monomeric
or oligomeric components such as amino acids, long-chain fatty acids, and simple
sugars. This process is carried out by extracellular enzymes that are owned by
microorganisms into the total volume of solution (Thanarasu et al. 2022). This initial
stage, known as biological pretreatment, will also produce intermediate products
such as ammonia and long-chain fatty acids that can affect the AD process. Thus, the
adjustment process in the microbial consortium as well as the bioreactor needs to be
72 S. Suhartini et al.

carried out. Operational strategies include co-digestion, pretreatment with enzymes,

chemically or mechanically, as well as dividing the AD stages into two major or other
stages. Hydrolysis is also known as one of the rate-limiting reactions in which compo-
nents of complex chemical compounds such as fats, polysaccharides, and proteins
are deconstructed into monomers. Hydrolyses are included as individual monomers
from suspended solids. The results of this modeling can also determine sufficient
exposure time to provide pretreatment as an effort to increase biogas production
(Hirmiz et al. 2019). Some preventive steps to avoid biogas production barriers are
carried out in several ways, namely, by adding inoculum and adjusting the pH at the
hydrolysis–acidogenesis stage. The result of this adjustment can increase bioenergy
production two times higher than usual.

2.1.3 Acidogenesis

The acidogenesis stage begins with the absorption of monomer and polymer prod-
ucts into the cell membrane of acidogenic bacteria into volatile fatty acids (VFA),
alcohol, and other inorganic compounds such as CO2 , H2 , H2 S, and NH3 (Richard
et al. 2019). VFA control is an important factor in biogas production, because the
accumulation of these compounds will reduce the effectiveness of biogas production
(Thanarasu et al. 2022). Microorganisms involved in this stage include Acetivibrio,
Bacteroides, Clostridium, Eubacterium, Lactobacillus, and Streptococcus. One of the
syntrophic microorganisms, namely, C. Cloacimonas, plays a key role at this stage.
This microbial ability is related to the production of methane gas from the hydrogen
chain. The coenzyme associated with it is Methanoculleus (Niu et al. 2022). The
main challenge in this stage is to avoid VFA conversion to methane gas. The focus of
research must emphasize this issue to avoid inhibiting biomethane production. The
study of (Al-Sulaimi et al. 2022) showed a negative effect of VFA acidification on
production efficiency and biogas biodegradation from waste sludge substrates, espe-
cially for bioreactor conditions that use thermophilic reactor conditions. The high
concentration of VFA will cause the pH to drop and cause an unfavorable reactor
environment for the methanogen process.

2.1.4 Acetogenesis

Substrate through subsequent breakdown which produces H2 and CO2 accompanied

by acetate is referred to as the process of acetogenesis. Acetogens are an important
step in the biodegradation process of organic matter to maintain the efficiency of
the biogas production process. Acetogenic bacteria will use production from hydrol-
ysis and oxidize pyruvate. Pyruvate is part of the intermediate product in the AD
process and will be converted to acetate (Thanarasu et al. 2022). Other results such as
the hydrogen gas produced will initiate syntrophic relationships in the AD-transfer
system between hydrogen species (Meegoda et al. 2018). Acetogen species can
be divided into several parts, namely, hydrogen generators and proton-to-hydrogen
Biomass Valorization for Bioenergy Production 73

reducers. Most of the known homoacetogen species are from the genera Aceto-
bacterium, Acetoanaerobium, Acetogenium, Butyribacterium, Clostridium, Eubac-
terium, and Pelobacter (Borja and Rincón 2017). In a single stage, microorganisms
that play a role in acetogenesis and methanogenesis develop simultaneously. The two
microorganisms require different environmental conditions. So, currently, another
study is being carried out regarding the optimum conditions for both bacteria in
single conditions or in two-stage conditions (Qian et al. 2019).

2.1.5 Methanogenesis

The end product of acetogenesis is acetate, H2 , and the methylated mixture is used by
methanogenic bacteria for gas production. Acetate used by methanogens can become
a methyl group and CO2 , and then this group is reduced to methane using electrons
provided by the carboxyl group (Thanarasu et al. 2022). This methanogenesis takes
place at the end and takes a long retention time between 15 days and 3 months
(Tabatabaei et al. 2020). Microorganisms that play a role in this stage are methanogens
which are a group of archaebacteria that are unicellular and very sensitive to the
presence of oxygen (Borja and Rincón 2017). One of the inhibiting factors in this
stage is the presence of excessive ammonia gas from a high nitrogen content substrate
as the main inhibitor. The toxic level of this gas is caused by un-ionized ammonia, due
to its ability to enter the bacterial cell membrane to disrupt the balance of potassium
and methane protons. The handling practice is to provide additional water, but this
also increases operational costs and is inefficient. Because of this, several research
and industrial projects have begun to focus on adapting environmental conditions
to these methanogenic microbes (Capson-Tojo et al. 2020). There are two pathways
involved in this step involving acetic acid and carbon dioxide, the two main products
from the previous step to produce methane gas (Kirk and Gould 2020):

Acetotr ophic Reaction CH3 COOH → CH4 + CO2 G0 = −30, 9 kJ/mol
H ydr ogenotr ophic Reaction 4H2 + CO2 → CH4 + 2H2 O G0 = −135, 4 kJ/mol

The main product resulted from AD is called biogas. Biogas is a mixture of

gases produced by methanogenic bacteria through an anaerobic digestion process
that transforms complex organic compounds with the help of microorganisms under
anaerobic conditions (Gonzalez-Gil et al. 2018). Complex organic compounds can be
found in biomass. However, biomass used as raw material for biogas generally has low
economic value or is waste, such as industrial agriculture waste (agro-industry), forest
product waste, and livestock waste. Many gases contained in biogas include methane
(CH4 ), carbon dioxide (CO2 ), ammonia (NH3 ), water vapor (H2 O), hydrogen sulfide
(H2 S), methyl siloxane, nitrogen (N2 ), oxygen (O2 ), volatile components (VOC),
carbon monoxide (C.O.), and hydrocarbons. The most extensive content in biogas is
50–80% CH4 and 20–50% CO2 from the amount of gas produced. Biogas’ energy
depends on methane concentration, where the higher the methane content, the greater
74 S. Suhartini et al.

the energy content or calorific value (Neves et al. 2004). Physically, methane is color-
less, odorless, and flammable. Chemically, methane is composed of one C atom and
four H atoms. This compound is very stable due to the presence of C–H bonds and
requires an energy of 438.8 kJ/mol to break it down (Park and Lee 2013). A simple
purification process can purify biogas by passing the gas through a NaOH solu-
tion to bind CO2 (one impurity) and converting it to precipitate sodium bicarbonate
(Na2 CO3 ) H2 O. This process will increase the purity of methane (CH4 ). Based on
research conducted by Lasocki et al. (2015) using 1 M NaOH for 10 min can increase
the concentration of CH4 and remove H2 S and CO2 altogether.

2.1.6 Enhancing ADPprocess: Pathways

As AD technology is primarily dependent on microbial activity, ensuring the feed-

stock has higher accessibility for the microorganisms is a must. Pretreatment on
biomass especially with higher lignocellulosic content should be considered before
treated with AD. There are common pretreatment methods, i.e., physical, mechan-
ical, biological, and chemical. Ampese et al. (2022) used the dried apple pomace
as AD feedstock VS that through physical pretreatment using a mill to reduce its
size resulted in 36.61 L CH4 /kg VS that was predicted to generate electricity, heat,
and carbon reduction in 1.92 kWh/ton, 8.63 MJ/ton, 0.62 kg, and CO2eq /ton, respec-
tively. Steam explosion (STEX), as part of physicochemical pretreatment, is being
investigated on many energy crops and ecological plants. A lignin-rich macrophyte
(Potamogeton maackianus) under semi-continuous AD is pretreated via STEX and
resulted in enhancing the hydrolysis efficiency from 19% (108 ± 31 L/kg VS) up to
50% (200 ± 36 L/kg VS) (Akizuki et al. 2022). The STEX gives an easier breakdown
for the microbial activity and enhances the biomethane yield.
Minimizing the energy input and faster reaction is part of merits of chemical
pretreatment, but another consideration that should be noted is the toxicity profile
from the materials that has to meet the principle of green chemistry. Lomwongsopon
and Aramrueang (2022) seek the biogas potential from cassava pulp pretreated using
mild-chemical with concentrations 0.1–0.3 w/v% of H2 SO4 , HCl, NaOH, and KOH.
KOH under 2.0 w/v% showed the highest methane yield of 324 ± 4 L/kg VS. Biolog-
ical pretreatment as one of the favorable methods to explore is generally utilized
microorganism activity to degrade and reduce the thickness of cell wall of lignocel-
lulose biomass. For instance, stover as the feedstock is being investigated via AD
and given the biological pretreatment using microbial consortium to positively affect
the activity of functional microorganisms. A 62.85% increment on the peak phase is
shown than the untreated stover and significant methane production is resulted (Zhao
et al. 2019).
Along with its development, AD with one feedstock produces low yields and is
inefficient (Beniche et al. 2021). The application of Anaerobic co-digestion (AcoD)
which is an AD system that uses two or more substrates to improve the process,
stability, or production of biogas is preferred because it can increase biogas produc-
tion by diluting toxic substances and provide a synergistic growing environment
Biomass Valorization for Bioenergy Production 75

for microorganisms and can also handle multiple wastes at once (Taboada-Santos
et al. 2019). For instance, the study of (Wang et al. 2022) the anaerobic co-digestion
between excess sludge and chicken manure has effectively increased the methane
yield in mesophilic and thermophilic temperatures up to 123.1 L/kg VS and 171.3 L/
kg VS, respectively. Proper mixing ratio to have the balance support factor (i.e. pH,
C/N) is the main key to enhance the biogas production.

2.1.7 Examples

Commodity of palm oil has been the major crops in some tropical countries, i.e.,
Indonesia, Thailand, and Malaysia as the three largest producers of biomass. From
the industrial process, empty fruit bunches of palm oil (EFB) are the generated
waste that could be valorized via AD route to produce biogas. (Suhartini et al. 2020)
evaluated the untreated EFB biomass with batch AD under mesophilic temperature
for 30 days and the specific methane potential was 0.110 m3 /kgVSadded. Similarly,
(Hidayat et al. 2020) investigated the biomass potential with fungal pretreatment to
manage the higher content of lignin which is one of the barriers of AD steps. The
pretreatment is capable to disrupt the lignocellulose content and enhance methane
production than the untreated along with increment in the organic matter.
Interest in utilizing the macroalgae along with other feedstock has gained more
studies with various perspectives either with co-digestion or additional of other bioac-
tive compounds to obtain higher methane yield. In our previous studies, using G.
verrucosa with the addition of tofu dregs substrate, a higher SMP was produced in a
20% tofu dregs mixture (compared to only 10%) with a value of 120 L CH4 /kgVS
(Suhartini et al. 2022a).
In line with the synergetic effect from co-digestion, a study by Wickham et al.
(2019) showed a positive effect of AcD, with a stable composition of methane gas
in the biogas of 60–65%. In this case, AcD does not show any negative implications
such as disrupting the stability of the reactor or other toxic gases. However, not all
AcoD can provide optimal results, in the study (T.A.S et al. 2020) compared mono
and co-digestion substrates and resulted in mono-digestion gave higher yields of
0.61 m3 /KgVSreduced compared to AcoD of 0.39 m3 /KgVSreduced . Therefore, AcoD
needs to be carried out to see the relation impact on biomethane production either
antagonistic or synergetic. Apart from the biomass potential on biogas production
and pretreatment work, for the pilot and industrial scales of AD implementation,
sometimes the foaming issues are the major concern. This condition could be due
to the presence of unwanted microorganisms and other operational factors. Trace
element supplementation, adjustment of organic loading rate, addition of anti-foams,
and water dilution could be the solution for the foaming issue (Suhartini et al. 2019).
76 S. Suhartini et al.

2.2 Transesterification

2.2.1 Definition and Mechanisms

Transesterification as the most common mechanism to produce biodiesel will be

discussed in detail in this section. Figure 3 shows the main process that starts from
long-chain fatty acids react with alcohol with catalyst and through three stepwise
that ultimately produce methyl esters as biodiesel and glycerol as the co-product
(Meher et al. 2006). In addition, this reaction is divided into two sections: catalytic
or non-catalytic. As shown in Fig. 4, catalytic process could be conducted with
homogeneous catalysis, heterogeneous catalysis, or combinatorial catalysis. Acids,
alkalis, and enzymes are the materials to be utilized for synthesizing biodiesel under
catalytic conditions. FAME produced from various substrates of waste is attracting
more researchers to utilize the low cost of feedstock and minimize the waste presence.
This study used chicken fat oil and waste cooking oil with acid catalyst and gained
the FAME yield of 90.8% and 92.3%, respectively, and categorized as the suitable
feedstock for biodiesel feedstock (Shatesh Kumar et al. 2020). Meanwhile, the non-
catalytic is carried out with supercritical fluid. This process could be produced more
than one product such as other value-added by-products, i.e., triacetin. Under the
economic and environment assessment, the supercritical fluid, the progress to address
the saponification issue and other unwanted waste generation through other steps,
which provides more advantages will have more feasibility if using the glycerol-free
process to gain high revenue (Ang et al. 2014).
Table 2 shows the advantages and disadvantages of each process of transesterifica-
tion under catalytic or non-catalytic process. It should be noted that every process will
work effectively under the specific feedstock. The heterogeneous catalytic process
emerged to combat drawbacks of homogeneous catalyst role, but if homogenous
catalyst coupling with other methods, i.e., ultrasound-assisted extraction using the
Annona squamosa seed oil will result in rich oil content of 94.7% yield (Sundarama-
halingam et al. 2021). The study revealed that the combined methods seem more
feasible from energy balance and have a positive impact on the economic view.
Transesterification of biomass or organic waste results in biodiesel. Biodiesel as
a green fuel produced from animal oil, vegetable oil, and algae oil has the physico-
chemical similarity with fuel diesel but has low energy density and generates lower

Fig. 3 Transesterification reaction (Meher et al. 2006)

Biomass Valorization for Bioenergy Production 77

Fig. 4 Overview of transesterification in biodiesel production processes. Adapted from Nayab et al.
(2022)

Table 2 Overview of transesterification in biodiesel production processes

Process Materials Advantages Disadvantages References
Homogeneous H2 SO4 , NaOH, – Faster reaction – Contaminated Sakai et al.
catalysis NaOCH3 , KOH rate the outcome, (2009), Kaur and
– Required mild affected the Ali (2011)
reaction separation
condition process
– High – Soap formation
corrosiveness – Low purity
– Non-reusability – Toxic waste
– Difficult to be generation
separated
Heterogeneous CaO, – Reusable – Partial Lee et al. (2014)
catalysis CaO–KOH, – Easy separation recoverability
CaMgO, CaZnO – Reduces and reusability
wastewater – Energy and
generation waste
– Lower toxicity conservation
problem
– Longer
retention time
Combinatorial Mixed catalysis – Increase the – High expenses Kim et al. (2008)
catalysis yield
– Decrease the
toxic product
Supercritical Variance in – Shorter duration – Operational cost Deshpande et al.
fluid pressure, progress (2017),
temperature, – Efficient Makareviciene
molar ration conversion step and Sendzikiene
between alcohol – Catalyst-free (2021)
and lipid, and operation
retention time – Lower quality
of feedstock can
be used
78 S. Suhartini et al.

Table 3 Biodiesel production and major feedstock (OECD-FAO 2021)

Country Production (%) Major feedstock
European Union (EU) 32.3 Palm oil, rapeseed oil, used cooking oils
United States 18.1 Soybean oil, used cooking oils
Indonesia 15 Palm oil
Brazil 12.2 Soybean oil
Argentina 5 Soybean oil

GHG emissions (Okechukwu et al. 2022). This substitution could tackle the climate
change issues due to reliance on fossil fuel-based sources. Biodiesel or fatty acid
methyl ester (FAME) is generated by transesterification and esterification, and is
produced of mixed fatty acid esters (biodiesel) and glycerol as the by-products.
Global biodiesel production is projected to reach 50 billion L in 2029, which was
53% higher than in 2016 (Rezania et al. 2019). EU has the first position as the largest
country in the production of biodiesel followed by the USA and Indonesia. The major
feedstock in detail is shown in Table 3. Biomass and its waste utilization in biodiesel
production could lead to bioeconomy concept which offers sustainability pathways
to generate renewable energy sources.

2.2.2 Pretreatment for Biodiesel Production

In order to enhance the efficient and effective result, biomass is pretreated either
with mechanical, physical, chemical, or biological. This pretreatment to assure the
extraction will give the maximum result. For instance, the lignocellulose biomass is
rich in lignin content, and high crystallinity which will hinder the process of biodiesel
production, especially with tolerance to microbe and chemical conditions. Biomass
pretreatment for biodiesel production has been investigated in several studies. Biolog-
ical pretreatment is being sought from other studies. It has been reported that the corn
straw is pretreated with Mycobacterium smegmatis LZ-K2 to prove the microor-
ganism’s capability in reducing the lignin content (Zhang et al. 2019) and the
enzyme system shown the possibility further to treat such lignin-rich biomass. In
other pretreatment, i.e., dilute acid could produce ethanol and biodiesel. This case of
wheat straw valorization under moderate conditions with 140–160 °C and 0.3–0.6%
of sulfuric acid concentration could degrade the lignin content more than the other
parts. Meanwhile, the physical pretreatment has also been investigated to enhance
the yield of FAME. Priyadarshi and Paul (2018) conducted the advanced microwave
technology (AMT) pretreatment in regard to achieve high yield of FAME with source
from kitchen waste. It resulted to have 96.89 wt% as the maximum FAME yield.
A similar pretreatment was also identified from the study to increase the yield of
FAME and reported that 93 wt% of the FFA conversion is achieved with AMT as the
pretreatment method to reduce water and the viscosity reduction (Idowu et al. 2019).
Biomass Valorization for Bioenergy Production 79

2.2.3 Bioeconomy Concept on Biodiesel Production

Majority of the world is facing crisis in energy, water, and food nexus. Most of all
technology still lack sustainability and viability from the economic and environment
view. For instance, in order to produce one renewable energy product, the energy
balance resulted in negative values which is not possible to proceed to the pilot
scale or industrial scale. This issue is affected by many factors, including feedstock
characteristic and technology itself. Hence, implementing bioeconomy concept into
the conversion technology is urged to be adopted. Not only produce one product
but also more value-added products to balance the energy balance or generate more
energy output. Same condition applied for this biodiesel production. Most studies
have investigated the chance of producing more products.
Angelaalincy et al. (2023) reported the investigation on managing the envi-
ronmental contamination from arsenic with phytoremediation and nanopartible-
mediated adsorption while co-produce the biodiesel and increase up to 125% the
lipid content and incline the FAME production. This technology combats the pollu-
tion issue and provides the new renewable energy outcome which promotes the
biorefinery system. Jeyakumar et al. (2022) studied the third generation of biofuel,
Padina tetrastromatica and Sargassum swartzii macroalgae, to produce bioethanol
and the solid waste generated is being sought the biodiesel production potential. The
result shows that both marine biomasses are capable of extracting ethanol yield and
FAME yield more than 80%. Another example of biomass valorization to promote the
circular economy is converting Macaúba husk into biodiesel and biomethane. The
generated waste from biodiesel production is firstly pretreated by subcritical water
hydrolysis to breakdown the lignocellulose compound and via anaerobic digestion
(AD) to have the clean energy, i.e., biogas (Ampese et al. 2021). The pretreatment
successfully improves the biodegradability of feedstock and the biogas production
could be further utilized in combined heat and power scenarios to enhance the func-
tionality. Another feedstock being identified with its potential to obtain the same
output (biomethane and biodiesel) is the energy crop, sunflower plant. The study
result shown the biodiesel yield and biogas production in 96.2% and 342.7 N mL/
g VS, respectively (Ebrahimian et al. 2022). This study explored hot water, sodium
carbonate, and phosphoric acid as the three physiochemical pretreatments to increase
the hydrolysis step that is beneficial for AD technology and showed a positive effect
to maintain the recalcitrant content of its biomass.

2.2.4 Examples

A study from Halim et al. (2022) regarding the extraction of oil and protein from
the species Nannochloropsis sp. (microalgae) developed a hypotonic autolytic and
osmotic incubation pretreatment method for easy penetration of microalgae cell walls
up to 4.9 times compared to only using conventional methods. After pretreatment, cell
disruption (CD) was continued with the mechanical disruption (HPH) or chemical
disruption (pH 12) method. From the experiments, the cell walls that did not undergo
80 S. Suhartini et al.

pretreatment only experienced 12% CD. The remaining more than 80% indicates that
the defense wall of Nannochloropsis cells is very thick and difficult to penetrate, so
it requires pretreatment to weaken this defense and also increase CD performance
(the next process) to maximize liberation products. The results of the pretreatment
of autolytic incubation made CD HPH and pH 12 increase, respectively, by 2.4x and
4.9x. The increase in CD performance is due to the high efficiency of disruption
which is characterized by the thinning of the cell wall due to its weak defense and
makes the biomass more sensitive to the treatment it receives. Other pretreatment
results from hypotonic osmotic shock also provide good output. The CD of HPH
and PH12 increased by 1.6x and 1.4x, respectively. The low result of this osmotic
performance is because this method further increases membrane permeability.
The capability of fixing CO2 by microalgae is very high and this process can also
synergize with each other to produce other energy products such as omega-3, biohy-
drogen, bioethanol, and biodiesel. The study of Srinuanpan et al. (2018) showed the
potential for synergy in the production of biogas and also biodiesel by cultivating
the microalgae Scnedesmus sp. CO2 gas reduction of >96% and CH4 levels obtained
>98% are obtained and shown the effective of elaboration of converstion technolo-
gies. Another study examining the addition of bacterial culture in microalgae culti-
vation to increase the FAME yield was carried out by Kumsiri et al. (2021). The use
of Picicocus intestinalis WA3 bacteria in the cultivation of microalgae Tetradesmus
obliquus gives a 1.3x increase in biomass production, 1.39x chlorophyll content, and
1.55x lipid productivity. Another way to enhance the biodiesel yield is feedstock
valorization. The mixed culture of microalgae (MC) and food waste have been used
as a feeding source for the black soldier fly larvae (BSFL) to enrich its nutrients and
the BSFL is utilized as the medium to extract biodiesel from it. The result shown
that FAME yield of BFSL with MC feeding is higher up to 140% comparing to the
food waste feeding (Mahmoud et al. 2022).

2.3 Densification

2.3.1 Introduction and Mechanisms

Densification, which defined as a way of developing material functions to increase the

energy content per unit volume, heat capacity, and reduce combustion ash (Qadry
et al. 2018). The densification process is the process of compacting biomass by
pressing or pressing thus increasing the potential energy and product mass density
(Pradhan et al. 2018). The densification process is mostly carried out on bulk mate-
rials or those that have irregular physical forms such as biomass. This is because
biomass has a low natural specific energy content. The densification process has
several advantages, including facilitating the handling, storage, and transportation
processes, controlled particle size distribution for product uniformity and density,
improved product composition quality, increased product calorific value per unit
volume, and increased energy content (Gong et al. 2021).
Biomass Valorization for Bioenergy Production 81

The densification is divided into extruding, briquetting, and pelleting. Extruding

process involves compressing the material on a screw (screw) or piston to produce
a solid and compact product (Istiani et al. 2021). Briquetting process produces a
product shaped like a tube with varying dimensions as needed. Pelleting process
involves the flow of material from a rotating roll accompanied by pressure into the
holes for the biopellets (dies) (Widjaya et al. 2019). According to Winata (2013), the
pelletization process is drying and forming biomass using high pressure to produce
cylindrical solid biomass with a maximum diameter of 2.5 cm. Pelletization is carried
out to produce denser biomass with a smaller volume and higher density. The quality
of solid biomass is influenced by the strength and durability of the particle bonds,
die diameter, die temperature, pressure, and preheating of the biomass mixture. The
pelleting process can be carried out using the cold method (or without a heating
process).
One of the products that resulted from densification is biopellet. Biopellets are a
form of renewable energy (Ulfa et al. 2021); in a pellet-shaped made from compressed
biomass materials (Munawar and Subiyanto 2014). Biopellets have a cylindrical
shape resembling a pipe and have a solid texture, but have a smaller size than
briquettes, and generally used for residential stoves, boilers, gasifiers, etc. (Rusdianto
and Choiron 2015). Biopellets are widely used by people in American and European
countries for space heating in winter and boiler fuel in industry. The size of the
biopellets is about 6–10 mm in diameter and about 10–25 mm in length. The phys-
ical characteristics of biopellets such as color depend on the raw materials used
(Hadiyane et al. 2021). An example of biopellets from coffee waste is shown in
Fig. 5.
The advantages of transforming biomass into biopellets include high fuel effi-
ciency and consistency (i.e., reduce NOx emissions, particulate matters, and volatile
organic matters), produce fewer emissions than wood, relatively easy to manufacture,
no risk of explosion (Rusdianto and Choiron 2015). Biopellets are a superior fuel due
to their higher energy content than the original biomass material. Also, biopellets

Fig. 5 Biopellet from coffee

waste (Hadiyane et al. 2021)
82 S. Suhartini et al.

have a denser structure, are easy to handle, environmentally friendly, and easy to
distribute (Qadry et al. 2018).
According to Frodeson et al. (2019), the two methods commonly used in the manu-
facture of biopellets are pelletization using screw pressing and hydraulic pressing.
Threaded pressing can be done using a single-or twin-screw press. Pressing using a
threaded press has several advantages including greater production capacity because
the process takes place continuously and saves production time. The way a threaded
press works is by applying the screw principle. The material is pressed using the thrust
of a rotating screw, then it will be pushed out as a result of the applied pressure. The
more the material goes to the end of the screw, the greater the pressure experienced
by the material. According to Damayanti et al. (2017), hydraulic pressing is done by
applying hydraulic pressure to the material of 2000 lb/in2 to allow the material to be
molded.
The process of making biopellets generally consists of several stages including
preparation of biomass raw materials, pretreatment in the form of initial drying
of raw materials and size reduction, densification process (pelleting), final drying
of biopellets, and packaging (Istiani et al. 2021). According to Raudhatul Jannah
et al. (2022), the size reduction is carried out with a chopping machine to make the
size of the raw material range from 3 to 5 mm and then flouring is carried out to
transform biomass material into powder. The pelletization process is carried out using
a ring die pellet mill. The powdered biomass is put into the machine to be molded
into biopellets according to the desired size. Drying of biopellets aimed to reduce
the water content to 10–15% before packaging (Damayanti et al. 2017). Factors
that affect the characteristics of pellets include characteristics and composition of
biomass, adhesive, particle size, moisture content, densification equipment, pressing
conditions, and post-production handling (Zikri et al. 2018).
The main parameter in determining the quality of fuel products is the calorific
value. The higher the calorific value, the better the quality of the biopellets (Ulfa
et al. 2021). High-quality pellets are generally obtained by densification under high
pressure with the addition of an adhesive. In the pelleting process in medium capacity,
effective adhesives are derived from natural and cheap ingredients such as sago flour
or tapioca flour (Damayanti et al. 2017). The physical criteria for good biopellets
include surface structure are smooth dense and solid, no moldy during long-term
storage, and do not emit excessive smoke (Lubis et al. 2016). Also, biopellets with
good quality must meet predetermined standards as regulated in SNI 8675:2018
(NSA 2018) about biomass pellets for energy, as can be seen in Table 4.

2.3.2 Examples

There are several studies that have examined the manufacture of biopellets from
various types of biomass, whether with the addition of adhesive or not. An example
is the research by (Damayanti et al. 2017), regarding the effect of sieve size and
the addition of tapioca flour adhesive on the characteristics of biopellets from cocoa
shells. The research design used was RAK with a factor of sieve size variation and
Biomass Valorization for Bioenergy Production 83

Table 4 Quality standard for

No Parameter Unit Requirement
biopellets in Indonesia based
on SNI 8675:2018 1 Density g/cm3 Min. 0.8
2 Moisture % Max. 12
3 Ash % Max. 5
4 Volatile matters % Max. 80
5 Bound carbon content % Min. 14
6 Calorific value MJ/kg Min. 16.5
Source National Standardization Agency (2018)

the percentage of adhesive addition of tapioca starch. Sieve sizes used were 20, 40,
60, and 80 mesh. The percentage of tapioca flour adhesive addition used was 0, 10,
and 20%. From the research, it was found that the optimal results were found in the
treatment of a 20 mesh sieve size and the addition of 20% adhesive. The resulting
quality for the parameters of moisture content and density complies with SNI 8675:
2018, while the parameters of ash content and calorific value do not comply with
SNI 8675: 2018 concerning biomass pellets for energy.
Qadry et al. (2018) study about the characteristics of biopellets from a mixture of
palm shells and sawdust. The method in this study is experimental. The composition
of the mixed ingredients is 100% palm shells, 100% sawdust, 30% palm shells and
70% sawdust, 70% palm shells and 30% sawdust, 50% palm shells, and 50% sawdust.
The sieve size used is 80 mesh. From the research, it was found that the best quality
biopellets were found in the combined treatment of a mixture of raw materials 70%
palm shell and 30% sawdust. Testing the characteristics of the resulting biopellets
complied with SNI 8675:2018 for the parameters of density, moisture content, ash
content, volatile matter content, and bound carbon content. Meanwhile, the calorific
value parameter does not meet SNI 8675:2018 in several treatments.
Istiani et al. (2021) studied biopellet production from candlenut shell with a
different mixture ratio of sago stem and sawdust. The treatment used was the compo-
sition of the ingredients including 65% candlenut shell, 5% sago stem, 5% sawdust,
25% tapioca adhesive; 55% candlenut shell, 10% sago stem, 10% sawdust, 25%
tapioca adhesive; 45% candlenut shell, 15% sago stem, 15% sawdust, 25% tapioca
adhesive; 35% candlenut shell, 20% sago stem, 20% sawdust, 25% tapioca adhesive.
From the research results, it was found that the average density of biopellets was
0.29 g/cm3 , the average moisture content was 10.31%, the average calorific value
was 4181 cal/g, the average ash content was 10.25%, and the average -the average
volatile matter content is 71.31%. The best treatment was obtained from a mixture
of 55% hazelnut shells, 10% sago bark, 10% sawdust, and 25% tapioca adhesive.
The parameters of density and ash content obtained did not meet SNI 8675:2018 for
several treatments.
Ulfa et al. (2021) studied the quality of biopellets from rice husk waste. This
study used a factorial RAL model with particle size factors and adhesive variations.
84 S. Suhartini et al.

Variations in particle size were 40, 60, and 80 mesh, with different adhesive concen-
trations at 25% and 30% tapioca flour. The results reported that the powder size and
the amount of added adhesive and the interaction between the two did not signifi-
cantly affect the density, moisture content, ash content, volatile matter content, and
bound carbon content. However, the powder size, the adhesive concentration, and
their interaction had a significant effect on the calorific value. The resulting density
values ranged from 0.7 to 0.95 g/cm3 , with an average value of ash content (16.5–
19.9%), moisture content (13.1–14.5%), volatile matter (57.3–63.6%), bound carbon
content (4.7–9.8%), and calorific value (2781–3378 cal/g). The calorific value from
the resulting biopellets still did not meet the parameter of SNI 8675:2018. Hence,
in-depth studies to improve the quality are required.
A study by Raudhatul Jannah et al. (2022) on biopellets from rattan shavings
and mixed sawdust biomass using sago adhesive. The research method uses RAL
with factors including powder size and powder composition. The powder sizes used
were 20, 40, and 60 mesh. The powder composition (on a wet weight basis) used
was rattan (100), sawdust (100), rattan:sawdust (70:30), rattan:sawdust (50:50), and
rattan:sawdust (30:70). The best biopellet results were obtained from the treatment
with a composition of 100% sawdust and 40 mesh size. The quality of the biopellets
produced for the parameters of density, volatile matter content, moisture content, and
calorific value complies with SNI 8021:2014, while the parameters of ash content
and bound carbon content do not comply with SNI 8021:2014.

2.4 Fermentation

2.4.1 Definition and Mechanisms

Fermentation is one of the main steps critical in the conversion of lignocellulosic or

sugars into bioethanol. There are several research methods and conversion design
configurations that are economical and environmentally friendly. The fermentation
steps of second-generation bioethanol can be carried out in four ways, namely,
separate hydrolysis and fermentation (Separate Hydrolysis and Fermentation or
SHF), saccharification and simultaneous fermentation (Simultaneous Saccharifica-
tion and Fermentation or SSF), saccharification and simultaneous co-fermentation
(Simultaneous Saccharification and Co-Fermentation or SSCF) and consolidated
bioprocessing (CBP) (Cardona and Sánchez 2007; Aditiya et al. 2016).
The Separate Hydrolysis and Fermentation (SHF) method is a method for making
bioethanol in which the cellulose hydrolysis and fermentation stages take place
in different reactors (Saini et al. 2015). Raw materials containing cellulose are
hydrolyzed or saccharified and then followed by pentose fermentation (C5). Ethanol
is then distilled and the remaining hydrolysate is flowed into the second reactor to
get the hexose component (C6) fermented. Bioethanol is also distilled after hexose
fermentation (Balat et al. 2008). This method has the advantage of optimizing the
Biomass Valorization for Bioenergy Production 85

operating conditions at each stage. The Simultaneous Saccharification and Fermen-

tation (SSF) method is a method for making bioethanol in which the cellulose hydrol-
ysis stage and the pentose fermentation (C5) stage are carried out simultaneously in
one reactor. Next, hexose (C6) fermentation will be carried out in another reactor
(Cardona and Sánchez 2007). In addition to the lower operating costs of this method
compared to the SHF method, the possibility of inhibition of enzyme-related prod-
ucts can also be avoided so that the bioethanol produced is higher (Saini et al. 2015).
In this method, the fermentation of pentoses (C5) and hexoses (C6) is separated
because microorganisms that utilize both sugars (pentoses (C5) and hexoses (C6))
are slower than microorganisms that only assimilate hexoses (C6). This method also
prevents the reduction of monomers formed after the hydrolysis process, so SSF is
claimed to produce higher bioethanol yields (Cardona and Sánchez 2007).
In the Simultaneous Sacharification and Co-Fermentation (SSCF) method,
different microorganisms are mixed for pentose (C5) and hexose (C6) fermenta-
tion in one reactor. This method allows mixed culture microbes to carry out saccha-
rification continuously without separation which is continued by the fermentation
process into bioethanol (Cardona and Sánchez 2007). However, the ability to ferment
pentoses (C6) together with hexoses (C5) is not widespread among microorganisms
and is one of the biggest obstacles in the industrial production of ethanol using this
method (Talebnia et al. 2010). The Consolidated Bioprocessing (CBP) method is the
conversion of lignocellulose using one or more microorganisms into bioethanol in
one unit operation without additional enzymes (Branco et al. 2018). The microor-
ganisms used can produce saccharolytic enzymes (cellulases and hemicellulases)
themselves, hydrolyze substrates, and are capable of fermenting both pentoses (C5)
and hexoses (C6) so that they are considered the most cost-effective method (Aditiya
et al. 2016). Despite its advantages that are more efficient in conversion and require
less energy, this method has disadvantages, namely, longer conversion times and
lower productivity (Miskat et al. 2020).
Bioethanol is a colorless liquid made through a fermentation process from carbo-
hydrates which involves biological processes. As much as 95% of ethanol in the
world is made with vegetable-based ingredients and the rest is made synthetically
(Lane and Morrissey 2010). In industry, bioethanol is used as a raw material for
industrial alcohol derivatives, mixtures of alcohol, basic ingredients for the pharma-
ceutical industry, and mixtures of vehicle fuels (Ishola et al. 2014). In developed
countries, bioethanol is widely used as an ingredient in gasoline mixtures to increase
the octane rating of fuel. Bioethanol is mixed with gasoline at volume fractions of
5, 10, and 85% (fuel name E5–E85). E85 fuel is used for vehicles with flexible fuels
(flexible fuel vehicles or FFV), while the E5 and E10 fractions can only be used for
vehicles without engine modifications (Bušić et al. 2018) According to data from
FAO Agricultural Outlook 2015, the two highest bioethanol producers are Brazil and
the United States. In Brazil, sales of FFV vehicles are sold consistently every year,
indicating that the demand for bioethanol in Brazil is always there.
The production of bioethanol from lignocellulosic biomass is a development
of second-generation biofuels. The use of lignocellulosic biomass has advantages
and disadvantages. The advantage is that the substrate is not competitive with food
86 S. Suhartini et al.

ingredients while the disadvantage is that it requires high efficiency for breaking
down lignin as a wrapper for cellulose and hemicellulose. Cellulose is the compo-
nent most responsible for the production of bioethanol from lignocellulosic biomass
(Prasad et al. 2019). In general, there are three main stages in bioethanol production,
including pretreatment, hydrolysis, and fermentation. However, the production of
bioethanol from biomass, one of which is OPEFB, requires longer stages, namely,
pretreatment, hydrolysis, filtration, fermentation, and purification. The stages of the
OPEFB conversion process into bioethanol can be seen in Fig. 6. Pretreatment is
used to produce monomer from OPEFB which then has the potential to be used as
raw material for bioethanol fermentation (Suhartini et al. 2022b). The choice of the
pretreatment method can greatly affect the economy because increasing conversion
efficiency tends to add significant overall costs (Rabemanolontsoa and Saka 2016).
Less significant lignin removal can reduce the rate of hydrolysis and decrease the
efficiency of the conversion process, therefore removal of lignin prior to hydrolysis is
essential to ensure higher production of C5 and C6 sugars (Wan Azelee et al. 2014).

2.4.2 Pretreatment for Bioethanol Production

Lignin as an aromatic phenolic compound can be attacked by laccase enzymes so

that its molecular structure is easily degraded. Laccase enzymes can attack phenolic
lignin to form phenoxy radicals but cannot oxidize non-phenolic lignin directly
because of its low redox potential. To this end, the laccase enzyme can oxidize
low molecular weight mediators which act as electron carriers and diffuse into the
insoluble lignin structure to oxidize them. Enzymatic pretreatment of biomass gener-
ally uses specific enzymes isolated from certain microbes. This pretreatment tends
to be used to degrade lignin which can be carried out by complex lignin enzymes,
consisting of lignin-peroxidase (Li-P), manganese-peroxidase (Mn-P), and laccase
(Lac). These enzymes are capable of converting and breaking down lignin compounds
into their constituent components, namely, conipheryl alcohol, p-cumaryl alcohol,
and synapcyl alcohol. After lignin is degraded into its constituents, cellulose, and
hemicellulose become more accessible (Ravindran and Jaiswal 2016).
Hendriks and Zeeman (2009) stated that pretreatment of lignocellulosic biomass
can also be carried out using combination, thermal, steam, and chemical methods
which include alkaline, acidic, and organosolv liquids. Physical pretreatment, i.e.,
cutting biomass into smaller sizes is widely applied to increase the surface area
and reduce the degree of polymerization (Taherzadeh and Karimi 2008). On the
other hand, chemical pretreatment, namely, the use of sulfuric acid (H2 SO4 ) and
sodium hydroxide (NaOH), showed excellent results for lignin degradation, reduced
crystallinity, and degree of polymerization so as to increase the digestibility of cellu-
lose (Tsabitah et al. 2014). In its application, H2 SO4 and NaOH are widely applied
in pretreatment of biomass to produce biogas and bioethanol although there are
drawbacks, namely, the emergence of chemical waste that requires further handling.
Biological pretreatments, such as ensiling using EM4 and ensiling using molasses
have also been widely studied as alternative options for producing biogas and
Biomass Valorization for Bioenergy Production 87

Fig. 6 Stages of the conversion process of OPEFB into bioethanol (Suhartini et al. 2022b)

bioethanol because of their economical and environmentally friendly advantages

(Lunsin et al. 2018). The relatively slow rate of biological pretreatment is the main
weakness of biological pretreatment. As for the use of the laccase enzyme, as a branch
of biological pretreatment, it is proven to be able to degrade lignin in lignocellulosic
biomass more effectively (Schroyen et al. 2015). The effectiveness and efficiency of
pretreatment in degrading lignin content is highly dependent on the composition of
the biomass and operating conditions.
88 S. Suhartini et al.

2.4.3 Examples

Releasing the sugar content from its biomass from the complex structure via pretreat-
ment should be facilitated with careful consideration into the economic viability and
sustainability regarding the chemical or materials used. Sugarcane bagasse (SB) has
been investigated as the feedstock for bioethanol production. Couple pretreatment
has been applied with chemical and biological to assure the delignification could
be higher. Imidazole, a green solvent, was used to take account for disruption in
the complex of cell wall structure of SB. Total bioethanol production of 30%, with
ethanol volume of 110.3 L/ton sugarcane has been reached and shows a great corre-
lation between the methods to manage its lignin content (Valladares-Diestra et al.
2022). Following the physicochemical pretreatment, other lignocellulosic biomass,
Miscanthus as part of energy crops is being exposed to some methods for having
much lower lignin content. After pretreated with a novel chemical surfactant, mild
chemical (H2 SO4 or NaOH) in steam explosion was used as the pretreatment for
mischantus and these combined methods have demonstrated an increment in ligno-
cellulose accessibility and will promote the enzymatic saccharification in its biomass
(Sun et al. 2020). Similarly, a study by Gao et al. (2021) coupling the steam explo-
sion with green-liquor pretreatment and leading to the highest bioethanol yield of
20.3% (%dry biomass) compared with other reported bamboo processes. Conclu-
sively, pretreatment is becoming one of the great strategies to enhance bioethanol
production by giving more accessibility to the biomass structure and increasing the
hydrolysis activity.

2.5 Thermochemical

2.5.1 Direct Combustion

Combustion is a chemical reaction between flammable matter and oxygen, resulting

in the release of heat (Glassman 1977). Direct combustion is one of the oldest methods
that is quite simple and is commonly used in thermochemical energy production.
Direct combustion is an energy conversion carried out by burning sufficient agro-
material in the air to produce heat, steam, or electricity (Mandø 2013). The hydrogen
and carbon contained in the fuel will react with oxygen and release energy. During
combustion, there are substances that vaporize together with a portion of the carbon
in the form of flammable gaseous hydrocarbons and the release of carbon monoxide
with thermal degradation. Carbon monoxide is formed due to the reduction reaction
of CO2 with C, with the following chemical formula:
Biomass Valorization for Bioenergy Production 89

In general, combustion is carried out in a furnace, steam turbine, or boiler (Ye et al.
2022) with a temperature range of 800–1000 °C. Three stages in the direct combustion
of agro-materials are evaporation of the water, then distillation and combustion of
volatiles occur after which carbon is bound and oxygen reacts at high temperatures
(Lackner 2013). Energy is produced in the last two stages; this process is suitable
for the conversion of all types of biomass that have a low moisture content (<50%).
Several factors affect the direct combustion process, such as the volatile content,
water, ash, and tar of the material (Lackner 2013). Direct combustion is considered
carbon neutral, although it produces pollutants in the form of nitrogen oxides, sulfur
oxides, carbon dioxide, and dioxins.
The heat generated from the direct combustion process is difficult to store, so it
must be used immediately. The production cost of the direct combustion process is
slightly higher than the pyrolysis and gasification processes. This is because the
preparation of raw materials for direct combustion requires drying, cutting, and
crushing before being fed into the boiler.

2.5.2 Torrefaction

Torrefaction is basically an improvement process from the direct combustion process

on a large scale. Torrefaction is the heat degradation of biomass in inert or nitrogen
(Wang et al. 2023). The temperature used for the torrefaction process ranges from
200 to 300 °C without oxygen within a few hours (depending on the condition of the
biomass) in non-oxidized conditions with the aim of improving the physicochemical
properties of biomass as a solid fuel. Torrefaction is a mild pyrolysis of the fibrous
structure of crushed biomass (Han et al. 2022). Its heating value and hydrophobicity
are increased to increase the stability of the biomass during storage. The main product
produced in this process is charcoal.
Torrefaction can improve the characteristics of the resulting fuel by reducing the
water content, increasing the heating value, reducing the oxygen/carbon ratio, and
increasing the energy density. There are two kinds of methods used in torrefaction,
the wet process and the dry process (Akbari et al. 2021). In wet torrefaction, agro-
materials are treated with compressed hot water and produce solid fuel products,
aqueous compounds, and gases. However, wet torrefaction has the disadvantage
that it is still necessary to separate the excess water contained in the biomass. Dry
torrefaction requires intensive drying of agro-materials at higher temperatures. The
temperature used for dry torrefaction ranges from 230 to 300 °C in the absence of
oxygen.
90 S. Suhartini et al.

2.5.3 Pulverization and Drying

In the pulverization and drying process, the biomass is reduced in size and dried with
the aim of increasing the quality of the raw material so that it is easy to burn (Sarnavi
et al. 2023). Besides that, reducing the size can make it easier for the biomass to be
put into the reactor/furnace, and drying can help the next process to be more efficient.
The result of this process is solid fuel.
Based on the crushing principle, the biomass pulverizer is classified into hammer,
blade, and combined types (Wei et al. 2014). According to the pulverization method
and approach, the biomass pulverizer is classified into chopping, rolling cutting,
and combined pulverizing types. According to the purpose and particle size of the
crushed material, it is classified into coarse, twisted, and fine types.
Drying is a process of removing the water content contained in a material using
evaporation to obtain a dry solid product (El-Mesery and El-khawaga 2022). There is
a process of transfer of heat and water vapor from the surface of the material to the air
without changing the shape of the material. There are two types of drying, namely,
natural and artificial drying. Natural drying can be done by placing the material in
the free air, and under the heat of the sun, the air is allowed to remove the moisture
contained in the biomass, while the sun’s heat can help evaporate water from the
material. Artificial drying is done by utilizing the heat generated from combustion.
This drying can be done using a dryer, such as using a tunnel dryer or oven. The
advantage of artificial drying over natural drying is that the temperature and drying
rate can be controlled according to needs and will not be affected by climate change.
However, artificial drying also has the disadvantage of requiring a higher cost when
compared to natural drying.

2.5.4 Liquefaction

Liquefaction is included in the thermochemical agro-material conversion technology

(Kavitha et al. 2023). In the process, agro-materials are converted to liquid fuel prod-
ucts through a complex sequence of physical and chemical changes. Agro-materials
are decomposed into small molecules. These small molecules are unstable and reac-
tive and can be polymerized into oily compounds. Liquefaction can be done directly
or indirectly. Direct liquefaction involves fast pyrolysis to produce tar and pyrolysis
oil (Folkedahl et al. 2011). Indirect liquefaction involves the use of a catalyst to
convert non-condensable gaseous products from pyrolysis or gasification into liquid
products.
In direct liquefaction (Folkedahl et al. 2011), the biomass is directly converted
into products without drying. Under these conditions, water remains in a liquid state
and has a low viscosity and high capacity to dissolve inorganic compounds. Indirect
liquefaction involves low temperature (300–350 °C) and high pressure (5–20 MPa)
with a residence time of about 30 min and is often carried out using a catalyst in the
presence of hydrogen, so this process can also be called catalytic liquefaction. The
composition of the resulting pyrolysis oil and char is influenced by the liquefaction
Biomass Valorization for Bioenergy Production 91

method used. The difference between liquefaction and pyrolysis is that pyrolysis
usually occurs at higher temperatures and lower pressures, and it is necessary to dry
the raw material first. This liquefaction technology is rarely used because it requires a
more expensive reactor. From the liquefaction process, liquid fuels, commonly known
as biofuels, such as ethanol and methanol, will be obtained. The most frequently used
biofuel is ethanol which can be obtained from sugarcane, corn, and other grains.

2.5.5 Pyrolysis

Pyrolysis is a process in which organic materials containing carbon are decomposed

thermochemically (Liu et al. 2022). The pyrolysis process involves little or no oxygen
in a closed vessel. Oxygen is only required in small quantities for combustion to occur
in order to produce enough heat to start the endothermic pyrolysis process. Then
slowly, the oxygen in the vessel will run out because there is no oxygen supply from
outside during the process, resulting in incomplete combustion in the vessel, and
charcoal is produced. The mechanism of the pyrolysis process of biomass generally
consists of hydrolysis reactions, primary reactions, and secondary reactions. Biomass
will decompose thermally without oxygen and involves many reactions of biomass
biomolecules.
Pyrolysis of biomass can be influenced by four factors, including water content,
decomposition of cellulose, lignin, and decomposition of hemicellulose (Vuppal-
adadiyam et al. 2022). Pyrolysis can be carried out at high temperatures up to
800 °C and low pressures up to 700 kPa. In extreme pyrolysis, carbonization will
occur, which will only produce charcoal. If the pyrolysis reactor is equipped with a
condenser (Fig. 7), there will be a change in the form of the resulting gas to liquid
commonly known as pyrolysis oil. The products produced during the pyrolysis are
charcoal, pyrolysis oil, tar, and non-condensable and flammable gases, such as CO,
CO2 , CH4 , and H2 (Samer 2017).
A pyrolysis reactor is an equipment to decompose organic compounds by the
heating process without direct contact with outside air at a temperature of 300–
600 °C. This reactor is sealed to prevent excessive heat from escaping. The pyrolysis
reactor is made of a cylindrical tank made of stainless steel, with rock walls installed
between the reactor covers to minimize heat escape. The heat source used is a gas
stove with LPG fuel, and there is a thermocouple connected to a pipe in the reactor
so that the temperature can be measured and adjusted as desired. The large volume
of the reactor will affect the amount of input that will be processed into pyrolysis oil.
The working principle of the pyrolysis reactor is to carry out the process of burning
biomass without air (O2 ). The decomposition of organic matter will occur as the
temperature used increases. There are several types of reactors used in the pyrolysis
process, namely, batch/semi-batch, fluidized bed reactors, fixed bed reactors, screw
kilns, and spouted beds. Batch/semi-batch-type reactors are the type of reactor that
is widely used by researchers because the design is simpler and easier to operate.
There are four stages of the pyrolysis mechanism based on the lignocellulose
contained in the material, including removal of water content, decomposition of
92 S. Suhartini et al.

Fig. 7 A small pyrolysis

reactor

hemicellulose, decomposition of cellulose, and decomposition of lignin. The dewa-

tering step occurs at a temperature of less than 200 °C. If the water content in the
material is too high, there will be a risk of producing large amounts of ash. The hemi-
cellulose decomposition stage occurs at a temperature of 200–280 °C at this stage to
produce syngas and minor pyrolysis oil. The cellulose decomposition stage occurs
at temperatures above 240–350 °C. At this stage, syngas, pyrolysis oil, and minor
biochar products are produced. Furthermore, the last stage is the decomposition of
lignin which occurs at a temperature of 280–500 °C (Samer 2017). At this stage, the
products of pyrolysis oil and biochar are produced.
The mechanism of the pyrolysis process can be seen in Fig. 8. Cellulose and
hemicellulose mainly form volatile products on heating due to the thermal splitting
of the sugar units. Lignin mainly forms char because it is not easily broken down
into smaller molecules.
Some factors that can affect pyrolysis products include temperature, type of agro-
material, heating rate, the proportion of oxygen, and equipment design. In general, the
resulting charcoal is up to 50% of the dry agro material, the gas product ranges from
5 to 20% depending on the temperature used, while the tar product can be up to 25%
by weight percent of the dry agro material. Charcoal has low sulfur and nitrogen
content, making it much easier to store and transport than the agro-raw materials
used. The resulting tar still contains water and can be separated by distillation or
extraction. Pyrolysis oil obtained from the condensation of the resulting gas needs
to be acid-neutralized so that it can be used as fuel. The quality and quantity of the
product produced are also influenced by the temperature used during the pyrolysis
Biomass Valorization for Bioenergy Production 93

Fig. 7 Biomass degradation temperature (Samer 2017)

process. There are three types of pyrolysis based on temperature levels (Weir et al.
2022), as seen in Table 5.
Slow pyrolysis is a process in which biomass is heated at a slow temperature
rate in an inert atmosphere to a maximum temperature (de Almeida et al. 2022).
Slow pyrolysis takes place at low heating rates (0.1–0.8 °C/s), longer times (5–30
min or even 25–35 h), and temperatures around 300–550 °C. This pyrolysis process
aims to obtain maximum bio-charcoal, syngas, and pyrolysis oil simultaneously.

Table 5 Characteristics of biomass thermochemical conversion technologies

Pyrolysis Liquefaction Gasification
Slow Fast
Feedstock
Feed Size Any Small Very small Mixed, large
Moisture content Low Very low Very low 50% max
Parameters
Temperature °C 400–600 450–900 250–400 1000–1500
Pressure, bar 0.01–1 1 100–200 Up to 20
Maximum throughput 5 0.05 0.1 40
achieved to date, dry/
year
Product (dry basis on dry feed)
Gas Yield, %wt Up to 40 Up to 70 20 100–250
HHV, MJ/NM3 5–10 10–20 2–6 May 15
Liquid Yield, %wt Up to 30 Up to 70 Up to 50 Up to 3
HHV, MJ/kg 23 23 30 23
Solid Yield, %wt 30 Up to 15 Up to 25 Nil (ash)
HHV, MJ/kg 30 30 30 –
94 S. Suhartini et al.

This process can also eliminate the content of smoke production and the forma-
tion of solid products. This method can produce a solid uniform product with low
water content and higher energy content than the initial biomass. Fast pyrolysis takes
place at higher heating rates (10–1000 °C), and very short residence times (0.5–2 s),
with temperatures around 850–1250 °C (Hu et al. 2022). This pyrolysis aims to get
more pyrolysis oil products than bio-charcoal and syngas. In general, fast pyrolysis
produces 60–75% liquid, 15–25% solid, and 10–20% gas. Flash pyrolysis is almost
the same as fast pyrolysis, with some modifications and improvements (Cornelissen
et al. 2009). This pyrolysis takes place at a very high heating rate of around 1000 °C,
a residence time of 0.1–1 s, with a temperature of 800–1000 °C. This pyrolysis aims
to get the maximum syngas product.
Pyrolysis technology has several advantages, including, namely, the process is
simple, so it does not require high expertise to operate. This technology can eliminate
carcinogenic compounds such as PAHs (Polycyclic Aromatic Hydrocarbons). This
technology is quite efficient. Namely, it can produce three products with only one
process required. The resulting pyrolysis oil product is able to overcome the problem
of traditional smoking of fish/meat. The main components of pyrolysis oil are organic
compounds and water (15–30%). Therefore, pyrolysis oil has the opportunity to
be converted into fuel and chemical sources. The biggest component of bio-oil is
hydrocarbons which are traditionally produced from petroleum. Given the dwindling
reserves of petroleum, pyrolysis oil has the potential to be developed as a substitute
fuel for petroleum. Pyrolysis oil can be used as fuel in furnaces or boilers. However,
the high oxygen content in pyrolysis oil components can cause undesirable fuel
characteristics, such as increased viscosity, corrosion, low heating value, and unstable
during storage (can form a precipitate). Several organic compounds of high economic
value found in pyrolysis oil are levoglucosan, toluene, xylene, limonene, and phenol.
The constituent components of pyrolysis oil are highly dependent on the composition
of the raw materials, the pyrolysis process, and the reaction conditions. Therefore,
research on raw materials and pyrolysis process variations is important to extract
chemical components with high economic value.

2.5.6 Gasification

Gasification is the partial (imperfect) combustion of solid fuels, which can produce
flammable gas and ash (Valizadeh et al. 2022). Gasification of agro-materials belongs
to the thermochemical conversion technology, in which solid agro-materials are
converted into gaseous fuels. Under controlled conditions, characterized by low
oxygen supply and high temperatures, most of the agro materials can be converted
into producer gas fuels consisting of carbon monoxide (CO), hydrogen (H2 ), carbon
dioxide, nitrogen, and methane (CH4 ). Producer gas is a mixture of flammable and
non-flammable gases. Basically, the gasification process begins with a pyrolysis
process at a temperature of around 150–900 °C, followed by an oxidation process
of the pyrolysis gas at a temperature of 900–1,400 °C, then a reduction process at a
temperature of 600–900 °C (Valizadeh et al. 2022).
Biomass Valorization for Bioenergy Production 95

The gasification process can produce relatively higher gas, which is around 85%,
compared to pyrolysis, which can only produce around 35% gas. Gas fuel from
biomass gasification can be used for cooking, both at the household level and in
small industries in undeveloped areas. In addition, the gas is used to drive turbines
and combustion engines, as fuel in steam boilers, and for lighting. The application
of biomass thermal gas is a more efficient alternative and produces lower pollution
than direct combustion, although it has the disadvantage of higher investment costs.
There are four different process stages during fuel gasification (Akhtar et al. 2018),
among them are:
a. Fuel drying
Agrofuel enters through the top of the gasifier and then moves down during the
process. Heat radiation from the combustion zone helps reduce fuel moisture entering
the drying zone. Temperatures in this zone range less than 120 °C.
b. Pyrolysis
Dry fuel moves downward and is exposed to heating at higher temperatures, namely,
above 200 °C. At this temperature, the fuel begins to lose its volatile content. In this
zone, no air is allowed to enter. When the temperature reaches 400 °C, an exothermic
reaction occurs, where the structure of the wood material or other organic solids
begins to break down.
c. Combustion/Oxidation
The combustion reaction is an exothermic reaction resulting in a theoretical oxidation
temperature of up to 1450 °C. The combustible substance of the fuel usually consists
of carbon, hydrogen, and oxygen. In complete combustion, carbon dioxide is obtained
from carbon in the fuel and water is obtained from hydrogen, usually in the form of
water vapour. This heating causes some of the charcoal to be oxidized, and the rest
undergoes a reduction process.
d. Reduction
Partial combustion products, such as water, carbon dioxide, and partially cracked
pyrolysis products, move through the hot charcoal bed, where subsequent reduction
reactions occur. The temperature in the reduction zone ranges from 800 to 1000 °C.
The lower the temperature in the reduction zone, the lower the heating value of the
gas.

2.5.7 Plasma Technology

Plasma is a gas that undergoes ionization in an electric discharge or a mixture of

electrons, radicals, and negative and positive ions. Plasma technology can be used to
decompose organic compounds from solid, liquid, or gas waste (Dimitrakellis et al.
2022). This technology is carried out by heating the waste using a device similar
to an oven/microwave. The heating principle used in plasma technology is that in
96 S. Suhartini et al.

the combustion chamber, two high-voltage electrodes (about 10,000 V) are given,
and then the waste is put into the furnace. The electrode is given an electric voltage
so that plasma will form, which will decompose the waste. Plasma technology has
the advantage that it does not require a large area, takes a short time, and does not
involve chemicals.
In wastewater treatment, plasma technology plays a role in various oxidizing
processes of the compounds (Dimitrakellis et al. 2022). A reaction occurs on ions
and electrons in the plasma in the liquid waste, and then UV light and shockwave are
formed. These ions and electrons contain very high energy causing the decomposition
of water (H2 O) and producing active species such as OH, O, H, and H2 O2 . Active
specifics are strong oxidants that can oxidize organic compounds and kill bacteria in
liquid waste.

3 Conclusion

Biomass can be derived from any organic material including agricultural crop
residues, agro-industrial waste, household waste, forestry waste, etc. Different types
of biomass has also unique characteristics, which may play a key factor in the selec-
tion of the best conversion routes. Lignocellulosic biomass, considered as third-
generation sources of bioenergy, is potential to be directly valorized into bioen-
ergy, either via physical/mechanical, biochemical, and thermochemical. However,
the results may not be optimum. Hence, pretreatment or other measures are often
required to improve the conversion process’ efficacy. Specific to anaerobic tech-
nology, it is widely adopted in the global world from small to commercial scale.
The resulted biogas can be converted into electricity (and heat) or biomethane and
the digestate into biofertilizer. Transesterification is the main process to transform
biomass into biodiesel, mostly suitable for biomass with high in lipid or oil contents.
While fermentation of lignocellulosic biomass to bioethanol remains a challenge,
therefore integration with pretreatment and an improved hydrolysis step are neces-
sary. Densification of biomass into biopellets can be one of the alternatives with
lower operating cost, however, the risk of carbon emission from burning the fuels
need to be considered. Thermochemical pathways offer greater and faster conversion
to bioenergy, yet a high cost for the capital investment and operation may hinder the
technology for commercial application in Indonesia.

Acknowledgements The authors would like to thank the Department of Agro-industrial Faculty
of Agricultural Technology, Universitas Brawijaya, for the support in writing this book. We would
like also to thank Pusat Kolaborasi Riset Biomasa dan Biorefinery for the opportunity to join in
this publication.
Biomass Valorization for Bioenergy Production 97

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Biomass Utilization and Biorefinery
By-Product from Palm Oil and Marine
Resources for Animal Feed and Feed
Additive

Ahmad Sofyan, Hendra Herdian, and Agung Irawan

Abstract Indonesia is the largest producer of palm oil. Biorefinery by-products from
palm oil can be classified into lignocellulosic and fiber-rich biomass. Palm kernel
meal (PKM), a by-product from palm kernel oil extraction contains crude protein
(13–16%) and there have been ongoing efforts to improve its utilization as animal
feed. The restriction of PKM used in animal feed is linked with the imbalance of
amino acids, high fiber content, shell, and other physical characteristics. On the other
hand, Indonesia is among the leading countries in marine industry, by-products of
fish, shrimp, crustaceans, and other marine processing industries are of high potency
for animal feedstuff. Chitin, the dominant by-product of shrimp production, has ahead
popularity in the last decade due to its large spectrum functions, especially as antimi-
crobial agent, non-toxic, biodegradable, and biocompatible. This chapter discusses
the recent advances in PKM and marine industry by-products availability status and
utilization, and novel technologies to improve their quality for animal feed and feed
additive. A practical and conceptual development of the bioproducts for implementa-
tion, especially in the context of Indonesia and other countries with similar character-
istics of nature. Biological processes including solid-state fermentation, mechanical
processing, and valorization techniques can be integrated to process the biomass
from palm oil industry. Chemical treatments including green chemistry techniques
could improve chitosan functionality. Implementation of biorefinery techniques of
biomass and by-products of palm oil and marine resources promise supporting raw
material stock and sustainability for the feed and feed additive of animals.

Keywords Agroindustrial by-product · Biorefinery · Feed technology ·

Livestock · Palm oil

A. Sofyan (B) · H. Herdian

Research Center for Animal Husbandry, National Research and Innovation Agency, Cibinong,
Bogor, West Java, Indonesia
e-mail: [email protected]
A. Irawan
Vocational School of Animal Production, Universitas Sebelas Maret (UNS), Surakarta, Central
Java, Indonesia

© The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2024 105
M. A. R. Lubis et al. (eds.), Biomass Conversion and Sustainable Biorefinery, Green
Energy and Technology, https://doi.org/10.1007/978-981-99-7769-7_5
106 A. Sofyan et al.

1 Introduction

Availability of feed ingredients in terms of quality, quantity, and continuity (sustain-

ability) is the key in supporting livestock productivity. Therefore, efforts to continu-
ously increase the availability of feed ingredients are paramount to support national
meat self-sufficiency. This is in line with the national program “Efforts for the Special
Acceleration of Increasing the Population of Pregnant Cattle and Buffalo (Upsus
Siwab)” in increasing the national livestock population. However, the problem arises
when conversion of land-producing forages or feedstock is continuously occurring.
On the other hand, increasing effluent residues is identified to be a major problem
in the biomass-generating industry (biorefinery). Therefore, integrating biorefinery
industries into the livestock industry coupled with implementing novel technologies
can help to aid the problem for both.
There are many underutilized biomass or waste generated from agricultural
processing and biorefinery industry that are plenty and economical. Based on
the biomass characteristics, they can be classified into lignocellulosic and non-
lignocellulosic biomass. It is well known that Indonesia is the leading producer
of palm oil (Safi et al. 2022). Many by-products of palm oil biorefinery are left-
over including oil palm trunks, empty fruit bunches, oil palm fronds, palm kernel
shells, and palm kernel meal, that can be classified into lignocellulosic and fiber-rich
biomass (Kahar et al. 2022). Those biomasses are produced up to 70 tons per hectare
of palm oil. Among those biomass, palm kernel meal (PKM), a by-product generates
from the extraction of palm kernel oil, receives growing interest due to its potential
quality and quantity, particularly in Indonesia and Malaysia that contributes to over
80% of the world’s PKM supply (Safi et al. 2022). PKM contains moderate crude
protein (13–16%) and there have been ongoing efforts to improve its utilization as
animal feed. The problem associated with palm kernel meal is related to the imbal-
ance of amino acids, high fiber content, and physical characteristics (Sundu et al.
2008). Lack of access to technology is a key problem to optimally utilize the PKM.
At present, most of them are exported to European countries and used as an econom-
ical feed ingredient for livestock (Azizi et al. 2021). For other biomasses of palm oil
biorefinery as mentioned above, they are generally burned and used as fertilizer in
the plantation which can increase pollution to the air (Kahar et al. 2022).
Valorization of PKM is a critical point to enhance the quality of valuable animal
feed. Several strategies that can be used to improve the nutritive value of PKM
include mechanical, biological, and chemical treatments. Using multiple alternative
processing technologies, PKM can be processed as a whole biomass or subjected to
protein extraction to obtain various functional protein concentrate. In addition, inte-
grating livestock into the palm oil crop system has also been introduced in Indonesia
as this approach is potentially more economically and environmentally beneficial
(Agus and Widi 2018). However, much more effort is needed to valorize the low-
quality biomass using novel technologies that can be implemented in the plantation
area, together with the livestock.
Biomass Utilization and Biorefinery By-Product from Palm Oil … 107

On the other hand, reflecting that Indonesia is among the leading countries for
its aquaculture industry, by-products of the fish processing industry are of high
potency for feed of livestock. In particular, Indonesia is the leading country for
shrimp production and is becoming the top five exporter countries for fresh shrimp
in the world due to the large production volume supported by nature and available
resources (Aneesh et al. 2020). Chitin is a major by-product of shrimp production and
a primary source of chitosan, biopolymer obtained from diacylation of chitin (Shan
et al. 2012). Chitosan is gaining popularity in the last decade due to its large spec-
trum of functions, especially as antimicrobial agent. It is also considered non-toxic,
biodegradable, and biocompatible (Anggraeni et al. 2022). In addition, chitosan has
been widely used in various industries including pharmaceuticals, foods, textiles,
and agriculture. Recently, the use of chitosan as an additive for livestock has been
extensively investigated for its potential as rumen modulator and methane suppressor
(Harahap et al. 2022).
Optimization of extraction process and efforts for improving the functionality of
chitosan have been introduced in the last decade, in addition to undergoing efforts to
optimize the bio-functionality. To produce chitosan, chitin is commonly subjected
to different steps including demineralization, deproteinization, and deacetylation.
Recent advances in processing chitin into chitosan using novel technology can
provide insight into the progress of technological development and future priorities
of product development, especially in the context of feed additives for livestock.
In this chapter, we discuss the recent advances of PKM and fish industry by-
products availability status and utilization, their potency, and novel technologies
to improve the quality of by-products as an animal feed. In addition, we provide
practical and conceptual development of the bioproducts for implementation, espe-
cially in the context of Indonesia and other countries with similar characteristics of
nature. Collaborative framework among stakeholders should be established to take
full advantage of such bioresources as a feedstock and to connect the farmers with
industry.

2 Biomass and By-Product Characteristic and Potency

As a country with comparison of the ocean and land area of 6.3 versus 1.9 M km2
makes Indonesia known as the largest archipelagic country in the world (Indonesian
Geospatial Information Agency 2013). So it is not surprising that Indonesia has
great potential for marine resources. Crustacean as one of the marine products has an
important meaning for Indonesia’s economic growth. No less than US $ 1.55 billion
in year 2021 the export values of crustacean products from Indonesia that make
Indonesia the sixth largest country as crustacean producer in the world, while the
total sales of crustacean products in the world reach US $ 33.2 billion (OEC 2023).
As much as 40–50% of these crustacean products can end up as waste (Muthu et al.
2021), so, the potential to disturb the environment is massive if there is no further
utilization process. Likely, waste from crustaceans still has the potential to be reused
108 A. Sofyan et al.

Table 1 Chemical
Compound Concentration (dry weight)
composition of shrimp shell
(Rødde et al. 2008) Protein 33 and 40%
Chitin 17 and 20%
Mineral (CaCO3 ) 34 ± 2%
Lipid 0.3–0.5%
Astaxanthin 14–39 mg kg−1

either as food or feed (Senel and McClure 2004; Özogul et al. 2019). Crustacean
shell waste (lobster, shrimp, crab) generally contains protein parts, chitin minerals,
lipids, and a small portion of astaxanthin (Table 1). This part of waste has a number
of potentials that can lead to certain functions either as human food or animal feed
(Özogul et al. 2019; Suryawanshi et al. 2018).
Protein portions from crustacean skin waste can be obtained through the fermen-
tation of lactic acid bacteria to the cephalothorax and exoskeleton sections, resulting
in hydrolysates ranging in proteins from 8.43 ± 0.22 to 46.73 ± 1.29 (Bueno-Solano
et al. 2009), using protease (alcalase) enzyme following pH-stat method (Dey and
Dora 2014). Crustacean skin extraction using the alcalase enzyme (Mizani et al.
2005) is described in Fig. 1. Protein sources from hydrolysate crustacean skin waste
have the potential in addition to provide a source of nitrogen from crude protein also
has the potential to contain amino acids that are quite complete for animal feeds
Penaeus shrimp shells have a fairly complete amino acid balance and this is almost
equivalent to soybean meal (Yan and Chen 2015). The use of hydrolysate protein in
addition to supplying protein and amino acids also improves other functions because
there is a compensatory bioactive peptide (Alvarez et al. 2015). Chitin is a polysac-
charide found in the exoskeleton of an insect, crustaceans, mushroom structures,
fungi, and yeast (Pighinelli et al. 2019), this condition causes chitin to be the most
common polysaccharide found after Cellulosa (Senel and McClure 2004).
Chitin is a polymer of d-glucosamine while its deacetylated form is known as
chitosan polymer N-acetyl-d-glucosamine monomers (Amiri 2022). Chitosan has
a higher binding activity with several molecules compared to chitin. As a natural
biopolymer, chitin is a material that has biocompatibility, biodegradability, and
non-toxicity, has antibacterial properties, and anti-coagulant, absorbent molecules
in nature, the form of chitin consists of αchitin, β chitin and ϒ chitin (Pighinelli et al.
2019). Conventionally to recover chitin and chitosan from the crustacean shell the
extraction process by the chemical compound is described in Fig. 2. In ruminants,
chitosan has extensive biological functions including antimicrobial, and protein-
protecting agents to improve silage quality with various mechanisms of action
(Fig. 3).
Although not made de novo by crustaceans, the pigment substance astaxanthin
is obtained by crustaceans from the supply of feed material that contains a lot of
carotenoids and astaxanthin, in crustacean astaxanthin obtained in free form or
binds to proteins in the form of carotenoproteins (Özogul et al. 2019). The astax-
anthin extraction method using acetone solvents yields the best results compared to
Biomass Utilization and Biorefinery By-Product from Palm Oil … 109

Shrimp Head

Grinding

Hard Part Remind in Grinder

Buffer (1:1, w/v)
Shrimp Head
With or without sulphite
Waste
Homogenizations

Alcalase and/or Triton x-100

Hydrolysis
T = 40˚ C, t = 1 h

PMSF ( 1mmol/L) Enzyme Inactivation,

Centrifugation
1000 x g, 45 min, 40˚ C

Protein Hydrolysate

Fig. 1 Scheme of extraction crustacean shell protein procedure (Mizani et al. 2005)

Crustacean By Products

Deproteination Demineralization

Protein Astaxanthin CaCO3 Chitin

Deacetylation Depolymerization Chito-oligosaccharides

Chitosan

Fig. 2 Biorefinery of crustacean by-product to valuable compound (Özogul et al. 2019)

other conventional polar solvents (Dalei and Debasish 2015). Giving astaxanthin to
Holstein Friesian dairy cows that are lactating can improve immune function and
livestock reproduction (Da Costa et al. 2021).
110 A. Sofyan et al.

Chitosan

Antimicrobial agent Protein Protecting Silage Quality Rumen Modifier Animal Productivity
Agent

Chelating metal ion Electrostatic Increase acetate Inhibition of Increase dry matter
interaction concentration biohydrogenation ingestion
Decrease protein
degradation
Suppression of mRNA Hydrophobic Reduce ethanol Lower Organic and Increase NDF digestibility
interaction concentration Dry Matter Increase nitrogen and
digestibility energy efficiency
Cell membrane Van der Waals Reduce mold Lower rumen Decrease meat fat
destabilization forces and spoiled protozoa Decrease milk yield
bacteria Increase FCM and Lactose
production in milk
Electrostatic contact in Chitosan Cross Reduce dry Lower methane Increase unsaturated fatty
outer membrane linking matter loss emission acid on free oil diet
Inhibition ruminal Increase dry Lower A/P ratio, Decrease glycogenolysis &
amino acid atter content Increase propionate glycolysis
deamination Increase fermented Increase biochemical
substrate condition of liver oil

Fig. 3 Applications and effect of chitosan as feed additive in ruminants (Anggraeni et al. 2022)

3 Method for Improving Quality

3.1 By-Product of Palm Oil Processing Industry

Major limitation of PKM as a feedstuff for both poultry and ruminants is related to
the imbalance of amino acids and high fiber content (Sundu et al. 2008). For ruminant
animals, such high fiber content is not an issue. Several studies have demonstrated
that inclusion of PKM or palm kernel cake in ruminant diets did not affect nutrient
utilization, ingestive behavior, growth performance, and meat quality (da Silva et al.
2020, 2021). However, pretreatment processing is needed when it is used as a feed
for poultry animals. The high content of cellulase is co-limiting the availability of
amino acids by blocking the access of enzymes to utilize them especially when
fed to poultry. Amino acid (AA) digestibility is considered as moderate in broiler
chickens (Suprayogi et al. 2022). Replacement of a soybean meal-corn-based diet
with 25% PKM resulted in low crude protein digestibility (41.6%) and moderate AA
digestibility (averaged 75%) (Reza Abdollahi et al. 2015). The average essential and
non-essential AA digestibility commonly falls into the value between 80 and 85%
(Kim et al. 2022). The low CP digestibility is particularly a problem that can impair
the growth of broiler chickens. To this end, methods to aid the low nutrient profile
of PKM are of prime importance. Multiple strategies can be used to improve the
nutritive value of PKM, such as fermentation, thermomechanical, thermochemical
treatments, and their combinations.
Biomass Utilization and Biorefinery By-Product from Palm Oil … 111

3.1.1 Enzymatic Treatment

Enzymatic hydrolysis is more practical when compared to protein isolation in the

context of its utilization as an animal feed. The main purpose of enzymatic treatment
is to break down resistant starch of the PKM mainly lignin, cellulose, and β-mannan.
PKM contains considerable amounts of β-mannan (up to 30%) (Navidshad et al.
2016) that can cause hyperplasia due to the high viscosity characteristics. A growing
number of studies have been performed for this purpose. For instance, treatment
with a mixture of Aspergillus niger and β-mannanase was reported to increase the
true metabolizable energy (TME) by 32.1% and crude protein by 9% and concomi-
tantly decrease the crude fiber content by 48.3% (Navidshad et al. 2016). Other
carbohydrase enzymes such as xylanase, α-galactosidase, and β-mannosidase were
also demonstrated to effectively enhance the molecular structure of protein of PKM
(Moreira and Filho 2008; Álvarez-Cervantes et al. 2013; Chen et al. 2013).
A comprehensive review is available for the enzymatic treatment effect of PKM
(Alshelmani et al. 2021). Overall, enzymatic treatment either using crude enzyme or
microbial fermentation resulted in significant improvement in the quality of PKM,
especially most of AA components. Compared to direct enzyme supplementation
to the feed, enzymatic treatment to the PKM is more effective to increase the
value of PKM which subsequently can improve productive performance of poultry.
When these above enzymes were supplemented to broiler feed, no improvement was
observed (Aftab and Bedford 2018; Chen et al. 2019). On the other hand, weight gain
of broiler chickens was improved when fed with PKM treated enzyme compared to
untreated PKM (Navidshad et al. 2016).

3.1.2 Solid-Sate Fermentation

Solid-state fermentation has gained more attention in the last few years due to its
ability to improve the value of biomass, including feed ingredients for poultry. The
main advantages of SSF are its effect to enhance protein quality, produce func-
tional peptides, decrease antinutritional properties, and thus increase functionality
(Suprayogi et al. 2021, 2022). SSF is known to enhance antioxidant activity of the
substrate. There are not many available reports regarding the application of SSF to
improve the value of PKM. There is a huge opportunity to implement this technology
for PKM. The process of SFF is generally consisted of several steps including (1)
preparation of microbial culture; (2) substrate incubation; and (3) harvesting. The
schematic process of the SSF is provided in Fig. 4.
To date, several reports related to the effect of SFF were available on the quality of
palm kernel cake (PKC) while the study on PKM is scarce. Alshelmani et al. (2017)
reported that SSF of PKC using cellulolytic bacteria Paenibacillus polymyxa ATCC
842 and P. curdlanolyticus DSMZ 10248 resulted in significant decrease of NDF
(−12.9%) and hemicellulose (−20.7%) and also increased several AA contents such
as isoleucine, histidine, phenylalanine, threonine, methionine, arginine, and glycine.
SFF of PKC using four fibrolitic bacteria Bacillus amyloliquefaciens DSMZ 1067,
112 A. Sofyan et al.

Fig. 4 Schematic representation of solid-state fermentation to enhance quality of PKM

Paenibacillus curdlanolyticus DSMZ 10248, P. polymyxa ATCC 842, and B. mega-

terium ATCC 9885 showed similar trends of nutritional profile enhancement (Alshel-
mani et al. 2014). In addition, PKC was also suggested as an economical substrate for
enzyme production through SFF process, as reported in Ong et al. (2004). Another
study reported that Aspergillus niger, Aspergillus oryzae, and Aspergillus awamori
were able to grow in PKC, and xylanase was successfully produced. The obtained
enzyme was able to improve the nutritive value of PKC as shown by the increase
of CP and NFC and the decrease in the non-starch polysaccharides (Mohamad Asri
et al. 2020). Studies in this area using PKM and also various by-products generated
from palm oil biorefinery are worth investigating.
Different from PKM which is generated from solvent extraction process in palm
oil processing, PKC is obtained from expeller press to extract the oil. Nevertheless,
the characteristics of PKM and PKC are relatively similar. They contain considerable
amounts of antinutritional factors (ANFs) such as β-mannan, cellulose, arabinoxylan,
and glucuronoxylan (Alshelmani et al. 2021), while the first was considered as a
prebiotic and is beneficial when added to the diet in low amount (Salami et al. 2022).
Results of SFF are different depending on the type of substrate, inoculants, water-
to-substrate ratio, temperature, and incubation time. To obtain the best conditions of
SFF for PKM, a response surface model is the best starting point.

3.1.3 Valorization of PKM into Concentrated Protein

Although the percentage is considerably low, the protein quality of PKM is highly
attractive. Various methods can be used for protein extractions from PKM including
fractionation or using alkaline solution to extract and separate soluble and non-
soluble protein contents. A series process of enzymatic treatment is effective to extract
and purify soluble protein from PKM. The hydrolysis can be conducted in a reactor
by utilizing 5–10% dry weight of PKM at 60 °C using a certain level of proteolytic
enzymes for up to 4 h of stirring. After enzyme inactivation, centrifugation is needed
to obtain the supernatant-containing protein. Next, the supernatant is purified using
Biomass Utilization and Biorefinery By-Product from Palm Oil … 113

ultrafiltration to eliminate non-protein components using a specific membrane size

with the aid of pressure and continuous stirring. Using this technique, soluble protein
fraction is obtained (Safi et al. 2022). Known for different hydrolysis capabilities,
selecting highly effective enzyme is an important decision. In their work, Safi et al.
(2022) reported that alcalase is the most superior enzyme producing the highest
purity and yield of protein from PKM, up to >50% and 70% of the supernatant,
respectively. They also reported that using this technique, protein with high solubility
was obtained. In addition, characterization of protein isolates and protein hydrolysate
of PKM processed using the Osborne-type method have demonstrated a significant
AA profile improvement than unprocessed PKM. In their study, protein fraction of
the PKM was obtained by extracting the PKM using cold distilled water at room
temperature with continuous stirring for 1 h.
After a series of extraction processes, supernatant and soluble protein from residue
were dialyzed where the hydrolysate (protein concentrate) was obtained. Protein
isolate, on the other hand, is obtained by further processing the protein concentrate.
The protein concentrate is obtained using a dispersion method in a NaOH solution
for 2 h stirring to obtain supernatant. The supernatant is then filtered, and the filtrate
is subjected to protein precipitation in pH 4.6 and the precipitate is centrifuged to
obtain protein concentrate. Protein hydrolysate can also be produced using microbial
hydrolysis, as described in Aluko and Monu (2003) and Chang et al. (2014). Protein
isolate from PKM can contain up to 75.6% protein with 15–50 kDa of polypeptide
mass (Chang et al. 2014). Such protein isolate can be used as a highly valuable protein
for poultry feed. Additionally, SSF can also be employed to produce low molecular
weight functional peptides by using PKC as the main substrate. The peptides exhibit
antifungal activity as shown in the inhibitory activity against Aspergillus flavus,
Aspergillus niger, Fusarium sp., and Penicillium sp. (Mohamad Asri et al. 2020).

3.2 By-Product from Marine Industry

The increase in the human population increases the need for food consumption, one of
which is the increase in crustacean consumption. This problem certainly has a linear
value with the increasing waste by-product of this product. The challenge is how to
increase reuse without increasing disruption to the environment. The conventional
Chitin chitosan manufacturing process always involves the use of chemical reagents
which besides being expensive can also interfere with the environment. The approach
is carried out using a number of enzymatic, physical, and biological processes.

3.2.1 Biological Process

Lactobacillus acidophilus SW01 from shrimp waste for deproteinated fermentation

of shrimp wastewas used. As a result, the minerals and protein were quickly removed
114 A. Sofyan et al.

with their contents decreasing to 0.73% and 7.8%, respectively, after 48 h fermenta-
tion (Duan et al. 2012). A protease-producing strain, Exiguobacterium profundum,
and a lactic acid-producing strain, Lactobacillus acidophilus, were used to extract
the chitin. The yields for the chitin were 47.82 and 16.32%, respectively (Xie et al.
2021). Two strains of bacterial BAO-01 and BAO-02 were isolated and observed
for the chitinase activity. These Salinivibrio spp. did not show bioamine production,
hemolytic activity, and mucin degradation. Therefore, the in vitro screening results
suggested that these bacteria could be widely used as new candidates for chitin
hydrolyzation and seafood fermentation (Le et al. 2018).

3.2.2 Physical Treatment

The extraction process could use ball milling processes combined with steam explo-
sion that worked for woody biomass or biorefinery action, or other uses converting
chitin into small nitrogen-containing chemicals—such as derivatives of ETA and of
the widely used organic solvent furan (Yan and Chen 2015).

4 Feed and Feeding Containing Biomass

As a feed additive in ruminants, chitosan administration has two functions, namely

directly affecting the microbial fermentation process of the rumen, digestive system,
and livestock metabolism and indirectly, namely improving the quality of feed to be
consumed by livestock.

4.1 Direct Effect

4.1.1 Anti-Inflammatory and Immunomodulator

The intrauterine infusion of a chitosan solution could speed up the healing process
of uterine after parturition in dairy cows (Okawa et al. 2021). It happened because of
the antimicrobial effect of chitosan. Diet supplementation of chitosan in dairy cows
diet reveals the response of suppressed the activity and expression level of gene
and protein of inducible nitric oxide synthase, and linearly decreased interleukin-
1 content and gene expression. The supplementation of CHI linearly increased
the proportion of CD4+ and CD3+ (T lymphocytes). The supplementation of CHI
linearly down-regulated the expression of nuclear factor-κBp65 gene and phosphory-
lation level in peripheral blood mononuclear cells and linearly inhibited the activity
of inducible nitric oxide synthase and the production of nitric oxide (Zheng et al.
2021). The infusion of Chito-oligosaccharides on peripheral blood mononuclear cells
(PBMCs) in dairy cows at a concentration of 160 μg/mL exhibited the strongest effect
Biomass Utilization and Biorefinery By-Product from Palm Oil … 115

decreased the content and gene expressions of interleukin-1β and tumor necrosis
factor-α, the mRNA and protein expression and activity of the inducible nitric oxide
synthase, and NO production compared with the values observed in the control group
(Zheng et al. 2020).

4.1.2 Protecting Agent

Rumen-protected amino acid coating chitosan on the particles of hydrogenated fat

entrapping amino acid could protect arginine particles coated with chitosan by the
coacervation using NaOH could retain about 80% of arginine, with the addition of
ethanol employed, the chitosan-coated particles could retain even 97% of arginine,
and the chitosan-coated methionine particles prepared by addition of ethanol led
to 85% (Chiang et al. 2009). Minimizing the ruminal biohydrogenation process of
unsaturated fatty acids by the use of encapsulation of flaxseed oil with chitosan (14
and 7%) to encapsulate fats and their effect on in vitro fermentation and fatty acid
biohydrogenation with the result increased oleic unsaturated fatty acid significantly
compare to control (Besharati et al. 2022).

4.1.3 Methane Reducer and Rumen Modulator

Related to the metabolism of energy supply from crude fiber sources, normally CH4
gas is releaseed from the rumen, losses in the form of loss energy and emission of
greenhouse gas causing this process to be attempted to be reduced. chitosan addition
was able to reduce enteric methane emissions such decrease was accompanied by
a decline in the protozoa population and a tendency of methanogen reduction also
(Harahap et al. 2020). The methanogens could react with the chitosan as the result
of different cell charges leading to leakage of the cell (Zanferari et al. 2018). The
proportion of VFA was affected by the addition of chitosan. While dry matter intake
was not affected the addition of chitosan changed the carbohydrate fermentation
resulting in the increase of propionate and decrease of acetate (Araujo et al. 2015) this
condition was the same result as monensin was used (Goiri et al. 2010; Zanferari et al.
2018).

4.2 Indirect Effect

Chitosan has a function as an antimicrobial, based on this concept, the addition of

chitosan is given to the silage-making process to control fermentation processes that
are not desired by microbes. Chitosan addition at 4.47–7.47 g/kg of dry matter (DM)
increased the efficiency process by reducing the fermentation losses and increasing
the nutritional content of sugarcane silage (Del Valle et al. 2022). The infusion of
chitosan and a microbial inoculum to soybean whole plant silage enhances nutritional
116 A. Sofyan et al.

and fermentative quality, increases all bacteria, and decreases yeast and mold on the
silage product (Gandra et al. 2018). The addition of 2% chitosan on the alfalfa silage
Neutral detergent fiber (NDF), neutral detergent insoluble crude protein (NDICP)
values increase a lactic acid and butyric acid contents decreased, in conclusion,
chitosan negatively influenced fermentation quality of alfalfa silage, but reduced
mold and clostridial development (Sırakaya and Büyükkılıç-Beyzi 2022).

5 Future Strategy for Biomass Utilization

Biomass transformation via novel bioconversion technology is new frontiers for

biorefinery concept, with the main objective to produce value-added underutilized
by-products. At present, large volume of palm oil and fish processing by-products
need a serious attention for further processing. Solid evidence from the capability of
various technological approaches to increase the quality of by-products provides a
realistic target for future implementation. From an industrial and economic point of
view, there is a need to design a business model for palm kernel cake and palm kernel
meal, prior to the implementation of technological processing. At this step, identi-
fication of stakeholders that are in need of the final products and potential market
volume is fundamental. Next, re-routing the biorefinery process can be determined
by considering the industrial-consumer chain characteristics.
For the palm oil processing industry, integrated applications of novel technology
to process PKC, PKC, and other potential biomass is the first critical step to produce
competitive products. Solid-state fermentation, coupled with mechanical processing,
and various valorization techniques can be integrated to process the biomass from
palm oil industry. Meanwhile, chemical treatments to improve chitosan functionality
are now promising. At the same time, on-farm trials are needed to generate data and
validate the quality of the products, that may also help with future improvement
strategy. In addition, life cycle assessment may also help to assess the economical,
environmental, and social impacts of the implementation of biorefinery technologies
for palm oil and fish processing industries. The successful implementation of inte-
grated biorefinery technology to produce valuable by-products from these industries
is promising to establish a better livestock industry that is environmentally friendly
while decreasing the demand for imported raw materials.

6 Conclusion

Utilization of biomass and biorefinery by-products as feedstock is emerging as a

potentially viable alternative to support livestock development and sustainability.
Feed ingredients in terms of quality, quantity, and continuity (sustainability) is the
key in supporting livestock productivity. Implementation of technology for improving
biomass and by-product quality could be carried out by several techniques. Biological
Biomass Utilization and Biorefinery By-Product from Palm Oil … 117

processes including solid-state fermentation, mechanical processing, and valoriza-

tion techniques can be integrated to process the biomass from palm oil industry.
Chemical treatments including green chemistry technique could improve chitosan
functionality. Consequently, utilization of biomass and by-products of palm oil and
marine resources promises supporting raw material stock and sustainability for the
feed and feed additive of animals. Further, life cycle assessment should be carried out
to evaluate the economic, environmental, and social impacts of the implementation
of biorefinery technologies for palm oil and marine processing industries and their
relationship with the livestock industry.

Acknowledgements The authors thank the Research Collaboration Center of Biomass and
Bioproduct Collaborated Between UNPAD and BRIN. Research Center for Animal Husbandry
for facilitating and supporting during manuscript preparation.

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Aquatic Biomass Conversion
and Biorefinery for Value-Added
Products

Novia Amalia Sholeha, Nova Rachmadona, Fajriana Shafira Nurrusyda,

Nanang Masruchin, and Khatarina Meldawati Pasaribu

Abstract The biorefinery concepts that merge technology and methods to trans-
form aquatic biomass require the efficient utilization of most of the components. The
presence of lipids, protein, and carbohydrates in aquatic biomass makes it a suitable
feedstock for biofuel generation. Aquatic biomass’s sugar and lignin components
might be used to produce gas, heat, and bio-oil using thermochemical processes.
The sugar component might be fermented to generate bio-butanol, bio-methanol,
and bioethanol. The aquatic biomass lipid component could be used to manufacture
biodiesel. Aquatic biomass might also be converted through biological processes into
bio-methane and bio-hydrogen. Thermochemical processing (hydrothermal, pyrol-
ysis, torrefaction) is a potential clean method for converting aquatic biomass and
lignocellulosic materials to high-added value chemicals and bioenergy.

Keywords Aquatic biomass · Torrefaction · Pyrolysis · Biochar · Biogas · Bio-oil

N. A. Sholeha (B)
College of Vocational Studies, Bogor Agricultural University (IPB University), Jalan Kumbang
No. 14, Bogor 16151, Indonesia
e-mail: [email protected]
N. A. Sholeha · N. Masruchin · K. M. Pasaribu
Research Center for Biomass and Bioproducts, National Research and Innovation Agency of
Indonesia (BRIN), Cibinong 16911, Indonesia
N. A. Sholeha · N. Rachmadona · N. Masruchin · K. M. Pasaribu
Research Collaboration Center for Biomass and Biorefinery Between BRIN and Universitas
Padjadjaran, Jatinangor 45363, Indonesia
N. Rachmadona · F. S. Nurrusyda
Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Padjadjaran,
Jatinangor 45363, Indonesia

© The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2024 121
M. A. R. Lubis et al. (eds.), Biomass Conversion and Sustainable Biorefinery, Green
Energy and Technology, https://doi.org/10.1007/978-981-99-7769-7_6
122 N. A. Sholeha et al.

1 Introduction

Bioenergy is an environmentally conscious approach for minimizing reliance on

oil, coal, and natural gas fuel. Energy is a commodity; hence a massive amount of
feedstock is necessary. The search for innovative feedstocks for bioenergy produc-
tion has been prompted by the rising energy demand and challenges associated with
traditional feedstocks (Malode et al. 2022). In the recent past, researchers, policy-
makers, and the energy business have shown considerable interest in identifying and
creating novel feedstocks. Current research focuses mainly on identifying acceptable
feedstocks, creating effective conversion procedures, and reducing production costs
overall. As biofuel feedstocks, lignocellulosic waste, municipal trash, microalgae,
fungus, and other biomass have recently received considerable attention (Fakayode
et al. 2023). These feedstocks have demonstrated biofuel production potential (Patel
et al. 2021).
As a biofuel feedstock, aquatic biomass, including macro-, micro-algae, aquatic
plants, and cyanobacteria, have the capacity to produce far more biomass per hectare
than terrestrial crops; certain species produce fuel directly (Biller 2018). Advantages
of such aquatic biomass include cultivating on the non-arable ground or even offshore
and employing industrial carbon dioxide as a carbon source or wastewater as a
fertilizer input (nitrogen and phosphorus). Aquatic biomass refers to energy crops that
do not thrive with food crops for land or other resources. Numerous factors influence
the productivity and composition of aquatic biomass, including nutrients, salinity,
dark/light cycles, pH, irradiance levels, temperature, CO2 , and O2 concentration. The
composition of aquatic biomass includes proteins, lipids, carbohydrates, vitamins,
and pigments, with lipids being the most interesting portion for biofuel production
(Azwar et al. 2022). The practical implementation of regulating aquatic biomass as
the raw material for diverse value-added products, as well as its biorefinery process
(Fig. 1), has increasingly attracted the attention of researchers worldwide.

Fig. 1 The aquatic biomass biorefinery and its products

Aquatic Biomass Conversion and Biorefinery for Value-Added Products 123

2 Aquatic Biomass as a Bioenergy Feedstock

Aquatic biomass has been identified as a viable renewable biomass feedstock in

the production of bio-ethanol due to its high area-specific yields and photosynthetic
efficiency. Microalgae have historically been explored for their potential high levels
of lipids for biodiesel production (Ruiz et al. 2013). Among the significant benefits
of microalgae versus terrestrial biomass is their elevated efficiency of photosynthetic
to enhance CO2 abatement and contributes to larger growth rates. Phototrophic and
heterotrophic are the two functional classes of algae. Photoautotrophic algae absorb
light and carbon dioxide for photosynthesis, while heterotrophic algae require oxygen
and organic carbon sources.
Seaweed refers to a category of eukaryotic, photosynthetic marine organisms
known as macroalgae (Alam et al. 2021). Both in terms of physical and biochemical
properties, they differ significantly from microalgae. There are numerous species
of them throughout the oceans and coastal waterways of the world. Aquatic weeds
contain considerable levels of biofuel-convertible lignin, hemicellulose, and cellu-
lose. The bulk of aquatic plant biomass is composed of lignin and carbohydrates.
Lignin might be utilized to create combustible gases, bio-oil, and heat energy through
thermochemical processes. The sugar component is immediately fermentable to
produce bioethanol (Khammee et al. 2021). There is also a substantial opportunity
to make bio-methanol and bio-butanol, among other alcohol molecules.
Additionally, aquatic weeds have lipids and a waxy covering composed primarily
of modified fatty acids. Through the process of transesterification, these lipids may
be converted into biodiesel. Through anaerobic digestion and biological processes,
aquatic weeds can be used to produce biomethane and biohydrogen, respectively.
Since aquatic weeds are fast-growing plants, they can provide greater biomass yields
than most terrestrial energy crops. Macroalgae generally consist of a stipe, lamina
or blade, and an anchoring and sustaining holdfast in marine conditions. Due to
their size, the bulk of macroalgae must be ground before pumping. For instance,
freshwater macrophytes are a diverse group of aquatic plants consisting of mono-
or multi-cellular forms that frequently contain chlorophyll but lack genuine roots
and stem in some cases. (Anyanwu et al. 2022). Algae and floating macrophytes
(submerged, floating, and developing) are tiny, fast-growing plants typically found
in watery habitat, and would not require agricultural land for agriculture, and several
varieties are capable of living in freshwater, so preventing competing for water and
land essential to produce food. Recent studies have revealed that the aquatic macro-
phyte Ledermanniella schlechteri (LS) and the green macroalgae Ulva lactuca (UL),
prevalent throughout the Democratic Republic of the Congo, may be utilized to
produce sustainable bioenergy (Mayala et al. 2022). Using biochemical methane
potential (BMP) assays to evaluate how their anaerobic digestion functioned, it was
revealed that the typical CH4 levels for LS and UL are 262 and 162 mL gVS−1 , respec-
tively. Moreover, LS’s average HHV is 14.1 MJ kg−1 and UL’s average HHV is 10.5
MJ kg−1 . Due to their negative ash behavior and high ash content, both biomasses
would be challenging to convert thermally. The biochemical analyses revealed a high
124 N. A. Sholeha et al.

percentage of anaerobically digestible proteins and carbohydrates and a low quantity

of lipids and lignin. The average biodegradability (BI) for LS was 76.5%, compared
to 43.5% for UL.
Other kinds of aquatic biomass that are not algae but instead aquatic plants include
water lettuce (Pistia stratiotes), water hyacinth (Eichhornia crassipes), cattail (Typha
latifolia), salvinia (Salvinia molassis), and duckweed (Lemnaceae). Typically, all
of those are invasive species that could colonize large bodies of water and prevent
sunlight from reaching the bottom. They must be physically or mechanically removed
from different rivers where they significantly damage the ecosystem to retain an
intact biological system in many parts of the globe. These aquatic plants may also
be grown expressly for biofuel generation; they use nutrients from wastewater and
can generate approximately 20 tons (dry)/ha/year in only 24 h. As a result of its high
moisture content, wet biomass cannot be burnt, making its disposal problematic and
potentially expensive.
One kind of aquatic biomass, cyanobacteria, uses photosynthetic processes to
convert carbon dioxide and solar energy into chemical compounds efficiently.
Gram-negative photosynthetic bacteria called cyanobacteria are essential for global
processes, including nitrogen fixation, carbon sequestration, and oxygen evolution.
The natural cyanobacterial host system must often be better understood to boost goal
output. In recent years, the accumulation of invaluable insights into the biochemistry
and metabolism of cyanobacteria has propelled the development of cyanobacteria
as cell factories for biochemical synthesis, including the synthesis of biofuels (Liu
et al. 2022). Among the cyanobacteria that have been extensively examined for their
ability to make biofuels are a marine species (Synechococcus sp. PCC 7002) and two
freshwater species (Synechocystis sp. PCC 6803 and Synechococcus elongatus sp.
PCC 7492) (Kumar et al. 2022).
That aquatic biomass (micro-, macro-algae, aquatic plants, and cyanobacteria)
was processed through liquefaction, hydrothermal carbonization, hydrothermal
torrefaction, and pyrolysis. The products from the process are summarized in Table 1.

2.1 Bio-Oil

A combination of several organic compounds known as "bio-oil" is often utilized as

a raw material to manufacture pure chemicals like phenol, alcohol, organic acids, and
aldehydes. Gasoline might be made from bio-oil via a processing step. Additionally,
it contains chemicals that may be utilized for various applications at economically
recoverable levels. Bio-oil has numerous manufacturing and selling benefits in the
areas of combustion, preservation, transportation, adaption, and refurbishment. The
literature is severely lacking in information on the creation of bio-oil from aquatic
biomass. Several researchers have tried using thermochemical methods to extract bio-
oil and other byproducts from aquatic biomass. Water hyacinth by pyrolysis method
at 450 °C produced carboxylic acids, aldehydes, ketones, alkenes, quinines, alcohols,
phenols, and aromatics, significant bio-oil components (Wauton and Ogbeide 2019).
Aquatic Biomass Conversion and Biorefinery for Value-Added Products 125

Table 1 Value-added products from biomass refinery (Boro et al. 2022; Chacon et al. 2022)
Products Properties Limitations
Biogas (bio-methane and –Approximately consists of CH4 –Large bioreaction tanks
bio-hydrogen) (70%), CO2 (25%), and other increase the land area needed
gases such as H2 S, NH3 (5%) –Contain contaminants that,
–Ignition temperature around when used as fuel, might
700 °C in anaerobic tanks damage the engine systems of
–There is no smoke or residue automobiles
produced during combustion –Maintenance energy, optimal
(carbon neutral) temperature, and a considerable
–It can be utilized as cleaner amount of organic biomass are
fuel to generate electricity in the required
form of compressed natural gas –Foul odor
(CNG) –The production procedure is
–Zero carbon dioxide and costly
greenhouse gas emissions –Need compression due to its
–Highly flammable and extremely low density
effective in producing energy
–The only byproducts produced
are water and heat
Bio-oil (bioethanol and –Utilized as an alternative fuel –Low efficiency compared to
bio-butanol) for automobiles by blending gasoline
with gasoline –Implementation in vehicles
–Must increase the combustion necessitates engine
rate while cleaning the modifications for older vehicles
emissions –Due to bioethanol’s low vapor
–A transparent, colorless liquid pressure, it is difficult to use it as
–As a result of the low vapor a fuel at low temperatures,
pressure in comparison to resulting in cold-start issues for
gasoline, the rate of evaporation vehicles
is low –It has a high capacity to absorb
–Can utilize any substrate moisture, which increases the
containing sugar; thus, risk of fuel pump corrosion
agro-based lignocellulosic waste
biomass usage is highly
regarded for reducing
challenges associated with the
disposal of such waste
–Nature-friendly and readily
dilutable
Biochar –Enhance soil permeability –Land loss due to erosion
–Increasing the water-holding –Compaction of the soil during
capacity makes it simpler for application
plants to absorb water, nutrients, –Elimination of crop residues
and oxygen
–Boost soil pH levels
126 N. A. Sholeha et al.

Moreover, duckweed at 350–700 °C of pyrolysis yields bio-oil around 35.5–45%,

char around 30–50%, and gas around 11–20% (Djandja et al. 2021). Various aquatic
plant biofuels’ calorific value, such as Azolla (38.2 MJ kg−1 ) and Salvinia molesta
(39.73 MJ kg−1 ), is more than that of biogas (30 MJ kg−1 ), shown in Table 2 (Arefin
et al. 2021).
In recent years, bioethanol has surpassed bio-oil as the primary alternative to fossil
fuels, contributing up to 75% of global biofuel demand with an approximate extensive
distribution rate of 86,000 kt/year. Aquatic biomass is handled using conventional
hydrolysis techniques, much like any other bioethanol feedstock, and the resultant
sugars are subsequently fermented to produce bioethanol. Aquatic vegetation is a
favorable feedstock for bioethanol synthesis due to its richness of both cellulose
and hemicellulose and the absence of lignin. Limnocharis flava was converted to
bioethanol using several alkaline treatments (0% alkaline, 2% NaOH, and 1–2%
CaO) to determine the most effective pretreatment for degrading cellulose, hemi-
cellulose, and lignin to sugars fermentation. Significantly, 1% CaO resulted in a
satisfactory total sugar, ethanol yield, and reducing sugar of 50.81, 0.72, and 28.88
g/L, respectively (Mejica et al. 2022). Prior research indicates the significance of
NaOH for bioethanol in terms of Brachiaria mutica (Para grass) and Alternanthera
philoxeroides (Alligator weed) (Aarti et al. 2022). In 12–96 h, the biomass from
pre-treatment process showed that saccharification degree increased by 44.46 0.7%,
55.53 0.8%, 73.26 0.7%, 94.41 0.8%, and 73.3 0.7%. Bioethanol production from

Table 2 Aquatic biofuel properties comparisons with conventional fuel (Arefin et al. 2021)
Fuel Aquatic plant biofuels Conventional fuel
properties Azolla Water Salvinia Water Duckweed Diesel Gasoline Biogas
hyacinth molesta lettuce
Calorific 38.2 35.8 39.73 24.93 21.7 45.5 45.8 30
value (MJ/
kg)
Density ~880 834 792.23 952 800 850 715–780 1.15–1.25
(kg/m3 )
Fire point 120 600–1370 300 – – 210 280 650–750
(°C)
Flashpoint 108 246 139 120 169 60 −43 −188
(°C)
Pour point 3 −5 1.4 17 6 −2 to −4 to − –
(°C) −12 20
Cloud 8 −1 1.5 – – −12 −22 –
point (°C)
Viscosity 4.3 9.85 3.657 26.4 ~4.9 2.40 0.48 0.01–0.06
(cP)
pH 3.5–10 2.93 6–7.7 6.6 7.8 5.5–8 5.9–6.8 6.8–7.2
Water 40 1.8 5 94.6 63.46 2 10 1–5
Aquatic Biomass Conversion and Biorefinery for Value-Added Products 127

pre-treated aquatic weeds was evaluated utilizing yeast cells immobilized in sodium
alginate for simultaneous saccharification and fermentation.

2.2 Biogas

Biogas in the aquatic biomass biorefinery comprises bio-hydrogen and bio-methane.

Bio-hydrogen is seen as a feasible renewable energy source and an alternative to fossil
fuels due to its higher energy content (122–142 kJ g−1 ) in contrast to biomethane (56
kJ g−1 ) and biodiesel (37 kJ g−1 ). Biohydrogen can be produced at ordinary pressures
and temperatures with low energy input, and its combustion simply creates water.
Biohydrogen is already used in fuel cells, gasoline, and automobile engines (Yu et al.
2020). Currently, fossil fuels, which are expensive and inefficient, account for 96%
of H2 production. Single-celled algal species, including blue- and green algae such
as Chlorella sp, Platymonas subcordiformis, and Chlamydomonas reinhardti, are
typically used in H2 production systems (enzymes such as the family Enterobacte-
riaceae). In anaerobic processes, the proton reduction by electronic hydrogenase of
ferredoxin is necessary for biohydrogen production. The release of electrons from the
breakdown of glucose to pyruvate leads to acetyl-CoA oxidation and carbon dioxide
(Debowski et al. 2021).
One of the most flexible and clean-burning biofuels is biomethane, which is
created through the anaerobic digestion of various feedstock materials (Zhang et al.
2021). Biomethane has advantages in easily transported and distributed by the same
pipes as natural gas due to its easy storage after liquefaction. The byproduct of the
manufacturing process may also be used on agricultural land as fertilizer. All types
of biomass can be used to make biomethane, which offers advantages over other
feedstocks, not just in terms of renewability but also in terms of waste manage-
ment. Therefore, aquatic biomass has significant potential as a feedstock because it
may be used immediately for biomethane production. AcD, also known as anaer-
obic co-digestion, is a promising strategy for boosting the biomethane manufacturing
process’s efficiency and overcoming the constraints of single digestion using cata-
lysts. One of the AcD investigations found that adding Co3 O4 -NPs (3 mg/L) to water
hyacinth (WH) increased biogas production by 27.2%. In addition, the production
of methane (CH4 ) was raised by 89.96% for the CD method and by 43.4% for the
co-digestion method. The techno-economic analysis reveals that this method would
generate 428.05 kWh of revenue based on the maximum net energy content of biogas,
with such a sales revenue of 67.66 USD per m3 of substrate (Ali et al. 2023).

2.3 Biochar

Biochar is black carbon or carbon-rich charcoal derived from organic matter through
pyrolysis process; however, it can also be formed from a feedstock a feedstock
128 N. A. Sholeha et al.

via flash carbonization, torrefaction, or gasification (Janiszewska et al. 2021).

Biochar has the capacity to hold carbon for millennia through enhancing water and
nutrient retention and reducing greenhouse gas emissions from fertilized soils, hence
enhancing the condition of the soils to which it is applied. As a feedstock for biochar,
lignocellulosic (“woody”) biomass has been the subject of most of the study. This
feedstock produces biochar with low mineral concentration and high fixed-C content.
Marine and freshwater macroalgae are alternate feedstocks for biochar manufac-
turing. Algal biochar contains considerable amounts of macronutrients and essential
trace elements, including nitrogen, phosphorus, calcium, magnesium, potassium,
and molybdenum, while having less carbon than lignocellulosic biochar. Due to
the nutrient retention effects of micronutrients (Mo) and macronutrients (Ca, N,
Mg, P, and K) on the soil, algal biochar has the potential to produce more signif-
icant increases in the quantity of certain types of soil than lignocellulosic biochar.
According to previous findings, the biochar of the freshwater macroalgae Oedogo-
nium formed at 750 °C has the most resistant carbon and leaches the least amount
of metal (Roberts et al. 2015). The retention of fertilizer nutrients (Mo, Ca, N, Mg,
P, and K) and the growth of radishes are both boosted by 35–40% when this biochar
is applied to poor-quality soil. Radishes grown in biochar-modified soil exhibited
comparable or lower metal concentrations than radishes grown in unmodified soil
but had significantly greater concentrations of essential macronutrients (Mg, K, and
P) and trace elements (Mo).

3 Thermochemical Process of Aquatic Weeds

3.1 Torrefaction

Torrefaction, one of thermochemical processes with slow heating, has been utilized
extensively to volatilize biomass and can be classified as dry and wet, with bio-
coal (biochar and hydrochar) as the main products (Yek et al. 2022). Without the
use of solvents, dry torrefaction (DT) takes place in oxidizing (flue gas or air) or
non-oxidizing (CO2 or N2 ) atmospheres between 200 and 300 °C. Compared to
non-oxidative torrefaction, oxidative torrefaction has a quicker reaction rate and
shorter torrefaction duration due to exothermic reactions in the biomass thermal
breakdown (Viegas et al. 2021). Additionally, the ultimate separation of nitrogen
and air is unnecessary for oxidative torrefaction. A large part of the ash content
remained in the torrefied aquatic biomass following dry torrefaction pretreatment,
leading to undesirable agglomeration, fouling, and slagging despite the good poten-
tial for biofuel production. Aquatic biomass has been pre-treated to lower its ash
content before torrefaction. At a reactor temperature of 440 °C, the pyrolysis process
was carried out after pretreatment of the water hyacinth biomass at 200, 250, and
300 °C. Torrefaction severity significantly impacted the yields of char classified as
brown coal (high quality) or peat. ST-Raw non-torrefied sample had a char yield of
Aquatic Biomass Conversion and Biorefinery for Value-Added Products 129

27.4%, whereas the ST-300, ST-250, and ST-200 torrefied samples had char yields of
59.4%, 51.2%, and 42.3%, respectively. However, when the torrefaction temperature
increased, syngas and bio-oil yield declined. GC-MS and FTIR analyses both showed
that the bio-oil acidity had significantly decreased and that the torrefaction tempera-
ture had increased. Torrefied bio-oils are therefore assured to be less corrosive than
un-torrefied bio-oils (Parvej et al. 2022).
Water causes wet torrefaction (WT) when it is present at temperatures between
180 and 260 °C for 10–24 min (Das et al. 2021). When later wet torrefaction happens
in a wet situation, the conventional pre-drying stage for thermal conversion processes
may be avoided, particularly for highly moist biomass such as manure, sewage, and
aquatic biomass. When water is heated to 180 °C, its properties (density, viscosity,
ion products, and dielectric constant) change in a manner that is favorable for ther-
mochemical conversion in the aqueous phase (Nazos et al. 2022). The dissolution
of the ash’s minerals in the liquid reduces the quantity of ash in the solid result. In
addition, steam torrefaction can operate at greater temperatures (200–260 °C) with
the assistance of a high-pressure steam explosion that expands the lignocellulosic
components and separates individual fibers. Carbon content and calorific value of
the biomass increase as low molecular weight volatiles are eliminated during the
steam explosion, although the product’s bulk density, equilibrium moisture content,
and mean particle size decline. The lowest production costs (without carbon credits)
were associated with grape pomace’s dry and wet torrefaction, at 2.29 and 4.14 $/
GJ, respectively. It is more difficult to create pellets from biochar than from raw
biomass because biochar is more brittle, dry, and volatile. Because hydrochar has a
higher concentration of oxygen functional groups than biochar, it has a higher water
affinity (hydrophilicity) on the surface, which enhances the soil’s ability to retain
water when immersed (Akbari et al. 2020).

3.2 Hydrothermal Carbonization (HTC)

Hydrothermal carbonization (HTC) or wet thermal process takes place at pressures

higher than 1 MPa and temperatures between 180 and 300 °C (Akbari et al. 2020).
Although HTC has a shorter residence time and a lower temperature than HTL, both
processes are carried out in subcritical water conditions. Furthermore, HTC produces
hydrochar with the same yield and energy content as a torrefied solid product at
far lower temperatures. Biomass/water ratio, temperature, and duration of 42 wt%,
232 °C, and 99 min, were determined to be optimal for producing high HHV (22
MJ/kg) and low char generation (47 wt%), respectively (Lynam et al. 2015). The
carbonization processes quicken as the temperature increases, leading to quicker
kinetics and less hydrochar generation. If the length of the stay is increased, the
temperature may yet have a distinct impact. The HTC research with fresh aquatic
plants such as cattail and water hyacinth use an autoclave reactor at 180–220 °C.
Following HTC treatment at 220 °C, the carbon content of aquatic biomass (cattail
and water hyacinth) increased by 30.2–41.7%. Greater H/C and O/C ratios in the
130 N. A. Sholeha et al.

feedstocks relative to the comparable hydrochars may have resulted from the disso-
ciation of the dehydration and decarboxylation processes that occur throughout the
HTC process. As the temperature rose, the H/C and O/C atomic ratios fell, and
the 220 °C hydrochar sample exhibited peat-like characteristics (Poomsawat and
Poomsawat 2021).

3.3 Hydrothermal Liquefaction (HTL)

Hydrothermal liquefaction (HTL) is among the most major advancement promising

processes for aquatic biomass upgrading, which directly converts biomass into bio-
oil (Guo et al. 2017). HTL has several advantages, including obtained bio-oil having
lower oxygen content and not requiring drying as the required microalgae concen-
tration is around 20 wt.% (Biller 2018; Biswas et al. 2021). The drying process was
known as the main economic and energetic obstacle before further processing of
aquatic biomass conversion into biofuel. However, in HTL, the cost of the drying
process can be reduced because water functions as a solvent in the system (Biswas
et al., 2021).
Various products, such as aqueous-phase product, bio-oil, volatiles, gas, and solid
residual, are the primary constituents of the HTL process’ hydrothermally decom-
posed biomass conversion (Guo et al. 2017). Species of feedstock and processing
parameters, including temperature, residence time, and solvent, determine HTL
product. Numerous studies have been investigating these various parameter effects
on different aquatic biomass. Due to its potential lipid content and enhanced photo-
synthetic efficiency, aquatic biomass has been recognized as a possible renewable
biofuel source (Biller 2018). Furthermore, aquatic biomass has a higher growth rate
than terrestrial plant biomass with less demanding cultivation and land use (Biswas
et al. 2022).
Some studies show that microalgae, as a species of aquatic biomass, had been
utilized in the production of biofuel using HTL, such as Chlorella, Nannochloropsis,
and Sargassum sp. (He et al. 2020; Moazezi et al. 2022). Microalgae with a high lipid
content will be completely converted to bio-oil; therefore, algae species with a high
lipid content will be more valued (Biller 2018). Table 3 demonstrates that Sargassum
sp. is rich in lipid and protein; hence, it is more susceptible to being transformed
into bio-oil. While, in Nannochloropsis sp., ash contents are much higher, mainly
contributing to solid residue production (He et al. 2020). As shown in Table 4, the
bio-oil yields of Sargassum sp. (16.3% wt.) were significantly less than those of
Nannochloropsis sp. (39.0% wt.).
Table 4 also presents bio-oil yields for different biomass species and opera-
tional parameters. Temperature has an important influence on the production of
bio-residues, gas, and bio-oil. At lower temperatures, the degradation of biomass
will be incomplete and unreacted. Thus, the bio-oil formation will be suppressed
while increasing the solid products. An increase in temperature should be beneficial
for bio-oil formation due to the acceleration of biomass decomposition. The yield of
Table 3 Composition data of algal biomass used for hydrothermal liquefaction
Biomass Component (%wt) References
Protein Lipid Cellulose Hemicellulose Lignin Poly-saccharide Carbo-hydrate Moisture Ash
Sargassum sp 9.9 0.80 9.04 38.6 13.0 – – – – He et al.
Nannochloropsis sp. 45.6 6.20 0.30 0.91 0.52 – – – – (2020)
Sargassum sp 7.5 1.33 – – – – 50.7 27 Ardiansyah
et al. (2018)
Gracilaria corticata 22.8 7.07 – – – 49.6 8.30 8.40 8.10 Rosemary
et al. (2019)
Azolla filiculoides 4.6 0.72 – – – – 0.82 91.8 2 Bhaskaran
and
Kannapan
(2015)
Azolla filiculoides 19.7 4.2 – – – 10.3 – – 18.5 Datta (2011)
Chlorella vulgaris 58.0 11.5 – – – – 19 5 6.5 Moazezi
Aquatic Biomass Conversion and Biorefinery for Value-Added Products

et al. (2022)
Chlorella vulgaris 45.0 20.0 – – – 5 20 – 10 El-Naggar
et al. (2020)
131
Table 4 Bio-crude yields and properties of distinct aquatic feedstocks under various HTL conditions
132

Biomass Description Solvent Operational parameter Product (%wt) References

T (o C) Reaction Time Solid Liq Gas
(min)
Sargassum tenerrimum Brown H2 O 260 15 61.2 11.5 11.6 Biswas et al. (2018)
macroalgae 280 15 32.3 16.3 12.1
300 15 24.2 14.7 9.0
Gracilaria corticata Red H2 O 260 15 21.7 3.9 11.8 Fernandes et al.
macroalgae 280 15 23.0 2.8 4.9 (2021)
300 15 26.0 5.2 11.2
Methanol 300 15 – 8.2 –
Ethanol 300 15 – 14 –
Acetone 300 15 44.0 16.2 –
Ethanol–water 300 15 – 13.3 –
Azolla filiculoides Aquatic plants H2 O 280 15 38.0 21.5 5.0 Biswas et al. (2021)
260 30 47.0 13.6 12.4
300 60 39.0 15.2 6.9
Methanol 260 15 41.2 28.7 12.9
260 30 37.5 26.3 7.6
280 60 36.2 24.3 11.9
Ethanol 300 15 29.5 26.5 2.1
300 30 33.8 26.3 14.8
280 60 36.7 28.8 15.5
(continued)
N. A. Sholeha et al.
Table 4 (continued)
Biomass Description Solvent Operational parameter Product (%wt) References
T (o C) Reaction Time Solid Liq Gas
(min)
Chlorella vulgaris High lipid, H2 O 287 40 – 56.2 Moazezi et al.
microalgae (2022)
Nannochloropsis sp. Low lipid, H2 O 260 30 11.0 39.1 15.9 He et al. (2020)
microalgae 280 30 6.7 43.5 18.8
300 30 4.8 45.3 23.9
320 30 3.1 54.1 20.8
340 30 4.39 41.73 36.97
Sargassum sp. Brown H2 O 260 30 40.04 3.11 22.22 He et al. (2020)
macroalgae 280 30 36.36 5.99 22.39
300 30 36.12 6.93 23.96
320 30 32.08 8.43 25.38
340 30 32.04 9.49 26.20
Aquatic Biomass Conversion and Biorefinery for Value-Added Products
133
134 N. A. Sholeha et al.

bio-residue and bio-oil decreases at the temperature above 280 °C, as shown in Table 4
(Biswas et al. 2018). The relationship between re-polymerization and hydrolysis has
a substantial impact on the HTL process’s temperature. Extensive depolymerization
will occur at high temperatures to activate the bond-breaking activation energy. The
bond-breaking increases free radicals and repolymerizes the pieces that have been
degraded (Moazezi et al. 2022).
Based on bio-oil yield using Gracilaria corticata as biomass, the relative effi-
ciency of solvent used during the HTL process could be reported as follows: water
< methanol < ethanol-water < ethanol < acetone. Table 4 demonstrates that the
type of solvent affects conversion yield. Organic solvents promote the solubility and
stability of chemical intermediates due to their lower dielectric constants, resulting
in a greater yield. Additionally, it will facilitate esterification and alkylation between
intermediate molecules and solvents (Fernandes et al. 2021).
In addition to being affected by the type of biomass employed, the duration of
reaction times can determine the products derived from HTL as well as the feedstock
conversion rate. As indicated in Table 4, a relatively short reaction time is suited
for efficient biomass breakdown since the HTL process rapidly hydrolyzes biomass.
In a longer reaction period, liquid products will undergo greater decomposition and
repolymerization, hence contributing to the creation of gaseous products and biochar
(Moazezi et al. 2022).

3.4 Pyrolysis

The thermochemical conversion processes can be split into four categories based On
the basis of operating features such as temperature, pressure, heating rate, and reac-
tion environment: gasification, combustion/incineration, liquefaction, and pyrolysis
(Vuppaladadiyam et al. 2022). Pyrolysis, often known as thermal decomposition in
an inert atmosphere, has been widely used to transform biomass into products with
added value (Gao et al. 2020). Pyrolysis is a type of thermolysis or carbonization that
employs intense heat in a low or oxygen-free (O2 ) atmosphere to thermally decom-
pose biomass into a number of pyrolytic chemicals (Tripathi et al. 2016; Lee et al.
2020). This thermochemical conversion yields biochar, bio-oil, and bio-syngas as its
principal by-products (Azizi et al. 2018). The features of the aquaculture biomass,
the operational parameters, and the kind of pyrolysis reaction influence the number
of products and the HHV (Chen et al. 2015).
Table 5 summarizes the experimental parameters for pyrolysis techniques. The
pyrolysis process has been classified into 2 categories; conventional and advanced
approaches (Lee et al. 2020), presented in Fig. 2. Conventional pyrolysis can be
divided into three distinct types: slow pyrolysis, fast pyrolysis, and flash pyrolysis,
depending on the operational parameters employed during the process. Slow pyrol-
ysis is a crucial synthesis technique that is mostly used to produce biochar with
byproducts such as syngas and bio-oil (Lee et al. 2017). Slow pyrolysis settings
emphasize slow heating rates (30 °C/min), moderate temperatures (550–950 °C),
Aquatic Biomass Conversion and Biorefinery for Value-Added Products 135

and slow reaction time. According to Table XZ, the yields of biochar, bio-oil, and
bio-syngas produced by pyrolysis at 600 °C in which bio-syngas is the dominant
product obtained in this technique (Maddi et al. 2011). Fast pyrolysis, the counter-
part of slow pyrolysis, is frequently utilized for biomass under the following pyrolysis
conditions: rapid heating rate (>60 °C/min), high temperature (850–1200 °C), and
brief pyrolysis period (0.5–10 s) (Campanella and Harold 2012; Ly et al. 2015).
Fast pyrolysis aims to optimize bio-oil synthesis, readily stored, or transported, and
contains less nitrogen and sulfur (Roddy and Manson-Whitton 2012).
To improve the pyrolysis process, advanced pyrolysis techniques are often modi-
fied to create new methods, e.g. co-pyrolysis, catalytic pyrolysis, and microwave-
assisted pyrolysis, that make the pyrolysis process superior to conventional tech-
niques and enhanced the yield, quality, and characteristics the pyrolysis products.
Under a catalyst, catalytic pyrolysis is a directed control method for obtaining high-
quality liquid fuel and high-value-added chemicals with a high yield (Qiu et al. 2022).
In a fixed-bed reactor, Pavlova microalgae were pyrolyzed at various temperatures
in the presence of titania-based catalysts. When Ni/TiO2 (22.55 wt%) was present at
500 °C, the bio-oil output increased by 20% (Aysu et al. 2017).
In parallel to catalytic pyrolysis, co-pyrolysis (Duan et al. 2015; Uzoejinwa et al.
2018, 2019) and microwave-assisted pyrolysis (Beneroso et al. 2013; Hong et al.
2017) have been identified as a promising strategy for enhancing the performance
of biomass pyrolysis processes through synergistic interactions. Co-pyrolysis is the
process of heating together two or more organic materials in the absence of oxygen
to produce the bio-oil, and it is also the synergistic effect in terms of gas, liquid, and
solid product distribution and product composition modifications (Ma et al. 2022).
(Duan et al. 2015) reported a good synergistic impact between the waste rubber tire
(WRT) and microalgae. The largest synergistic impact value (37.8%) was recorded at
a mass ratio of 1:1 R:M. During co-pyrolysis, the interaction between microalgae and
WRT promoted denitrogenation and deoxygenation, hence enhancing the quality of
the bio-oil. The heating values of bio-oils derived from the co-pyrolysis of microalgae
and WRT were between 35.80 and 42.03 MJ/kg.
On the other hand, microwave-assisted pyrolysis is regarded as a straightforward
processes with direct control (Zhang et al. 2016). Hong et al. found that porphyra
was a more ideal raw material for syngas-rich gas production (85.6–87.1 wt%) by
using microwave-assisted pyrolysis because of its high carbohydrate content (47.7
wt%), but spirulina and chlorella were more advantageous for oil production due to
their higher protein levels. Scenedesmus almeriensis was also found to be an appro-
priate feedstock for microwave-assisted pyrolysis to create gas products (Beneroso
et al. 2013). By reducing CO2 and light hydrocarbons, it has been claimed that the
maximum output of syngas at 800 °C with the highest H2 /CO ratio can approach
94% by volume.
The pyrolysis of algal biomass generates and disperses a variety of organic
and inorganic chemicals. As pyrolysis fuel, the chemical components of aquatic
biomass such as cyanobacteria, duckweed, micro- and macroalgae are acceptable.
As measured by pyrolysis, they may affect the HHV values, viscosities, pH, densi-
ties, and product composition (Bharathiraja et al. 2015). By a significant margin,
Table 5 Biofuel production from different aquatic feedstocks under various conditions of pyrolysis
136

Algal biomass Type of Operational reaction parameters Product (wt.%) HHV (MJ/kg) References
Pyrolysis T (°C) t (min) Carrier gas Heating Solid Liquid Gas
flowrate (mL/ rate (°C/
min) min)
Lyngbya sp. Slow 600 20 He: 200 30 17 12 44 Bio-char: 25.6 Maddi et al.
Cladophora Slow 600 20 He: 200 30 26 20 38 Bio-char: 22.7 (2011)
sp.
Saccharina Fast 350 2s N2 : 4500 – 34.2 45 20.8 Bio-oil: 24.8 Ly et al. (2015)
japonica
Green algae Fast 500 1.5 s N2 : 250 – 26 58.6 17.8 Bio-oil: 26.7 Campanella and
Green-blue Fast 500 1.5 s N2 : 250 – 28.4 54 19.9 Bio-oil: 26.8 Harold (2012)
algae
Chlorella Fast 500 1.5 s N2 : 250 – 29 53.9 17.3 Bio-oil: 25.5
Pavlova sp. Catalytic 450–550 60 N2 : 545 100 35.9–49.0 14.1–22.5 36.5–46.3 Bio-char: 4.8–6.9 Aysu et al.
(Titania) Bio-oil: 33.3–37.1 (2017)
Saccharina Fast catalytic 500 – – – 22.3 39.1 39.3 Bio-oil: 27.2 Ly et al. (2019)
japonica (HZSM-5)
(continued)
N. A. Sholeha et al.
Table 5 (continued)
Algal biomass Type of Operational reaction parameters Product (wt.%) HHV (MJ/kg) References
Pyrolysis T (°C) t (min) Carrier gas Heating Solid Liquid Gas
flowrate (mL/ rate (°C/
min) min)
Enteromorpha Co-pyrolysis 400–600 – N2 : 100 5–25 22.8–31.4 39.2–47.2 28.7–31.4 Bio-char: Uzoejinwa et al.
prolifera (Rice Husk) 26.9–31.6 Bio-oil: (2018)
25.5–30.6
Chlorella Co-pyrolysis 290–370 10–120 – – 19–49.7 37.5–65.4 4.6–14 Bio-oil: 33.7–42.9 Duan et al.
pyrenoidosa (waste rubber (2015)
tyre)
Scenedesmus Microwave 400 30 He: 100 – – – 87.7 wt.% Bio-syngas: 3.36 Beneroso et al.
almeriensis Wh/g (2013)
Chlorella Microwave 400 30 N2 : 100 – 8 8 84 Bio-syngas: 5.6 Hong et al.
Spirulina Microwave 700 30 N2 : 100 – 10 6.3 83.7 Bio-syngas: 2.9 (2017)
Porphyra Microwave 700 30 N2 : 100 – 10.4 2.5 87.1 Bio-syngas: 3.1
Aquatic Biomass Conversion and Biorefinery for Value-Added Products
137
138 N. A. Sholeha et al.

Fig. 2 The classification of the pyrolysis process in algal biomass

aquatic biomass confirms its suitability as pyrolysis feedstock for the eventual
commercialization of energy-dense goods.

4 Conclusion

Aquatic biomass is emerging as a resource to produce biofuels and other goods with
added value. Biomass derived from aquatic organisms offers significant potential for
biomethane, bio-oil, and bioethanol production. However, the scientific community
must address the following concerns.
• Research is necessary to develop an effective pre-treatment and conversion
process.
• Collecting biomass, high processing costs for scaling up, poor hydrolysis, and
conversion are challenges that must be overcome.
• Biological and other hybrid pretreatment approaches, as well as the intensification
of the process, can be utilized to increase biofuel output.
• To achieve economic viability, the whole potential of aquatic weed biomass must
be utilized.

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The Production of Microalgae
and Cyanobacteria Biomass and Their
Valuable Bioproducts

Awalina Satya, Azalea Dyah Maysarah Satya, Tjandra Chrismadha,

Nofdianto Nofdianto, Gunawan Gunawan, Ika Atman Satya,
Souvia Rahimah, Efri Mardawati, Sara Kazemi Yazdi, and Pau-Loke Show

Abstract Microalgae and cyanobacteria produced abundant high-valued bioprod-

ucts in small arable land in a short time. The bioproducts range from their biomass
for food, feed, and biofuels to extractable fine bioproducts. The growing market
and techno-economical aspect support the viability of this biomass production.
Microalgae and Cyanobacteria are also highly diverse thus progression of its current
usage in biomass production served as a challenge of its own but also an opportunity.
In this book chapter, progression in cultivating and screening of technologies on
bioprocess engineering of microalgae and cyanobacteria will be discussed with their
high-demands on food, feed, and energy industry. This chapter further discusses the
advance and manufacturer of different valuable bioproducts through technologies
and production platforms for Microalgae and Cyanobacteria.

A. Satya (B) · T. Chrismadha · N. Nofdianto · G. Gunawan

Research Centre for Limnology and Water Resources, National Research and Innovation Agency,
KST Soekarno, Cibinong, Indonesia
e-mail: [email protected]
A. D. M. Satya · S. K. Yazdi · P.-L. Show
Department of Chemical and Environmental Engineering, Faculty of Science and Engineering,
University of Nottingham Malaysia, Jalan Broga, 43500 Semenyih, Selangor Darul Ehsan,
Malaysia
I. A. Satya
Research Centre for Computation, National Research and Innovation Agency, KST Soekarno,
Cibinong, Indonesia
S. Rahimah · E. Mardawati
Department of Agro-Industrial Technology, Faculty of Agro-Industrial Technology, Padjadjaran
University, Jatinangor, Indonesia
P.-L. Show
Department of Sustainable Engineering, Saveetha School of Engineering, SIMATS,
Chennai 602105, India
Zhejiang Provincial Key Laboratory for Subtropical Water Environment and Marine Biological
Resources Protection, Wenzhou University, Wenzhou 325035, China

© The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2024 143
M. A. R. Lubis et al. (eds.), Biomass Conversion and Sustainable Biorefinery, Green
Energy and Technology, https://doi.org/10.1007/978-981-99-7769-7_7
144 A. Satya et al.

Keywords Microalgae · Cyanobacteria · Cultivation technology · Fine

biochemicals

1 Introduction

Microalgae and cyanobacteria consist of a broad spectrum of photoautotrophic

microorganisms which grow through photosynthesis. The conversion of chemical
energy from solar as a unicellular form made them known as the oldest life form/
thallophytes (primitive plants) known (Abreu et al. 2022). As primitive plants, they
exhibit an absence of roots, stems, and leaves and possess chlorophyll-a as the main
photosynthetic pigment for energy conversion. This characteristic enables them to
adapt toward the predominant environmental circumstances, hence allowing said
lifeforms to flourish for a long period of time (Kumar et al. 2020). As for the types
of the cells, cyanobacteria—as the oldest of the two—have prokaryotic cells which
are characterized by the lack of membrane-bound organelles usually present around
plastids, mitochondria, nuclei, Golgi bodies, and flagella. Meanwhile, microalgae
are eukaryotic cells which have these organelles that control the function of cells.
Microalgae are mainly eukaryotes grouped into many classes defined accordingly to
their pigmentation, life cycle, and basic cellular structure; with green algae (Chloro-
phyta), red algae (Rhodophyta), and diatoms (Bacillariophyta) posing as the three
most important classes in microalgae. Both microalgae and cyanobacteria can be
either autotrophic (inorganic compounds such as CO2 , salts, and light as prerequi-
sites) or heterotrophic (with an external source of organic compounds and nutrients
due to their non-photosynthetic nature). Several photosynthetic algae or cyanobac-
teria are mixotrophic (capable to perform both photosynthesis and acquire exoge-
nous organic nutrients). Autotrophs rely on photosynthesis for their survival, as they
convert solar irradiance and absorbed CO2 by chloroplast into adenosine triphos-
phate (ATP) and O2 . Both ATP and O2 are usable energy packages at the cellular
level, which are then used in respiration to produce energy to support their growth
(Farhan et al. 2017).
Recently there are numerous studies related to techniques and large-scale produc-
tion of microalgal and cyanobacterial biomass (Ugoala et al. 2012). In general, there
are two types of cultivation techniques, namely open pond system and closed photo-
bioreactor system. Both systems have advantages and disadvantages, therefore pref-
erence for the being used system depends on the characteristics of targeted products
(Milledge 2011). Difficulties in controlling the contamination and predation in open
pond systems frequently occur. On the contrary, photobioreactor systems enable
to control of nutrients for growth and operating parameter such as pH, tempera-
ture, dissolved CO2, and contamination/predation. Unfortunately, photobioreactor
systems need a high investment cost and are quite specific to strains of microalgal and
cyanobacterial physiologies which being cultivated (Acién Fernández et al. 2013).
Therefore, a consideration of the production to facilitate an optimum production for
specific microalga or cyanobacterium is quite important. The harvesting process
The Production of Microalgae and Cyanobacteria Biomass and Their … 145

is conducted for dewatering the algal and cyanobacterial biomass. Methods for
this purpose are flocculation, centrifugation, and filtration. A favorable dewatering
process for harvesting must apply to a wide range of microalgal and cyanobacterial
strains. This aims to concentrate biomass recovery and cost-effective production. The
important matter to consider for mass biomass production is combining cultivation
and dewatering processes at the possibly lowest cost while maximizing the microalgal
and or cyanobacterial biomass production. Bioprocess engineering in microalgal
and cyanobacterial biomass production provides various downstream products for
commercial purposes. Microalgal and cyanobacterial biomass contains an abundant
bioactive compound that useful being used in many important industries such as
pharmaceuticals (for the manufacture of antioxidants, antibiotics, immunomodu-
lators, etc.) (Mobin and Alam 2017; Kholssi et al. 2021). Meanwhile, for human
consumption, microalgae biomass can be extracted to obtain its high protein contents,
vitamins, and polysaccharides (Catone et al. 2021; Hernández et al. 2015). Some
microalgae and cyanobacteria are known to contain high lipids which are then
extracted (oil press, solvent extraction, supercritical fluid extraction and ultrasound)
and converted (trans-esterified) into biofuels (Castro et al. 2021; Felix et al. 2019).
Furthermore, the residue of lipid-extracted microalgal and cyanobacterial biomass
can be converted into other forms of biofuels, such as biomethane, bioethanol, and
biohydrogen (Felix et al. 2019; Nitsos et al. 2020).
Microalgae and cyanobacteria also display a capability to overcome emerging
environmental issues, for example, the greenhouse effect and water (industrial,
domestic, and agricultural) pollution (Gil-izquierdo et al. 2021; Liu et al. 2021).
These microorganisms can sequester CO2 from flue gas for their photosynthetic
activity and reduce aquatic nutrients efficiently from wastewater at minimal cost
(Song et al. 2019). Some species of microalgae and cyanobacteria show the capa-
bility to fix nitrogen and absorb phosphorus as well as heavy metals from wastewater
(Gonçalves et al. 2017; Singh and Ahluwalia 2013; Satya et al. 2017, 2021a, b; Vija-
yaraghavan and Balasubramanian 2015). Those facts demonstrated that microalgae
and cyanobacteria can provide a promising solution to address emerging environ-
mental problems and concomitantly generate many valuable consumer products.
Conceptually, the production of microalgal and cyanobacterial biomass can be
generated from the carbon recycling process (Fig. 1).
This chapter book discusses the different cultivation, harvesting, and processing
methods of microalgae and cyanobacteria for producing several bioproducts. These
materials involve biofuels, fine biochemicals, and food/functional food. The prospect
of microalgae and cyanobacteria overcoming emerging environmental problems is
also delivered in this chapter book.
146 A. Satya et al.

Fig. 1 Conceptual diagram for producing microalgae and cyanobacteria biomass adapted from
Chisti (2007)

2 Microalgae Cultivation

2.1 Open System

An open system for cultivating microalgae and cyanobacteria is generally found as

an open pond in a variety of shapes and sizes. There are some advantages and disad-
vantages related to the implementation of this cultivation system. Examples of open
pond types are raceway pond with paddle wheels (Fig. 2a), open tanks (Fig. 2b),
shallow big ponds (Fig. 2c), circular ponds, etc. The place where the pond is located
becomes a determining factor for choosing the proper type of pond the selected strain
of microalgal or cyanobacterial being used and the availability of light for photosyn-
thesis. The open pond is a function of the local climate; therefore, the chosen location
will affect the achievement of cultivation. Several key growth parameters (solar irra-
diance, temperature, pH, and concentration of dissolved oxygen) are limiting the
performance of the open pond (Ashokkumar et al. 2014). Another critical problem
for an open pond system is the occurrence of predation due to the higher risk of
contamination. Only several microalgal or cyanobacterial can be grown successfully
in an open pond even in severe environmental conditions (such as Dunaliella in
high salinity, Arthrospira in high alkalinity and Chlorella in high aquatic nutrients).
Dunaliella salina was cultivated for producing carotenoids, this compound in nature
protects this microalga against the high intense sunlight during grown in open pond.
Other reports mentioned that Chlorella sp achieved a decent photosynthetic activity
in raceway system, while Muriellopsis sp. was cultured for producing lutein in an
open tank equipped with a paddle wheel (Blanco et al. 2007). These biomasses can
The Production of Microalgae and Cyanobacteria Biomass and Their … 147

Fig. 2 Open ponds system for microalgal and cyanobacterial cultivation: raceways with paddle
wheel (a); open tanks (b); shallow big pond (c). Both (a) and (b) locates in RC for Limnology and
water resources-NRIA, Cibinong while (c) locates in PT. Albitech, Semarang Regency-Indonesia

be used as food colorant, feed additives in aquaculture, and poultry. Cost for culti-
vating microalga and cyanobacterium is an important factor for considering a choice
between using an open system and photobioreactor. The investment and maintenance
costs in open pond construction is less than photobioreactor, even so the biomass
productivity in open pond is lower than photobioreactor and biomass quality in open
pond is more variable compared to photobioreactor (Ugoala et al. 2012; Debeni Devi
et al. 2022). Consequently, for providing the needs of bulk requirement of biomass
(e.g., for producing biofuel), an open pond form is preferable.

2.2 Photobioreactor System

Photobioreactor provides better control on most operational parameter compared to

open pond system, therefore extensive research on designing photobioreactors for
cultivating microalgae and cyanobacteria is a must (Huang et al. 2017; Duan and
Shi 2014). Higher biomass productivity can be achieved in a controlled environment
which is the advantage of using this system. Careful considerations, however, are
needed since the performance of a bioreactor is assessed from its productivity. These
considerations stem from difficulties in comparing the productivity among photo-
bioreactors because the difference in microalgae strains and scales of the photobiore-
actors used. In the principle, photobioreactor types are differentiated into tubular and
148 A. Satya et al.

plate shapes. The tubular reactors are considered to be more suitable for outdoor culti-
vation. It is because of the use of transparent material for configuring the tubes, which
exhibits large illuminated surfaces. Various configurations can be made depending
on the specification of the system and its purposes. Generally, those tubing configu-
rations may be found in straight-line forms and or coiled forms (Johnson et al. 2018;
Nwoba et al. 2019). Geometry of the photobioreactor also determines its perfor-
mance, as tubular reactors can be configured in inclined, horizontal, or vertical planes.
The vertical design enables better mass transfer and requires lower energy supply.
In the case of the horizontal photobioreactor design, a larger area is required than
vertical design. In terms of scalability, horizontal design is more preferred (Sirohi
et al. 2022; Xia et al. 2013).
Tubular photobioreactors for culturing microalgae and cyanobacteria can be
configured as vertical, horizontal, and helical forms. Another configuration is flat-
plate photobioreactors which are characterized by narrow lightpath and therefore
able to maintain a higher cell concentration up to an order magnitude than another
configuration. Moreover, this configuration type of photobioreactor is favorable since
it allows a lower power energy consumption and high mass transfer capacity. It
also allows a reduction in oxygenic accumulation, providing no dark volume and
high photosynthetic efficiency. A proper photobioreactor design is needed to obtain
the maximum biomass cell production. The flat-plate photobioreactor for culturing
microalgae and cyanobacteria may be constructed in the form of glass, thick trans-
parent polyvinyl chloride materials, V-shaped, and inclined. The translucent mate-
rial gives a maximum light penetration meanwhile other materials for designing are
inexpensive and easy to construct. Figure 3 describes the basic diagram of a tubular
photobioreactor (3a) and a real view of a tubular photobioreactor system on a pilot
scale.

Fig. 3 Schematic basic diagram of a tubular photobioreactor (3a) and a real scene of tubular
photobioreactor at a pilot scale. After Fernandez et al. (2014)
The Production of Microalgae and Cyanobacteria Biomass and Their … 149

The two existing cultivation systems present their own advantages and disad-
vantages with few similarities in operation as seen in Table 1. Optimization of
the two systems can be done by either change of several operative conditions or
even combining the two systems. The goal of combining the two systems is to
gain higher biomass yield with few design parameters. Important design param-
eters for tubular photobioreactors are mixing, gas hold-up, bubble diameter, and
intensity of light and dark cycles. Meanwhile, important design parameters for flat-
plate photobioreactors are mixing, gas hold-up, bubble diameter, light-to-dark cycle
efficiency, and illuminated surface-to-volume ratio (Cui et al. 2021). The factors
affecting missing in flat-plate photobioreactors are much more than that in tubular
reactors. In flat-plate photobioreactors, aside from resident time distribution (RTD)
and circulation time which are present in a tubular reactor’s mixing factor, shear rate
played a bigger role as sheer stress affects cells, nutrients, temperature, and eventu-
ally toxic levels of dissolved oxygens and carbon demands with novel system dealing
with the particular problem showed significant improvement of biomass production
up to 61% (Yaqoubnejad et al. 2021). There are also significantly more hardware
designs needed in developing a tubular reactor. These harder designs in addition to
sparger designs are methods of mixing and pumping. However, the tubular reactor
offered much more sparger designs (orifice, ring, foam types) to allow some enhance-
ment methods. These enhancement methods are the incorporation of static mixers,
static kinetics mixers, helical mixers, or swirl flow for a tangential inlet (Sirohi et al.
2022; Sung et al. 2022). Flat-plate photobioreactors provide easier control in massive
microalgae biomass production but its maintenance and large light areas are their
major bottleneck. For outdoor cultivation, solar-irradiation filtration technologies
should be considered for garnering multiple advantages in addition to combining the
existing systems (Wu et al. 2023; Huang et al. 2023).

3 Harvesting of Microalgae

3.1 Flocculation

Flocculation is the first stage in the bulk harvesting process. This stage aims to
aggregate the microalgae and cyanobacteria cells to increase the effective particle
size for harvest. This method is usually applied as the initial step in harvesting (dewa-
tering) which significantly facilitates the next processing steps. Their effectiveness
depends on their ionic charge. Microalgal and cyanobacterial cells pose a negative
charge, therefore, repulsed themselves from aggregating into suspension. The surface
charge of microalgal and cyanobacterial cells can be neutralized by adding chemi-
cals such as flocculating agents (flocculants). These cationic compounds coagulate
the suspended microalgal and cyanobacterial cells without affecting the composition
and harming the cells and not poisoning the product as can be seen in Fig. 4. The
typical flocculants are multivalent salts such as FeCl3 , Al2 (SO4 )3, and Fe2 (SO4 )3 .
150 A. Satya et al.

Table 1 Advantages and Disadvantages of Tubular and Flat-panel photobioreactors

Types of Tubular reactors Flat-plate reactors
PBRs
Advantages • Lower land needs to produce a • Higher photosynthetic efficiency due to
considerable amount of biomass its more surface-per-volume ratio
• The photostage loop and mixing • Possible and easier optimization with a
ensure higher concentrations of reduction in energy consumption by
biomass cultures factoring in environmental conditions
• Reducing power consumption (inclination, panel spacing, and light
and prevention of cell injury is pathways)
possible by controlling liquid • Comparatively minimal deposition of
velocity from 0.1 to 0.8 ms−1 dissolved O2
Disadvantages • Transmission of mass is lower • Lower biomass per areal yields
with the increase in size • Scale-up process requires a safely
• Prone to oxygen build-up laminated surface
• Optimizing scale-up between • Requires a larger area and one more
width and height design parameter
Similarities • Use of transparent material is • Use of transparent material is preferred
preferred to ensure illuminance to ensure illuminance
• Sparger design is applicable to • Sparger design is applicable to enhance
enhance their mixing effectivity their mixing effectivity
Combined information obtained from Sirohi et al. (2022), Yaashikaa et al. (2022)

Cationic polymers (those are polyelectrolytes) also can be used as flocculants by

physically linking cells together, thus advancing little to no disruptions on the cells.
Key polymer characteristics involved are charge, molecular weight, and concentra-
tion. The preference for polymer types depends on the properties of microalgae or
cyanobacteria cultures such as charge in suspension, pH, and biomass concentration
(Wu et al. 2012). Harvesting using Fe [III] flocs induced with pH improved efficiency
up to 80% (Knuckey et al. 2006). Flocculation using FeCl3 can be suppressed by
exopolymers released by Aphanotece halophytica therefore extra addition of this
flocculant is needed (Chen et al. 2009). Chlorella was found better to be flocculated
using cationic polyelectrolytes, while anionic polyelectrolytes gave no flocculation.
The use of organic flocculants is advantageous due to their stability by being
less sensitive to pH, allowing them for a wide range of applications and in most
cases needing a lower dose of flocculant depending on the presence of ions in
mediums. Brackish and saline waters, for example, needed more chemical floccu-
lants due to the presence of competing cations (Abbaslou et al. 2020). Aside from
cationic ions, natural flocculants can also be used such as in the case of Oscilla-
toria, Spirulina, Chlorella, and Synechocystis flocculated using chitosan. The dose
of given flocculants depends on algal or cyanobacterial species. For example, dose of
40 mg/L chitosan is effective to flocculate Tetraselmis chui, Thalassiosira pesudo-
nana, and Isochrysis sp, while Chaetoceros muellaris required 150 mg/L of chitosan
(Divakaran and Sivasankara Pillai 2002). Auto-flocculation can also naturally occur
by interrupting the CO2 supply in culture microalgal or cyanobacterial broth. This
fact attributes to elevated pH through photosynthetic CO2 consumption related to
The Production of Microalgae and Cyanobacteria Biomass and Their … 151

Fig. 4 Flocculants in aggregating microalgal and cyanobacterial cells. The neutralization provided
by flocculants allowed for microalgae to aggregate

salt precipitations containing calcium, magnesium, phosphate, and carbonate. The

positive charge of calcium phosphate is prone to react with the negative charge of
microalgal or cyanobacterial cells then leads to flocculation (Sukenik and Shelef
1984).

3.2 Centrifugation

The most preferred method for harvesting microalgal and cyanobacterial cells is
centrifugation. Centrifugation is conducted by using centripetal acceleration to sepa-
rate the microalgal growth medium into sections depending on their densities which
correlate with their growth. Mature cells will be obtained in the lower part of the
centrifugation vessel. The separated mature cells of microalgae or cyanobacteria
biomass (supernatant) can later be obtained by simply draining the medium solution
from the saturated supernatant at the bottom of the centrifuge cells. This method
is reasonable for microalgae harvesting with one drawback: the shear forces during
the spinning can disrupt cells. That prevents the faster centrifugation speed which
would generate higher separation capability. There are several key parameters to
look for in applying centrifugation for large-scale harvesting of microalgae. These
factors are concentration which determines the rate of rotation needed to separate;
energy consumption which correlates with the amount of energy needed to power the
rotation; the cost which is how much of a percentage the whole harvesting process
152 A. Satya et al.

would require; operation mode which determines the size of the operation; concen-
trating method which determines whether or not there needs to be a prior concen-
trating method such as cooling or addition of binding agents; and last but not least
is the reliability of the centrifugation method to harvest the biomass. Centrifuga-
tion is the most efficient method for microalgal and cyanobacterial harvesting (95–
100% with 88–100% cell viability) compared to drum filtration and dissolved air
floatation. Centrifugation on a laboratory scale is suitable when cell concentration
is about 30 mg/L. The centrifugation method, however, is not cost-effective when
implemented on a large scale. This is because of its high-power consumption. Other
limitations are its higher gravitational and shear stress frequently damage the cell
structure (Ashraf et al. 2020; Shao et al. 2015).

3.3 Filtration

The filtration method is preferable in harvesting microalgae and cyanobacteria

compared to other harvesting methods. Filtration methods consisted of several types
such as dead-end filtration, microfiltration, ultra-filtration, pressure filtration, vacuum
filtration, and tangential filtration. In the principle, filtration is implemented by
passing through the culture broth through filters then biomass will accumulate on
the filter membrane while the medium passes through the filter continuously until
the filter contains a cake of algal or cyanobacterial biomass (Mohan and Sivasub-
ramanian 2010). To further suitably concentrate the biomass, filter presses under
pressure or vacuum are added into the system for microalgae or cyanobacterium
with larger sizes for example Arthrospira (Spirulina) platensis, but not for smaller
sizes like Chlorella and Dunaliella. Tangential flow filtration and pressure filtration
are considered energy-efficient methods for separating microalgal or cyanobacterial
biomass from its medium culture. This fact is suggested by the amount of output and
input of the feedstock (Shao et al. 2015). Back mixing becomes a drawback to the
use of dead-end filtration; however, this simple filtration method can be combined
with centrifugation for improving the separation process. In general, the filtration
method is frequently associated with wide-ranging running costs and concealed
pre-concentration requirements (Senatore et al. 2022; Morais et al. 2020).

4 Bioproducts from Microalgae

4.1 Biodiesel

Generation of biodiesel is a process of breaking down vegetable oils or animal fats that
contain triglycerides which comprise of three fatty acid chains linked with a glycerol
molecule. In the process of generating biodiesel, glycerol substitutes with methanol
The Production of Microalgae and Cyanobacteria Biomass and Their … 153

which forms fatty acid methyl ester more commonly known as biodiesel. A phase
separation method was then implemented to separate glycerol (as a by-product) from
biodiesel. The process is denoted as transesterification which is a process of replacing
methanol for glycerol in a chemical reaction with an acid or alkali catalyst. The
encounters of replacing conventional diesel with biodiesel are: (1) biodiesel feedstock
must be sufficient at a commercial scale, (2) must have a lower price than conventional
fossil fuel, and (3) meet standard specifications of fuel quality. Those reasons are
met by the microalgal and cyanobacterial biomasses for biofuel since it can provide
raw material at a cheaper yet faster biomass productivity rate reaching up to 50 times
magnitudes than ordinary terrestrial plants with adequate lipid contents fraction for
biodiesel. Their lipids are mostly neutral lipids with a lower level of unsaturated
grade. According to Chisti (2007) Botryococcos braunii, Nannochloropsis sp, and
Schizochytrium sp. respectively contain lipids ranging between 25–75%, 31–68%,
and 50–77%, respectively.
Many methods for algal/cyanobacterial lipid extraction are known, but the most
commonly practiced methods of algal/cyanobacterial lipid extraction are by the use
of an expeller, supercritical fluid extraction (SFE), solvent extraction (liquid–liquid
extraction), and various ultrasound techniques. In the method of expeller/oil press, the
biomass of microalgae must be dried for effective extraction, cell breaking was then
conducted by pressure which squeezed the oil out. In addition to that, this method was
only capable to extract 75% of oil with a longer extraction time. Solvent extraction
is excellent to extract from microalgae and cyanobacteria, faster and simpler than
the SFE method (Jacob-Lopes and Franco 2013).
In the solvent extraction method, organic solvents (benzene, cyclohexane, hexane,
acetone, or chloroform) are added to algal/cyanobacterial paste. The solvent’s func-
tion is to destroy algal/cyanobacterial cell walls which allows the extraction process to
ensue. The extraction will result in the formation of a lipid layer on top of the aqueous
medium since their higher solubility in organic solvents than in water (medium). Then
the solvent extract can be distilled to separate the oil from the solvent. The solvent
can later be reused. Among the reusable solvent in this process, Hexane is the most
efficient solvent due to its lower cost and higher extraction capacity. Lipid extrac-
tion also can be conducted in two steps using ethanol then followed secondly with
hexane. This procedure is aimed to purify extracted lipids with a yield recovery of
80%. Temperatures can also improve this extraction method. Some studies reported
that fatty acids were always nearly extractable at 100 °C compared to ambient temper-
ature mainly saturated acids (16:0, 18:0), but polyunsaturated fatty acids (18:2;18:3)
or PUFA resulted in lower yield with hot propanol-water (3:1 v/v). However, fatty
acids content varied with microalgal strains, and the solvents for extraction (such as
chloroform, and methanol) were also hazardous and destructive to the environment
and human health (Slade and Bauen 2013).
Supercritical extraction (SFE) ruptures the microalgal cells through high pres-
sures and temperatures. This method is extremely time efficient and is commonly
used. The implemented high pressures and temperatures did not give any effect on the
yield of the extracted compounds but affected the rate of extraction. It was observed
in lipid extraction from Nannochloropsis sp for obtaining PUFA using SFE at 45 and
154 A. Satya et al.

55 °C, 400–700 bar. A higher yield result was found when SFE was used in Spir-
ulina platensis for obtaining PUFA compared to extraction using solvent (Amorim
et al. 2020; Bleakly and Hayes 2017). Ultrasound method for lipid extraction from
microalgae and cyanobacteria is also promising. This method treats microalgae and
cyanobacteria with high-intensity of ultrasonic waves which form minute cavitation
bubbles around cells. The shockwaves resulting from collapsing bubbles will shatter
and rupture the cells which then released desired compounds into solution. Fatty acids
and pigment extraction from Scenedesmus obliquus using ultrasound showed over
90% without any changes or breakdown in the product (related to time storage), while
almost complete extraction of lipid was achieved in Chaetoceros gracilis. Ultrasonic
can increase the rate of extraction of oil content in microalgae at a laboratory scale,
further study on its feasibility for commercial production in this method is needed
(Vandamme et al. 2013; Kumar et al. 2017).

4.2 Bioethanol

Bioethanol is commonly produced through a biochemical process (fermentation)

and thermochemical process (gasification) of biomass sources. The conventional
biomass sources (sugar cane, corn, and bit) have a general acute problem that is
high value for food utilization and the requirement on land to be cultivated. There-
fore, this problem is a constraint for expanding biofuel production. Alternatively,
microalgal and cyanobacterial biomass as feedstock for the fermentation process
in bioethanol production eludes those problems. These biomasses contain carbo-
hydrates and proteins (Table 2) which enable being used as carbon sources in the
fermentation process.
Other microorganisms such as fungi, bacteria, and yeast (Saccharomyces
saravesei) are ordinarily used for fermenting the microalgal and cyanobacterial
carbohydrates under anaerobic conditions for the production of bioethanol. Theo-
retically, the maximum yield is 0.51 kg ethanol and 0.49 kg CO2 per kg of glucose.
The produced bioethanol then can be purified for producing biofuel while produced
CO2 can be recycled for cultivating microalgae as a growth nutrient source. In the
second stage, the remaining biomass after fermentation can be used as feedstock for
the anaerobic digestion process resulting in methane (CH4 ) gas which can later be
converted into electrical power (Demirbas 2010, 2011).
Hon-Nami (2006) mentioned that Chlamydomonas periglanulata can be
fermented for producing ethanol, butanediol, acetic acid, and CO2. They also reported
that the recovery of H2 and carbon was attained by 139 and 105%. Some advan-
tages of microalgal and cyanobacterial fermentation for producing bioethanol are
the less requirement for energy consumption and more simplicity of the process
compared to the conventional biodiesel production system. Even so, the production
of bioethanol from microalgal and cyanobacterial biomass still needs further research
for commercialization.
The Production of Microalgae and Cyanobacteria Biomass and Their … 155

Table 2 Carbohydrates and Proteins (%dry weight) in several Microalgal and Cyanobacterial
biomasses
Cyanobacteria Carbohydrates (% dry weight) Proteins (% dry weight)
Spirulina platensis 8–14 46–63
Spirulina maxima 13–16 60–71
Synechoccus sp. 15 63
Anabaena cylindrica 25–30 43–56
Microalgae
Scenedesmus obliquus 10–17 50–56
Scenedesmus quadricauda – 47
Scenedesmus dimorphus 21–52 8–18
Chlamydomonas reinhardtii 17 48
Chlorella vulgaris 12–17 51–58
Chlorella pyrenoidosa 26 57
Spirogyra sp. 23–64 6–20
Dunaliella bioculata 4 49
Dunaliella salina 32 57
Euglena gracilis 14–18 39–61
Prymnesium parvum 25–33 28–45
Tetraselmis maculata 15 52
Porphyridium cruentum 40–57 28–39
Modified from Becker (1994)

4.3 Biomethane

The methane fermentation technology on microalgae Chroococcus sp. and

Tetraselmis sp. Chlorella sp. (Koutra et al. 2018) and cyanobacteria (Aphanizomenon
ovalisporum and Anabaena planktonica) biomass are perspective since they can
produce economical by-products. One of these by-products is biogas (Catone et al.
2021). Biogas consists of a mixture of 55–75% of CH4 and 25–45% of CO2
during microbial anaerobic digestion. The CH4 can later be converted into elec-
tricity and fuel gas. As for the residual biomass that is left after the process,
they can be processed into biofertilizer. Therefore, these processes yield support
renewable and sustainable agricultural production systems by improving efficient
practices and lowering microalgal/cyanobacterial production costs. Microalgae and
cyanobacteria are considered to have no lignin and lower cellulose ingredient, thus
processes of turning their biomass would be considered faster than conventional
biomass sources. This character gives excellent conversion efficiency and stability
for the anaerobic digestion process. In the anaerobic digestion method, the biogas
production from microalgal/cyanobacterial biomass is determined by its organic
loadings, temperature, pH, and retention time in the bioreactor. In the principle,
156 A. Satya et al.

long solid retention and high organic loading can significantly affect CH4 yield. This
method can be performed in either mesophilic or thermophilic conditions. Integration
between microalgae and cyanobacteria cultivation in a wastewater treatment pond
and harvested its biomass anaerobically digested for producing biogas can offer good
potentials in overcoming environmental problems (water pollution) and commercial-
izing biogas production from microalgal and cyanobacterial biomass (Ramos-Suárez
et al. 2014; Tijani et al. 2015).

4.4 Fine Biochemicals

Carbohydrate content in microalgae and cyanobacteria biomass has the potential in

Acetone-Butanol fermentation (fermented using bacteria such as Clostridium sp) for
producing biobutanol with bio acetone as a by-product. These fine biochemicals are
valuable organic solvents. Biobutanol belongs to renewable transportation fuel, while
bio acetone is utilized as a multi-purpose solvent such as a cleaning agent, an extrac-
tion solvent, and other laboratory works. Applying biobutanol as fuel for vehicles is
reported to not require any engine modification as it can be directly blended in higher
concentrations with gasoline compared to other biofuels. Blending biobutanol with
gasoline is aimed to lower vapor pressure. Production of butanol with Neochloris
aquatica CL-M1 was done by using wastewater medium to yield 0.89 g/(L.h) of
butanol with 96.2% efficient removal of NH3 -N. This process’ success in yielding
butanol meant circular usage for extraction is possible by using the produced butanol.
Meanwhile, on the genus of Dunaliella (D.tertiolectra, D.primoelectra, D.parva,
D.bardawil, and D.salina) fermented by Clostridium pasteurianum was found to
yield four different kinds of organic solvents. These four organic substances namely
propanediol, acetic acid, ethanol, and n-butanol were present as a mixture with a
concentration of 14–16 g/L. However further study still needs to be conducted related
to the mechanism of the process (Veza et al. 2021; Nakas et al. 1983).

4.5 Food and Functional Foods

4.5.1 Omega 3 Oil

Naturally, microalgae and cyanobacteria contain omega-3 fatty acids that can be
purified into high-value added bioproducts such as food supplement. The sources of
omega-3 fatty acid in microalgae and cyanobacteria biomass are eicosapentanoic acid
(EPA) and docosahexaenoic acid (DHA). These compounds are widely found in fish
oil, but due to its low supply, unpalatable taste, and inadequate oxidative stability
of fish oil made it not a convenient source for omega-3 fatty acids. Microalgae
and cyanobacteria are self-producing omega-3 fatty acids, therefore processing on
microalga biomass is simpler than fish biomass. The use of EPA is widely known
The Production of Microalgae and Cyanobacteria Biomass and Their … 157

in human health purposes for curing heart and inflammatory diseases (asthma,
arthritis, migraine, and psoriasis). According to Hu et al. (2008) outdoor cultivation
of Pavlova viridis gave lower total fatty acid but higher EPA than of indoor cultiva-
tion system, hence concluded that outdoor cultivation is more favorable in producing
EPA. Nannochloropsis sp. also show similar result according to Cheng-Wu et al.
(2001). Production yields of EPA are also determined by the season. It was found
that the production of EPA is higher as 35% in summer than in winter. Temperature
and irradiance were not significantly impacting on yield of EPA (4% of dry biomass)
produced by Nannochloropsis sp. in an outdoor tubular reactor, and this report showed
the potential of this eustigmatophyte as an alternative source of EPA (Chini Zittelli
et al. 1999). Vazhappily and Chen (1998) reported that the highest EPA proportion
(% of total fatty acids) was produced by Monodus subterraneus UTEX 151 (34.2%),
followed by Chlorella minutissima UTEX 2341 (31.3%) and Phaeodactylum tricor-
nutum UTEX 642 (21.4%). But further studies are still needed to ensure the feasibility
of this EPA production system. In terms of DHA, this compound is also useful to
fight against cancer, AIDS, and heart diseases by reducing cholesterol, boosting
immune system, and detoxification of the body. Production of DHA depends on
the cultivated species of both microalgae and cyanobacteria. Marine species of both
microalgae and cyanobacteria have significantly higher DHA (mainly consist of satu-
rated or monosaturated fatty acids) content than freshwater species (Patil et al. 2007).
Marine microalga, Schizochytrium mangrove, contains DHA in the range of 33–39%
of total fatty acids (Jiang et al. 2004), whileVazhappily and Chen (1998) reported
that the highest DHA proportion (% of total fatty acids) was obtained in Crypthe-
codinium cohnii UTEX L1649 (19.9%), followed by Amphidinium carterae UTEX
LB 1002 (17.0%) and Thraustochytrium aureum ATCC 28211 (16.1%). Another
report by Patil et al. (Patil et al. 2007) mentioned that Isochrysis galbana contained a
significant amount of DHA with a specific productivity of around 0.16 g/(L.d). The
amount of CO2 , light intensity, and operation modes (batch and continuous) signif-
icantly affect the productivity of DHA (which was found of 1.29 mg/(L.d) under
optimized conditions in the cultivation of microalga Pavlova lutheri (Carvalho and
Malcata 2005).

4.5.2 Chlorophyll-a

In microalgae and cyanobacteria, there are mainly two main types of chlorophyll
being produced. They consist of chlorophyll a and chlorophyll b. The presence of
chlorophyll as a photosynthetic pigment is found in all photoautotrophic organisms.
Chlorophyll has been widely used as a medicinal drug due to its ability to stimulate
liver function recovery and increase bile secretion. It also possesses the ability to
repair damaged cells, increase the amount of hemoglobin in blood and encourage
rapid cell growth. Chlorophyll has also been reported to have various properties
(antimutagenic, anticarcinogenic, and antioxidant). Traditionally, chlorophyll has
been used in the food industry as a natural pigment due to the increasing consumer
demands for natural foods. Its use as a natural pigment possesses the same properties
158 A. Satya et al.

as listed previously. Chlorophyll is best extracted by the use of the supercritical fluid
extraction (SFE) method. High-performance liquid chromatography was found to
be the most accurate and sensitive technique to fractionate and quantify chlorophyll
along with its derivatives (Silva and Sant’Anna 2016).
Microalgae (Chlorella sp, Scenedesmus sp) and cyanobacteria (Spirulina sp.)
seem to be promising alternative sources for chlorophyll. The growth stage of a
particular algal species was found to be highly linked with the amount of chlorophyll
extracted. Microalgae extracted during the stationary growth phase were shown to
have a substantially higher amount of chlorophyll as compared to the same species
extracted during the logarithmic phase. Chlorophyll a has been recently revealed to
be a key compound in the treatment of ulcers which makes it vital in the postoper-
ative treatment of rectal surgery on humans. During the removal of large areas of
tissue, recovery can be difficult and the area near said removed tissue tends to be
painful. With the application of chlorophyll, the stimulation of cells in the host and
the consequent acceleration in tissue formation increases the rate of recovery, in many
cases up to 25%. Moreover, the use of chlorophyll was also found to eliminate foul
odor emanating from the wound after a few administrations. Chlorophyll’s non-toxic
nature, antibacterial properties, and ability to deodorize make it a prominent product
in treating oral sepsis (Stirbet et al. 2018). In conclusion, a downstream process needs
to be developed to purify chlorophyll a and b from microalgae and cyanobacteria.

4.5.3 Phycocyanin

Phycocyanin is a colorant present in cyanobacteria and red microalgae. When

purified, phycocyanin shows a brilliant blue color in the solution. Phycocyanin is
composed of two different subunits α and β combined into one. Its presence in
nature exists as monomers, trimers, or hexamers; small quantities of oligomers have
been found as well. In general, phycocyanin includes C-phycocyanin and allophy-
cocyanin. Both possess different maximum absorption peaks, which is at 620 and
650 nm, respectively. Studies have suggested that both C-phycocyanin (C-PC), and
allophycocyanin each have their own ratio of absorbance to indicate their purity;
with C-PC possessing a ratio of 620 and 280 nm, and allophycocyanin possessing a
ratio of 650 and 280 nm. Phycocyanin shows numerous special bioactivities which
are gradually recognized to have potential as raw materials for healthy food prod-
ucts. Studies have indicated that phycocyanin also has various bioactivities such as
anti-neoplasm, antioxidant, and anti-inflammatory effects. Several reports have also
suggested that C-PC has anticancer bioactivity.
The high content of protein in Arthrospira (Spirulina) sp. (~70% in dry cell weight)
poses as a valid reason to consider cyanobacteria as an alternate source of protein,
which is vital due to its presence at several cell locations as enzymes, structural
component, linked carbohydrate, among others under various forms. Phycobilipro-
teins correspond to nearly 60% of all the soluble protein in cyanobacteria among
the protein pool, while about 20% corresponds to C-PC. Pigment purity within the
The Production of Microalgae and Cyanobacteria Biomass and Their … 159

cultivation of microalgae is of utmost importance, especially when it’s used as a fluo-

rescent marker in biomedical research where the presence of impurities can severely
impair the quality of the extract. Extract of C-PC obtained from A. platensis which
was grown in a nitrogen-reduced medium indicated higher levels of purity (0.80)
followed by the extract cultivated within a control group (0.55) and lastly within a
nitrogen-free medium (0.21). Several studies have demonstrated crude extraction of
C-PC (with varying levels of purity between 0.19 and 1.4) extracted from A. platensis.
What is considered to be food grade C-PC has a purity of 0.7 (with a commercial
value of ~US$ 0.13 per mg), while 3.9 is considered as reactive grade C-PC (value
varies ~US$ 5 per mg) and a purity level of greater than 4.0 as analytical grade C-PC
(value can be as high as US$ 15 per mg. All in all, C-PC obtained from A. plantesis
grown within a nitrogen-reduced medium presents a very promising use within the
food industry (Qiang et al. 2021; Pagels et al. 2019).

4.5.4 Carotene

Carotenoids (Carotene) are known as one major class of photosynthetic pigments.

In microalgae, there are generally three known pigments which are chlorophyll,
phycocyanin, and carotenoids. Carotenoids, like chlorophyll, are water-soluble. They
consisted of terpenoid pigments derived from a 40-carbon chained polyene. This
distinctive molecular structure is associated with their chemical properties which
allowed electron transfers induced by light-absorption which is essential in photo-
synthetic activities. This pigment may be complemented by cyclic groups and oxygen
functional groups. The oxygenated derivatives are particularly known as xanthophylls
as there is the presence of hydroxyl groups (e.g. lutein), oxy groups (e.g., canthaxan-
thin), or both combinations (e.g. astaxanthin). However, carotenoids are usually typed
as two, namely primary and secondary carotenoids. Primary carotenoids come from
their structural and functional components in the cellular photosynthetic apparatus
(i.e., xanthophylls). Meanwhile, secondary carotenoids consisted of those produced
at a large level after exposure to specific environmental stimuli. Relatively, xantho-
phyl are hydrophobic therefore, they are typically found linked on the membranes or
noncovalently bound to specific proteins (Srivastava et al. 2022; Begum et al. 2016).
There are more than 400 variants of carotenoids within nature. Among them, β-
carotene is considered to be the most prominent. Moreover, some carotenoids contain
provitamin A and possess a broad range of biological functions and actions, most
notably in relation to human health (Pisal and Lele 2005). Researchers have reported
the benefits of β-carotene for the human body as the human body converts β-carotene
to vitamin A via the body tissue. The necessity of vitamin A in the immunity of the
human body is to prevent cataract, night blindness, and skin diseases. In the context
of multivitamin preparations, β-carotene is often used as pro-vitamin A (retinol) and
as an ingredient in the formulation of healthy foods. Alternative use of β-carotene
within the context of food production, is as a food colorant to improve the appear-
ance of margarine, cheese, fruit juices, confectionary, and other food products to
increase appeal toward customers, such as the case of the β-carotene cultivated from
160 A. Satya et al.

Dunaliella. β-carotene has also been reported to decrease the hazard of several degen-
erative diseases such as cancer (Nethravathy et al. 2019). Studies have found that
β-carotene from Dunaliella sp. contains 40% 9-cis and 50% all-trans stereoisomers
which play a crucial role in lowering incidence of several varities of cancer and other
degenerative diseases. Furthermore, an investigation of the antioxidant properties of
β-carotene was found allowing it to help mediate the harmful effects of free radicals
thus preventing life-threatening diseases such as arthritis, coronary heart diseases,
premature aging, and various forms of cancer. Another study has also shown that
β-carotene has the ability to stimulate the immune system, potentially preventing
various kinds of life-threatening diseases. In addition, it can also reduce the cogni-
tive impairment linked with Alzheimer’s which is caused by persistent oxidative
stress within the brain (Nethravathy et al. 2019; Murthy et al. 2005).

4.6 Microalgae for Phytoremediation

Phytoremediation is a process of remediating environmental contaminants or excess

nutrients through the use of plants. Phytoremediation usually focuses on controlling
Total Phosphorus (TP), Total Nitrogen (TN), and their related constituents such as
− +
PO3−4 , NO3 , NH4 , and much more which can be seen as Total Dissolved Salts
(TDS). Conventionally, complex plant systems available in the environment espe-
cially in aquatic environments are used to remediate environmental contaminants.
Phytoremediation often uses available plants with little value in the market such
as common reeds, water lilies, and pteridophytes (Pandey 2012; Wang et al. 2022;
Riggio et al. 2015). The downside of the conventional route is the process of devel-
oping enough biomasses to effectively remediate an area required years of accli-
mation and cultivation in the environment. In the wake of sustainable development
and circular economy, phytoremediation using microalgae and cyanobacteria fits
the criteria. In developing a sustainable business, the technology applied should not
further factor into the depletion of arable lands and not compete with the existing
Food-Energy-Water nexus (Olabi et al. 2023). The adaptive capability and structural
simplicity of microalgal and cyanobacterial cells make them a perfect phytoremedia-
tion agent. Owing to their adaptive capability, microalgae can live in numerous condi-
tions depending on the strains and their evolutionary pathways in combating extreme
conditions. A class of microalgae is even equipped with an additional protective layer
called frustules to accommodate their cells to live in extreme heat or sudden temper-
ature changes (Kooistra et al. 2007; Kim et al. 2017). This adaptability of microalgae
and cyanobacterial cells also resulted from their cellular simplicity which allowed
for an optimum growth rate attained in a shorter period than complex plants.
In determining the process for microalgae or cyanobacteria phytoremediation,
there are three main factors to consider. These factors are interconnected and serve
as important determinants in the growth of algae and thus the success of phytoreme-
diation. These considering factors are Environmental, Biological, and Operational
factors (Nie et al. 2020). The connections between them go as follows: Environmental
The Production of Microalgae and Cyanobacteria Biomass and Their … 161

conditions provide biological activities, biological activities ensure the optimum

operating conditions are chosen, and the chosen operating conditions are chosen
to sustain the environmental conditions (Fig. 5). Nine different genera of Chloro-
phyta phylum: Asterarcys, Chlorella, Chloroccoum, Chlorosarcinopsis, Coelastrella,
Desmodesmus, Micratinium, Parachlorella, and Scenedemus are often considered
the native to a freshwater environment involving aquaculture (Couto et al. 2022).
Their removal capacity has been studied in Galicia and continental Spain (without
Coalastrella, Asterarcys or Parachlorella genera as they are not usually present in
their freshwater streams) and showed removal efficiencies of 99, 92, and 49% for
ammonium, nitrite, and nitrate, respectively, on aquaculture-derived effluents in a
raceway pond with microalgae biomass production around 30–40 mg/L on the 7th
day.
Aside from Chlorophyta, Bacillariophyta (Diatoms), Cyanophyta (blue-green
algae), and Chrysophytae (golden algae) are the most abundant microalgae classes
in aquatic ecosystems (Vieira et al. 2020). In a present study of treating munic-
ipal wastewater with three native microalgae species (Navicula veneta -Diatom-,
Chlorella vulgaris -Chlorophyta-, and Nostoc muscorum-Cyanophyta-), the Navicula
veneta treatment was found to produce reusable effluent with high-rate removal of
COD, TP, and TN by 95.75%, 99.8%, and 96.96%, respectively (Sisman-Aydin
2022).
Microalgae phytoremediation does not require large areas with easier controlla-
bility as the internet of things (IoT) can be incorporated into monitoring its growth and
even the environment in a working system (Peter et al. 2021; Abdul-Hadi et al. 2013).
Recent research also suggested a phytoremediation system that included energy

Fig. 5 Factors at play in microalgae & cyanobacteria phytoremediation

162 A. Satya et al.

production. This was done by the construction of microbial fuel cells (MFCs) where
microalgae functioned as biocathode (Mathuriya et al. 2016). The energy conver-
sion efficiency of microalgae MCFs showed a maximum output of up to 9% while
other photosynthetic plants were at 4.6–6% (Shukla and Kumar 2018). Generally,
the external resistance of this system is at 1000 Ω with pollutant removal focusing on
Chemical Oxygen Demand (COD). There seems to be a relationship between working
volume and the type of wastewater playing a role in the maximum power density
as seen in recently published data regarding wastewater treatment and maximum
bioelectricity in Table 3 (Sharma et al. 2022). This type of phytoremediation is
regarded as a complete recycling machine (Greenman et al. 2019) with the potential
for high-yield hydrogen gas production (Logan et al. 2008). Further improvement
in phytoremediation using microalgae and cyanobacteria cells was also done by the
introduction of immobilized systems in wastewater treatment to improve retention
time in optimizing nutrient capture (Han et al. 2022; Shen et al. 2017).
Phytoremediation of microalgae usually utilized the use of an open pond cultiva-
tion system. That, however, resulted in lower biomass yield which is why the develop-
ment of tubular and flat-panel photobioreactors in bioremediation is of interest (Luo
et al. 2017). The system would require flue gas submersions into the photobioreactor
allowing for the capture of CO2 from the environment-utilizing the sequestration or
CO2 fixing pathways which have been done in a consortium with Clostridium sp.,
E.coli, and Saccharomyces cerevisiae (Hu et al. 2019). Sequestration of CO2 for the
valorization of waste mitigation has also been analyzed for massive algal biomass
production which showed positive outcomes with the integration of (Ma et al. 2022;
Yadav et al. 2019). In addition to sequestration, microalgae are also reportedly able
to interact with known emerging contaminants such as heavy metals, antibiotics, and
microplastics which all resulted in oxidative stresses that either boost or decrease
their lipid content or antioxidant levels to adapt to the surrounding (Satya et al. 2023).
These positive outcomes are overall improved cradle-to-gate approach of microalgae
biorefineries to renew the current economic model, sustainable recycling of water
and supporting food security as well as energy with their biorefineries, and helping
economic growth in tropical countries while improving or saving their environment
(Hosseinizand et al. 2017; Wu et al. 2018; Hossain et al. 2019).

5 Conclusion

Production of microalgae and cyanobacteria biomass is the new key to a sustain-

able future. The biomass generation is higher than that of conventional plants yet
requires smaller land to cultivate with their applicable closed cultivation system
using photobioreactors. The photobioreactors also ensure the quality of the biomass
and biorefineries are safe for even consumption levels. There are many potentials in
the biomass of microalgae. As bioproducts (bioethanol, biomethane, and biodiesel),
microalgae and cyanobacterial biomass have unique properties which are their higher
lipid levels with a faster rate of growth than conventional biomass sources. As they are
Table 3 Microalgae-MFCs for Phytoremediation
Wastewater type Location Microalgae-electrode Working volume (mL) External resistance Pollutant removal Maximum power
( Ω) density (mW m−3 )
Industrial Denmark Chlorella vulgaris with 200 1000 COD (66.8%), TN –
wastewater Ti-electrode mesh (69%), TP (48.5%)
coated with platinum/
carbon
Synthetic India Chlorella sorokiniana 300 1000 COD (95%) 2320
wastewater with carbon felt
Synthetic India Chlorella vulgaris with – 700 COD (96%), NH4 + 33.14
wastewater graphite plate (85%), PO3− (69%),
4
NO−
3 (68%)
Synthetic Thailand Chlorella vulgaris with 1000 1000 COD (71%), NH+
4 199.12
wastewater carbon cloth (79%)
Domestic Iran Chlorella vulgaris with – 1000 – 126
wastewater stainless steel
Pharmaceutical India Scenedesmus abundans – 100 COD (97.24%), TN 838.68
wastewater with Graphite rods (97.12%), TP (93.71%)
The Production of Microalgae and Cyanobacteria Biomass and Their …

Kitchen India Synechococcus sp. and 250 – COD (68%) 41.5 (Synechococcus
wastewater Chlorococcum sp. with sp.)
plain graphite plate 30.5 (Chlorococcum
sp.)
Adapted from Sharma et al. (2022)
163
164 A. Satya et al.

simple cells, the cost of separating the required biomass from contaminants (stems,
leaves, etc.) is relatively cheaper. The same can be said in applying microalgae as a
living cell in phytoremediation, replacing the conventional phytoremediators which
needed a longer time to acclimate to the conditions. The water conditions coming
from microalgae phytoremediation have also been shown to be within the permissible
limit of water reuse. The cradle-to-gate approach of using microalgae and cyanobac-
teria products also showed higher interest in the availability of omega-3 oils and
colorants (Phycocyanins) which are in high demand for food industries and the new
sustainable industries that follow.

Acknowledgements The authors would like to acknowledge PKR-Biomassa and Biorefinery

UNPAD-BRIN for funding the publication of this chapter book.

Author Contributions Awalina Satya, Azalea Dyah Maysarah Satya, and Tjandra Chrismadha
were the main contributors (data curation, writing original draft); others were supporting contribu-
tors: Nofdianto Nofdianto (review), Gunawan Gunawan (review), Ika Atman Satya (review), Souvia
Rahimah (review), Efri Mardawati (review), Sara Kazemi Yazdi (review), and Pau-Loke Show
(review).

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An Overview on Managing Minute
Duckweed (Lemna Perpusilla Torr)
Cultivation for Fish Feed Purpose

Tjandra Chrismadha, Awalina Satya, Ika Atman Satya,

Azalea Dyah Maysarah Satya, Sara Kazemi Yazdi, and Efri Mardawati

Abstract Feed is currently becoming a major problem in developing aquaculture

sector. The use of aquatic plants has been pointed out for the resolution, as they
can be easily produced while the nutrition value is reasonably good. One aquatic
species, minute duckweed (Lemna perpusilla Torr.), is believed to be the solution.
This species is abundantly distributed in tropical eutrophic waters. Although there has
been some success of feeding fish on aquatic plants reported since a long time ago,
little progress has been made for field implementation, as it has been hampered by the
lack of knowledge on the proper and harmonious management of plant production
to fulfill the fish requirement. This paper discusses a strategy to optimize the use of
minute duckweed for feeding Nile tilapia fish. A scheme of integrated multitrophic
aquaculture (IMTA) is proposed as a means of enhancing low-cost feed produc-
tion, while simulations are performed to figure out the quantitative interconnection
between the two commodities. Mass implementation of this integrated aquaculture
scheme in Indonesia, however, faces a major problem of short land ownership, so
institutional development is strongly needed, to encourage a segmentation farming
activity as well as to set a fair play regulation that assures their business continuity.

Keywords Aquaculture · Alternative feed · Minute duckweed · Lemna perpusilla

Torr

T. Chrismadha · A. Satya (B)

Research Centre for Limnology and Water Resources, National Research and Innovation Agency,
KST Soekarno, Cibinong, Indonesia
e-mail: [email protected]
I. A. Satya
Research Centre for Computation, National Research and Innovation Agency, KST Soekarno,
Cibinong, Indonesia
A. D. M. Satya · S. K. Yazdi
Department of Chemical and Environmental Engineering, Faculty of Science and Engineering,
University of Nottingham Malaysia, Selangor Darul Ehsan, Jalan Broga, 43500 Semenyih,
Malaysia
E. Mardawati
Department of Agro-Industrial Technology, Faculty of Agro-Industrial Technology, Padjadjaran
University, Jatinangor, Indonesia

© The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2024 171
M. A. R. Lubis et al. (eds.), Biomass Conversion and Sustainable Biorefinery, Green
Energy and Technology, https://doi.org/10.1007/978-981-99-7769-7_8
172 T. Chrismadha et al.

1 Introduction

The current major problem of fisheries aquaculture development is the high price of
artificial feeds that lowers the benefit as well as the economic feasibility. It is mainly
associated with a limited supply of protein sources while the price is considerably
high, and up to now becomes the strategic issue that remains unresolved (Ogello et al.
2014; Journey W et al. 1993; Hartog and Plas 1970). Finding out alternative feed
sources, particularly those related to low-cost sources of protein is very important
to encourage the development of a sustainable and environmentally sound fisheries
aquaculture (Ogello et al. 2014; Chrismadha et al. 2012; Pradhan et al. 2019).
Efforts to reduce the production cost can be directed by the utilization of any
resources that cannot directly become human consumptions or marginal products
with lower prices than the feed being produced (Arief et al. 2011; De et al. 2010).
There have been many explorations works carried out, on some marginal resources
with high biomass productivity and suitable nutrition value for alternative feeds (De
et al. 2010; Satya et al. 2022; Said et al. 2022; Elangovan et al 2017). The most
important fish feed purpose criteria are preferable and digestible, as well as good
nutrition value, which generally assessed in terms of high protein substances with
complete amino acids composition, while the fiber and antinutritional content are
low (Helfrich and L a. 2002; Chakrabarti et al. 2018) One among resources that has
many attentions for alternative feed is minute duckweed (Lemna perpusilla Torr.)
(Chrismadha et al. 2012; Andriani et al. 2019).

2 Minute Duckweed (Lemna Perpusilla Torr.)

Minute duckweed belongs to the family of Lemnaceae (Goopy and Murray 2003; Les
et al. 2002). There are two subfamilies in this group: Lemnoideae and Wolffiodeae are
distinguished by the root existence, whereas the group of Wolffiodeae don’t have any
root. Lemnoideae consists of three genera: Lemna, Landoltia, and Spirodella which
are all known as ‘duckweeds’, while Wolffiodeae consists of two genera: Wolffia
and Wolffiella which are commonly known as water meals. Up to this time, there are
14 Lemna species identified worldwide (Les et al. 2002; Sree et al. 2016) and the
species commonly found in Indonesia is L. perpusilla, which is recognized under
local name of ‘Matalele’, while the global name is ‘minute duckweed’ (Goren et al.
2021; Ekperusi et al. 2019). This individual plant consists of only one flatted-oval
form fake leaf, which is actually a composite of stem and leaf and is commonly called
‘frond’, and a root line prolongs from the frond base (Sree et al. 2016; Hartog and
Plas 1970). The frond is composed of chlorenchymatous cells structure separated
each other by aerial space providing buoyancy for floating on the surface of water.
The major components distinguishing minute duckweed from other Lemna
species are the occurrence of three thickening lines spread out from the base near
root to the opposite edge of frond which is called ‘venae’, and also the existence
An Overview on Managing Minute Duckweed (Lemna Perpusilla Torr) … 173

Fig. 1 A clump of minute duckweed (left) and root sheath and venae as the specific marks
(Chrismadha et al. 2012)

of triangle shaped membrane called ‘root sheath’ at the root base (Fig. 1). This
species has a relatively small size, with maximum frond length of only 8 mm and
get smaller under unfavorable growth condition. The root is very short under good
growth condition and prolonged as long as 5 cm when suffering from nutrient limi-
tation. One individual plant can produce up to 20 broods during 10 days of life
span. The offspring frond usually emerges from the bud pockets located at the two
opposing side just near the root base, and attach to the mother frond for several days,
so the plant commonly looks like a clump of 2–3 fronds. Minute duckweed actually
belongs to flowering plants (Spermatophytes), reproduces generatively by emerging
flowers irregularly depending on the growth condition, and delivers seeds that are
tolerant to long severe dry condition that will sprout up soon when the condition gets
favorable.
Minute duckweed distributes broadly in fertile water of low- to middle-altitude
tropical area (Acosta et al. 2021) Although it is also found in a relatively high inland
water, such as in Samosir District of North Sumatera (>900 m above sea level),
it possibly grows slower than that in the low land area. This tropical species has
a close taxonomic relationship with common duckweed (L minor) which grows
in cosmopolitan around the temperate area. They are only differentiated by the root
sheath occurrence in minute duckweed, while no such root sheath is found in common
duckweed (Pancho and Soerjani 1978). Those two species love highly fertile stagnant
water, and are frequently found to form a green mat on the surface of organic contam-
inated waters, including ponds, swamps, irrigation and drainage channels, as well
as slow moving river waters. It is commonly found to have a symbiotic growth with
other floating aquatic plants, such as water meals, water ferns, and water hyacinth.
174 T. Chrismadha et al.

3 Integrated Multitrophic Aquaculture (IMTA)

Integrated multitrophic aquaculture (IMTA) is aquaculture activities involving

various aquatic organisms set together in a harmonious way according to their trophic
properties, and directed to enhance more effective space and water resources, as well
as feed utilization. IMTA uses an ecological approach to encourage bio circular
concept, which is to utilize wastes from aquaculture activities to produce some
commodities that can provide added values while diminishing the negative envi-
ronmental impacts in an integrated way (Barrington et al. 2009). IMTA has an objec-
tive to increase the rate as well as the efficiency of biomass productivity per unit
of water column at any determined time period (e.g. kg/(m3 year). IMTA consists
of various components of cultivated organisms with different but complementary
trophic state, arranged to be a uniting production system based on the inner circular
mass and energy transfers to obtain internal ecological balance resulting in a higher
production efficiency.
Research of IMTA in Research Center for Limnology and Water Resources—
National Research and Innovation Agency of Republic of Indonesia (RCLWR-
NRIA) is focused to utilize waste from cat fish (Clarias sp.) cultivation—trophic
status of omnivore—carnivore—for growing floating aquatic plant minute duck-
weed (L perpusilla)—trophic status of autotrophic or producer—that can be used
for feeding Nile tilapia fish (Oreochromis nioloticus)—trophic status of omnivore—
herbivore (Fig. 2). The advantage value of this IMTA system is that the minute
duckweed converts waste materials from fish culture into biomass that can be put
back into fish culture as a valuable feed source. This implementation encourages the
minute duckweed to deliver a double strategic function, as the water phytoremedia-
tion agent that keeps the water condition to suit the fish growth requirement, and as
an additional feed source which reduces the cost for feeding expenses (Chrismadha
et al. 2021). Accordingly, IMTA implementation will simultaneously enhance a better
production efficiency and environmentally sound cultivation practice (Fig. 3).

4 Experimental for IMTA Implementation

Trials for implementation of IMTA scheme have been conducted in RCLWR-NRIA,

including utilizing cat fish cultivation waste water to grow minute duckweed and
using the biomass product for alternative feed in Nile tilapia culture. Observation
on growth and biomass productivity of the plant based on cat fish cultivation waste
water medium was carried out in a water closed recirculation ponds placed in an
open space, consisting of a cat fish cultivation pond in which the water media was
pumped in to three serial order minute duckweed cultivation ponds before it was
recirculated back into the cat fish pond (Chrismadha and Said 2019a). The pond size
was 2.4 × 5 m2 each with 50 cm depth, while the fish density was 1,500 fishes/pond
An Overview on Managing Minute Duckweed (Lemna Perpusilla Torr) … 175

Fig. 2 Concept of integrated multitrophic aquaculture (IMTA) in RCLWR-NRIA

Fig. 3 IMTA trial ponds at RCLWR-NRIA, KST-Soekarno, Cibinong-West of Java

176 T. Chrismadha et al.

Fig. 4 Growth and biomass productivity of minute duckweed in IMTA ponds with cat fish
aquaculture waste water as the growth medium (Chrismadha and Said 2019a)

(125 fishes/m2 ), provided with commercial feed containing 30–33% protein at 3%

of progressive grown body weight under assumption of 6% specific growth rate.
Continual four cultivation period observation from October 2017 to July 2018
showed the suitability of waste water from cat fish aquaculture for growth medium
of minute duckweed. In the meantime, the plant can effectively control the water
quality, so there was no water replacement required during the cultivation time, except
in dry season when the evaporation rate was considerably high while there was no
input of rain water. It saved the water requirement up to more than 80%. Almost all
throughout the observation period the average duckweed biomass productivity was
above 100 g/(m2 day) or equal to 328 ton/(ha year) of the fresh weight (Fig. 4). The
optimal harvesting period for the minute duckweed culture was 3–4 days.
Figure 4 also shows the fluctuation of the duckweed growth and biomass produc-
tivity in response to the cat fish growth period and the progressive amount of
feed delivery. The waste oversaturation level was visible just close before the fish
harvesting time, where the load of waste was too high and inhibited the minute duck-
weed growth. The recovery situation was there, however, several days after the fish
was harvested and the fish cultivation was restarted back from the fingerling size.
The cat fish culture productivity itself was in the range of 70–100 kg/(pond. harvest)
or equal to 233–333 ton/(ha. year), with feed conversion value of 0.8–1.4.
Nutrition state of minute duckweed grown in IMTA ponds has been reported by
Sutrisno et al. (2021), whichpointed out the advantage of high protein content up to
32.90% of the dry weight, while total lipid content was 9.73% DW, carbohydrate
8.75% DW, and ash 15.32%. Under a better growth condition, the plant has a higher
protein content up to 38.10% DW or more (Ilyas et al. 2014; Hasan and Chakrabarti
2009). Sutrisno et al. (2021) also showed a complete amino acids composition in
minute duckweed biomass. HPLC analysis with 15 reference amino acid standards
An Overview on Managing Minute Duckweed (Lemna Perpusilla Torr) … 177

revealed the existence of all the amino acids in the biomass (Table 1). Duckweeds are
naturally widely known to have a complete amino acids composition, close to animal
amino acids composition (Hasan and Chakrabarti 2009), so that it is considered to be
very suitable for animal feed purpose, including for fishes. Chakrabarty et al. (2018)
reported the occurrence of 20 amino acids species in common duckweed (L minor).
The main problem in utilizing minute duckweed for fish diet is the low energy
density, which is mainly caused by limited lipid content in the minute duckweed
biomass. Lipid is the most efficient energy source for fish, but the content in the
minute duckweed biomass ranges from 4.15 to 9.73% DW, whereas the ideal lipid
content in fish diet is 7–15% (Helfrich 2002). Beside its function as energy source,
lipid also has an important role as media transport for fat dissolved vitamins, such
as vitamin A, D, and E (Elangovan et al. 2017). Lipid in fish diet has also to contain
appropriate content of essential fatty acids, particularly those belonging to omega-
3 group. These polyunsaturated fatty acids (PUFAs) cannot be synthesized by fish
itself, so the availability completely depends on the external supply from the diet,
with an ideal portion of 0.5–2.0% of the total diet. There is no information yet
available for fatty acid composition of the minute duckweed, but Chakrabarti et al.
(2018) reported the omega-3 content of the closest related species, which is common
duckweed was up to 46% of the total fatty acids.
Under the assumption that fatty acid content of minute duckweed is similar to that
of common duckweed, the lipid supply can be directed for fulfilling the need of energy
so that the available protein input can be more obtained for growth. It is mainly due
to the carbohydrate content of minute duckweed that is also considerably low, which
is 7.27–8.75% DW only. Carbohydrate component in fish diet is generally high of
more than 20%, although it is not an efficient energy source for fish, carbohydrate has
considerably lower price compared to other components. The important notes related

Table 1 Proximate and amino acids composition of minute duckweed (L. perpusilla Torr) grown
in IMTA ponds
Proximate* Amino acids**
% DW Essentials % BK Non-essentials % dry weight
Moisture 88.2–94.3 Histidine 0.54 Alanine 1.98
Ash 6.70–16.50 Isoleucine 1.53 Arginine 1.71
Protein 27.3–38.10 Leucine 2.61 Aspartic 2.97
Lipid 4.15–9.73 Lysine 1.53 Cysteine
Carbohydrate 7.25–8.75 Methionine 0.54 Glutamic Acid 3.51
Phenylalanine 1.53 Glycine 1.44
Threonine 1.35 Proline
Tryptophan NA Serine 1.44
Valine 1.98 Tyrosine 1.62
*Hasan and Chakrabarty (2009), Ilyas et al. (2014), ** Sutrisno et al. (2021)
NA not analyzed
178 T. Chrismadha et al.

to the duckweed carbohydrate content is the crude fiber fraction that has been reported
to compose up to 44% DW (Ilyas et al. 2014), while in the group of duckweed the
nitrogen free extract (NFE) which generally represent the nonstructural carbohydrate
content, was 27.2–66.4% DW (Chakrabarti et al. 2018; Goopy and Murray 2003;
Hasan and Chakrabarti 2009). Crude fibers are materials that cannot be digested
by fish and become a source of inefficiency in terms of feed metabolism. Trials in
RCLWS–NRIA, KST-Soekarno-Cibinong also show that minute duckweed biomass
can be dried, to make it have a longer storage time as well as for a more flexible
distribution purpose. This drying practice, however, might give a consequence of
decreasing the nutrition value.
The next trial was utilization of the minute duckweed biomass for alternative feed
in fish cultivation. In line with the IMTA conception, it was a direction to employ the
fresh biomass for the feeding purpose. The fresh harvested biomass was delivered for
fish feeding as soon as possible, so that it eliminates the requirement for any post-
harvest processing as well as the storage places that might need some additional
energy and cost expenditure. There have been some experiments to feed various
fishes on fresh minute duckweed biomass, and the results show that almost all tested
fishes are ready to forage on it. There is a group of fishes that strongly like to forage
on minute duckweed, even when there is regular pellet available. This group consist
of Nile tilapia, common carp, and cat fish. Other tested fishes, including gourami
and pomfret fish consume minute duckweed when no other feed source is available.
Investigation to figure out fish growth performance fed on minute duckweed
biomass was carried out in two serial trials of Nile tilapia culture. Firstly, it is
conducted at a nursery level, involving new born fish of 1–2 weeks of age for the
starter and grown up for about 8 weeks to size of 40–50 g/fish, that is ready for
starter in the next round of grown-up cultivation. The second was the grown-up
level, involving these starting fish of 15–17 g/fish size and cultivated up to about 100
g/fish size for around 4 months.
The nursery level trial was carried out in aquaria of a closed water recirculation
system with water quality maintenance mainly directed for controlling the dissolved
oxygen content >3 mg/L, with culture density of 100 fishes/m2 (Sutrisno et al. 2021).
The minute duckweed employed for feed was the fresh biomass obtained from the
nearby IMTA ponds culture based on cat fish cultivation waste water medium. The
observation result is summarized in Table 2, showing a great potential of minute
duckweed biomass for the replacement of pellet commercial feed in the nursery-
level fish cultivation activity. Although the fish fed on minute duckweed biomass
had slower growth rate, but a simulation according to the specific growth rate values
showed that the fish only needs as long as 12–15 days additional time to achieve
the same size as that fed on commercial pellet. It can be concluded that minute
duckweed can completely replace commercial feed for growing Nile tilapia fish with
a consequence of a longer culture period.
This might become an interesting advantage when the minute duckweed biomass
can be produced at significant lower cost than that of purchasing the commercial
feed.
An Overview on Managing Minute Duckweed (Lemna Perpusilla Torr) … 179

Table 2 Growth performance of Nile tilapia fish at nursery level. After Sutrisno et al. (2021)
Time
Initial Week-2 Week-4 Week-6 Week-8
Pellet
Length 35,00 ± 0,00 57,03 ± 1,59 87,03 ± 0,76 123,27 ± 3,62 138,60 ± 4,88
(cm)
Weight 0,86 ± 0,00 3,42 ± 0,18 11,56 ± 0,28 30,96 ± 4,56 47,91 ± 5,62
(g/fish)
CF (g/ 0,24 ± 0,00 0,59 ± 0,02 1,30 ± 0,03 2,49 ± 0,23 3,40 ± 0,35
cm)
SGR 9,89 ± 0,38 7,62 ± 0,48 6,54 ± 0,68 3,36 ± 0,28
(%/
day)
WG 0,18 ± 0,03 0,51 ± 0,03 1,13 ± 0,16 1,30 ± 0,19
(g/fish/
day)
TF (g) 120,00 ± 0,00 730,00 ± 0,00 670,00 ± 0,00 990,00 ± 0,00
FCR 0,47 ± 0,03 0,89 ± 0,05 0,81 ± 0,12 1,19 ± 0,18
Minute duckweed
Length 35 ± 0,00 51,07 ± 3,43 76,15 ± 2,68 106,40 ± 3,36 124,75 ± 3,65
(cm)
Weight 0,86 ± 0,00 2,62 ± 0,24 8,42 ± 1,04 20,47 ± 2,31 32,18 ± 3,27
(g/fish)
CF (g/ 0,24 ± 0,00 0,50 ± 0,03 1,07 ± 0,11 1,90 ± 0,16 2,57 ± 0,25
cm)
SGR 7,98 ± 0,64 7,27 ± 0,41 5,93 ± 0,25 3,48 ± 0,39
(%day)
WG 0,13 ± 0,02 0,34 ± 0,03 0,69 ± 0,06 0,65 ± 0,06
(g/fish/
day)
TF (g) 191,67 ± 29,30 956,67 ± 40,41 983,33 ± 136,50 1100,00 ± 185,20
FCR 10,83 ± 0,30 17,56 ± 1,83 18,99 ± 0,95 27,78 ± 7,68
Notes CF = Condition Factor; SGR = Specific Growth Rate; WG = Weight Gain; TF = Total Feed;
FCR = Food Conversion Ratio

Experiment of the grown-up level cultivation was carried out in larger vessels,
which were 1 m3 provided with a closed water recirculating system by means of
submersible pumps to generate a water flow to maintain the dissolved oxygen level
>3 mg/L (Chrismadha and Mulyana 2019b). There was a variation in culture density
applied for the experimental treatment. At the lowest culture density, where less
space and any other resources competition was occurred, a single feed of minute
duckweed was able to stimulate fish growth up to 3%/day (Table 3). This growth rate
decreased with the culture density.
 180

Table 3 Growth performance of Nile tilapia fish at grown-up level. After Chrismadha and Mulyana (2019b)
Parameters Stocking density (fishes/m2 ) Days
0 40 75 115
Length (cm) 5 9.80 ± 0.00 14.22 ± 0.25 16.55 ± 0.27 18.30 ± 0.14
10 9.92 ± 0.00 13.65 ± 0.07 15.79 ± 0.86 17.24 ± 0.61
20 10.28 ± 0.25 13.16 ± 0.48 24.53 ± 1.91 16.28 ± 0.73
Weight (g/fish) 5 16.57 ± 0.63 55.30 ± 2.91 87.91 ± 4.99 126.36 ± 5.29
10 16.83 ± 0.13 49.55 ± 3.71 76.29 ± 7.25 107.09 ± 5.85
20 17.34 ± 0.20 43.26 ± 5.78 63.63 ± 13.66 88.03 ± 11.88
Condition factor (g/cm) 5 1.69 ± 0.07 3.89 ± 0.14 5.31 ± 0.22 6.90 ± 0.24
10 1.70 ± 0.01 3.63 ± 0.29 4.83 ± 0.20 6.21 ± 0.12
20 1.69 ± 0.01 3.28 ± 0.32 4.35 ± 0,37 5.40 ± 0.49
Specific growth rate (%/day) 5 3.09 ± 0.04 1.32 ± 0.31 0.98 ± 0.04
10 2.76 ± 0.21 1.23 ± 0.06 0.92 ± 0.11
20 2.33 ± 0.39 1.08 ± 0.24 0.90 ± 0.22
Weight gain (g/fish/day) 5 0.99 ± 0.06 0.84 ± 0.20 0.99 ± 0.01
10 0.84 ± 0.10 0.86 ± 0.09 0.79 ± 0.04
20 0.66 ± 0.16 0.52 ± 0.20 0.63 ± 0.05
Total feed (g) 5 3,501 ± 634 4,500 ± 1061 7,450 ± 354
10 6,550 ± 2192 8,800 ± 1556 12,100 ± 1131
20 12,225 ± 884 13,800 ± 2687 18,400 ± 424
Food conversion ratio 5 18.02 ± 2.22 27.62 ± 0.19 38.76 ± 2.14
10 20.56 ± 9.11 32.81 ± 1.47 39.41 ± 5.46
20 24.04 ± 3.94 35.25 ± 7.05 37.78 ± 1.90
T. Chrismadha et al.
An Overview on Managing Minute Duckweed (Lemna Perpusilla Torr) … 181

The growth rate in this trial was found to be lower than previously reported by
Rejeki et al. (2013). But it was in the range of that reported by Khalil et al. (2011).
which was 0.31–1.44%/day for the same fish species grown in floating cages in
reservoir water. Fish generally has a higher growth rate when cultivated in floating
net placed in reservoir waters compared to that cultivated in inland pond water. As
it has a better water circulation obtained from big water volume of the reservoir.
Accordingly, the growth performance of Nile tilapia in this experiment, which used
vessels of closed water recirculation system can be considered to be suitably good
and can reflect the ability of duckweed biomass to support fish growth even when it
is delivered as a single died.
From this experiment, it can also be figured out the pattern of fish consumption
rate on the duckweed biomass, which tends to progressively follow the body weight
development with the rate ranging from 38.91 to 40.50% of the body weight in which
the efficiency of feed conversion ratio was tend to decrease to form a logistic model on
the body weight development. Calculation of the minute duckweed biomass require-
ment to grow fish up to a certain size can be conducted by plotting the progressive
daily FCR values along the body weight development and multiply this daily FCR
value with the daily weight gain. It is estimated that to produce a fish of 250 g size the
required fresh biomass of minute duckweed is as much as 12.591–12.946 g which
is equal to the FCR value of 50.08–50.60. According to this FCR value, it can be
estimated that the production rate of 328 ton/(ha year) as mentioned above is suitable
to fulfill the feed requirement for producing 250 g size fish as much as 7–8 ton.

5 IMTA Criterions

IMTA has many types of implementations as its design is tailored to the environ-
mental and ecological aspects of the location it is implemented. It usually involves
the integration of many species consisting of fed species (usually fish) which provide
waste or detritus to be consumed by the extractable species such as marine inverte-
brates and/or algae (Alexander et al. 2016). The synergistic culture will attribute to
the trophic level which showcased the relationship between the components within
the pond. IMTA is an evolution from the concept of polyculture which has a long route
back to 2300-1700 BC in Southern China, long time ago in a form of ponds for water
storage during the Hans dynasty (Li 1994). Meanwhile, the modern type of poly-
culture with a quantitative and scientific approach close to the current system stems
from the work which evaluated the bioremediation work of organisms (shellfish)
and simple phytae consisting of microalgae and seaweeds in a systematic municipal
water treatment (Ryther et al. 1975). It demonstrated the combined tertiary sewage
treatment using an aquaculture system where the solid wastes from shellfish were
then fed to small invertebrates and other small creatures which were regarded as
high-quality foods to lobsters, flounders, and other commercially valuable secondary
crops. While the dissolved waste excreted was then used as nutrients for unicellular
algae applied as the final polishing step. The system gave a final effluent free of
182 T. Chrismadha et al.

inorganic nitrogen and did not contribute to eutrophication. However, the work was
considered to not be quantitative and practical enough thus it was further revised
by the addition of phytoplankton as a biofilter which further supports the uptake of
nutrients done by algae, pioneered by Gordin et al. (1981) and worked on continu-
ously by Harlin et al. (1982). Advances toward ecological engineering would only
be done to further improve IMTA. The pioneering work of Troell et al. enforced this
by stressing and eventually landing the basis of IMTA where it should factor in the
sustainability of aquaculture. The sustainability can also be measured by counting in
the economic, societal, and environmental benefits thus including recycling of waste
nutrients from higher trophic-level species into commercially valued lower trophic-
level crops (Troell et al. 2009). IMTA can either be land-based or open water-based
with species configurations. The basic set-up of this system is by utilizing ponds.
The typical set-up known to date is based on varied species systems run in open
water as first developed by Troell et al. (2003). The interconnected ponds are set
up as two structures placed close to each other. These also include protection from
the fed aquaculture and the extractive components to the existing environmental
conditions by the use of a caging system. The use of IMTA is additionally referred
to as a provider of ‘rich food for impoverished people’ as it provided a good envi-
ronment for fish with particularly high proteins, Lipids, Minerals, and vitamins (Ye
et al. 2017). In 2022, rapid fisheries and aquaculture production was observed with a
record-breaking of 214 million tons of production with approximately 112.6 million
tons attributed to aquaculture. As sludge of aquaculture is high in nutrients, removal
efficiencies must be paid attention to avoid environmental stress one of which is by
recirculating water (Couto et al. 2022). Closed water circulation is part of IMTA as it
ensured the system will not affect the environment uncontrollably. In terms of setting
up this closed system, careful consideration of choosing IMTA types has to consider
the following choices provided in Table 4. Table 4 shows that the selection criteria
for IMTA all fit well with the implementation of Duckweed.

6 The Economic Potential

The main objective of IMTA development is enhancing fishery aquaculture produc-

tion efficiency. In the implementation, IMTA involves alternative feeds that can
become a new commodity and needs a further direction for its cultivation practice in
the field. The important thing to encourage minute duckweed culture development
is the feature of its economic value. Referring to the FCR value as mentioned above
(≈50), it means that to produce 1 kg Nile tilapia fish it requires about 50 kg fresh
duckweed biomass. If it makes equal to the tilapia feed price, where the current
market price is IDR 13,500/kg and the commercial feed conversion value is 1.5, the
selling price of the fresh minute duckweed biomass can be calculated as much as IDR
405/kg. Accordingly, the potential of increasing land value by using it for minute
duckweed cultivation can be determined.
An Overview on Managing Minute Duckweed (Lemna Perpusilla Torr) … 183

Table 4 Criteria of selecting species for IMTA

Species selection Definition In our trial IMTA
criteria for IMTA implementation
Complementary This is to ensure that the extractive species Duckweed presented
co-cultured species can be eaten by fed aquaculture to ensure biomass that supports fish
improvement in water equality and good growth in the pond
economic output
Native species The choice of native species will avoid Duckweed is native to the
invasion and save acclimatization time and natural system where
cost too catfish grows
Consistency of waste Consistent waste material is to ensure that The growth of duckweed
material the system will be able to run sustainably and catfish can be seen to
grow side by side in
previous studies
Choice of the In choosing a biofilter organism. It should Duckweed also serves as a
bio-filtering organism have a high growth potential which can be bio-filtering organism and
periodically harvested. This is also to even can live in consortia
avoid IMTA’s biological bottlenecks which for improving water
include the lack of seed, long periods of quality. The growth of this
production, lack of knowledge in organism is also quite fast
cultivating, Biofouling, and predation
between the feed and bio-filtering
organisms
Established market In developing IMTA, the economic value Although duckweed is not
species should be co-cultured with buyers to invite well-established in the
larger investments market. The need for this
species will grow with the
growing food demands for
aquaculture
Commercial In choosing a species for IMTA, other Microalgae and duckweed
hindrances similar species must be considered to have almost the same
avoid commercialization hindrances growth and simpler
structure. Further research
can be done in comparing
these two or even consider
them as co-culture
Socio-ecological and In ensuring a sustainable set-up, Indonesia, for example, is
political compatibility socio-ecological and political one of the largest island
compatibility must be well-thought before countries with high
implementation. This includes the IMTA diversity in their waters.
bottlenecks such as licensing and Therefore the use of IMTA
regulations with duckweed and local
catfish can improve the
currently existing
aquaculture businesses
Source Nissar et al. (2023), Chen et al. (2018)
184 T. Chrismadha et al.

Food and Agricultural Organization (FAO) of United Nations Organization (UNO)

uses the parameter of land equivalent ratio (LER) for the evaluation of land use
efficiency in polyculture farming activities. In principle, the value of land used for
a certain purpose can be directly compared between two commodities or indirectly
by referring to a certain main reference commodity. This approach can be used
for the evaluation of potential land use for minute duckweed cultivation from the
perspective of economic development. Taking paddy field cultivation land as the
standard reference with an assumption of productivity level 24 tons/(ha year) and
the selling price of unhauled rice is IDR 5,000/kg while the selling price of minute
duckweed refers to tilapia feed value as described above (IDR 13,500/kg and FCR =
1.5) the land use value for minute duckweed cultivation is considerably higher than
that of paddy field purpose (Table 5). Therefore minute duckweed cultivation can be
considered to be more beneficial and potentially becomes an alternative activity for
earning life, particularly in the locations where highly fertile water is available.
The important note regarding the above economic evaluation is that the minute
duckweed selling price is presumed to be equal to the feeding cost in Nile tilapia
cultivation activity. The minute duckweed selling value has actually not yet been
performed and will be self-revealed to follow the market mechanism when the mass
cultivation has been developed. As an example, the minute duckweed selling price
determination refers to its utilization for fertilizer purpose will possibly lower down
the value compared to the above description for Nile tilapia fish feed. However, the
value will increase if it refers to the purpose for feeding cat fish. Lower price will
stimulate the segment of consumer but becomes a disincentive for the producers and
on the contrary, higher price will encourage the producer but lowering the consumer’s
enthusiasm. The current duckweed selling price on online sites ranged from IDR
20,000–40,000 per kg and is more directed for the purpose of ornamental fish feed.
As an additional note, trials in RCLWS–NRIA have also revealed that duck-
weed biomass can be utilized for additional feed in cat fish cultivation to reduce

Table 5 Land equivalent ratio of land used for various aquaculture commodities
Units Nile Tilapia Cat fish Minute duckweed Rice field
Wide m2 100 100 100 Land productivity
stocking Fishes 2000 15000 30 assumption of 24
Density ton/Ha/year
Survival rate % 90 90
Specific growth %/day 1.76 4.47 30
rate
Culture period day 157 63 4
Harvesting kg/m2 /day 0.029 0.214 0.1 0.0066
quantity
Unit price IDR/kg 22.000 18.000 405 5000
Land values IDR/m2 day 630.5732 3857.143 40.5 33
An Overview on Managing Minute Duckweed (Lemna Perpusilla Torr) … 185

the commercial pellet, need up to 30% without significant harm to the fish growth
(Chrismadha et al. 2021).

7 Challenges

It has been pointed out that challenges in minute duckweed culture development
are mainly associated with its position as a new commodity where the technical
aspect needs to be adapted to landscape suitability and socio-cultural of the local
farmers (Journey et al. 1993). Minute duckweed culture needs a large more intensive
maintenance compared to rice planting or even fishery aquaculture. This is mainly
due to the production cycle of minute duckweed that can be counted only in several
days while that of rice field is in 2–4 months. This is so for fisheries aquaculture even
though there is a need to have daily feeding activity, the production cycle is generally
more than two months. Beside the need for more intensive work a short harvesting
period also makes the farmer to have a close interaction with the users to market due
to the minute duckweed biomass property that contains high level of protein to make
it more susceptible to damage. In the tropical condition it is relatively warm and fresh
biomass of minute duckweed cannot sustain for more than 24 h. Whereas as a new
commodity the market place for minute duckweed has not yet been established so
that for the initiation minute duckweed culture development is only feasible when it
is conducted in an integrated mode. And this integrated mode must be done with any
fisheries aquaculture so that the resulting minute duckweed biomass can be directly
distributed as fish feed such as under the scheme of IMTA.
Implementation of integrated cultivation of minute duckweed and fish however
requires a considerably wide land area. Meanwhile, the land ownership level of
Indonesian farmers is relatively short. By average in the year of 2007, the land
ownership level of Indonesian farmers was 0.36 ha (Susilowati and Maulana 2012).
As has been mentioned above minute duckweed biomass conversion ratio to be Nile
tilapia flesh is ≈50, which means that to support Nile tilapia fish production it needs
an extensive minute duckweed production system.
A simulation of the integrated aquaculture ponds as shown in Table 5 shows that an
optimal utilization of minute duckweed biomass can be enhanced by the arrangement
of Nile tilapia fish cultures in a time serial segmentation in which a regular seeding
and harvesting are performed to keep the total population in a constant number so
that the feen requirement can also be managed in a steady amount. The extent of
segmentation which then determines the demand on the pond number depends on
the fish growth rate, while the pond large of the ponds has to follow the tonnage
target. As shown in Table 6, there are 14 culture time serial segmentation required
to accommodate an optimal minute duckweed feeding scheme of Nile tilapia fish
grow at a rate of 2%/day and the target harvesting size of 250 g/fish. To install
a fish production capacity of 90 kg/week with the minute duckweed productivity
of 0.1 kg/m2 /day there will be a need of totally 3,058 m2 pond area consisting of
458 m2 fish ponds and 2,600 m2 minute duckweed ponds. Considering the relative
186 T. Chrismadha et al.

shortness of Indonesian farmers land ownership the implementation of IMTA has to

bring up a communal activity by forming a group of fish farmers with an integrated
farming segmentation controlled by a predefined production target. According to
this predefined target the farming activities will divide into two groups of minute
duckweed and Nile tilapia fish cultivations in which each group will have a guarantee
for their business continuity where the fish farmer will obtain an assurance of feed
supply that at the same time gives a commitment to definite market for the product
of the minute duckweed farmers.
Regarding to this issue an institutional means has to be developed to organize all
the components of the involved production system including farmers, infrastructures,
science and technology, and markets. An authority has to be established to collect
farmers and divide them into segments of farming activities with accountable roles
and targets and gives a financial commitment as well as supply of any production
requirements to encourage the success of cultivation works.
Some important issues regarding this institutional aspect are as follows:
(1) Minute duckweed is a new farming commodity that is not yet widely known.
In this case. The emerged institution has to have a capacity to provide a bridge
for science and technology to enter and have an adaption to the local farmer
with their available resources. The new institution has also to have a close
interaction with the relevant R&D institution so that all the newest technology
can be received and implemented. At the same time all the actual problems in the
field can be communicated and have a appropriate assistance for the resolutions.
(2) As a new commodity minute duckweed has not been well-defined in the market.
So that the emerged institution has to have a capacity to create an integrated
relationship between producers and users to guarantee the market availability for
the minute duckweed biomass product. The simplest pattern is a combination of
minute duckweed cultivation and fish farming in which a farming segmentation
is designed to define the fish production target and its feed requirement. This
market issue is very important particularly from the minute duckweed cultivation
side as it has to be conducted in an intensive way with harvesting time every
3–4 days and biomass product susceptible to damage so that the product has to
be delivered as soon as possible to the users.
(3) Fair price making that gives incentives to all the parties. In this case the institution
takes a role as a mediator for price determination that gives advantages to both
the minute duckweed producer and the fish farmers.
(4) Incentive assurances of payment and financial capital credit. The institution has
to be able to deliver financial services providing payment upon the delivered
minute duckweed product as soon as possible, which is credited to the users
that will be billed at the harvesting time. This direct cash payment is believed to
become a good incentive for minute duckweed farmers to carry on the cultivation
activity.
(5) To become a feed buffer agency at the time of minute duckweed cultivation fails
to provide suitable feed quantity. This failure moment has to be considered as the
minute duckweed is an autotrophic organism and the growth is largely affected
Table 6 Simulation on time serial segmentation of Nile tilapia fish culture for harmonizing use of minute duckweed culture
Weeks Weight Weight Daily Weekly feed requirement
(W) gain feed
(WG) intake
(F)
g/fish (W * G) (WG * (F * P/1,000)
FCR)
g/fish/ g/fish/ kg/day
day day Pond numbers Total
1 2 3 4 5 6 7 8 9 10 11 12 13 14
1 42.05 0.84 15.14 0.84 0.84
2 47.36 0.95 17.05 0.95 0.84 1.79
3 54.40 1.09 19.58 1.09 0.95 0.84 2.88
4 62.49 1.25 22.50 1.25 1.09 0.95 0.84 4.13
5 71.78 1.44 25.84 1.44 1.25 1.09 0.95 0.84 5.56
6 82.45 1.65 29.68 1.65 1.44 1.25 1.09 0.95 0.84 7.21
7 94.71 1.89 34.10 1.89 1.65 1.44 1.25 1.09 0.95 0.84 9.11
8 108.80 2.18 39.17 2.18 1.89 1.65 1.44 1.25 1.09 0.95 0.84 11.28
9 124.97 2.50 44.99 2.50 2.18 1.89 1.65 1.44 1.25 1.09 0.95 0.84 13.78
10 143.56 2.87 51.68 2.87 2.50 2.18 1.89 1.65 1.44 1.25 1.09 0.95 0.84 16.65
An Overview on Managing Minute Duckweed (Lemna Perpusilla Torr) …

11 164.90 3.30 59.36 3.30 2.87 2.50 2.18 1.89 1.65 1.44 1.25 1.09 0.95 0.84 19.95
12 189.42 3.79 68.19 3.79 3.30 2.87 2.50 2.18 1.89 1.65 1.44 1.25 1.09 0.95 0.84 23.74
13 217.58 4.35 78.33 4.35 3.79 3.30 2.87 2.50 2.18 1.89 1.65 1.44 1.25 1.09 0.95 0.84 28.09
14 244.95 4.90 88.18 4.90 4.35 3.79 3.30 2.87 2.50 2.18 1.89 1.65 1.44 1.25 1.09 0.95 0.84 32.99
(continued)
187
Table 6 (continued)
188

Weeks Weight Weight Daily Weekly feed requirement

(W) gain feed
(WG) intake
(F)
g/fish (W * G) (WG * (F * P/1,000)
FCR)
g/fish/ g/fish/ kg/day
day day Pond numbers Total
1 2 3 4 5 6 7 8 9 10 11 12 13 14
15 42.05 0.84 15.14 0.84 4.90 4.35 3.79 3.30 2.87 2.50 2.18 1.89 1.65 1.44 1.25 1.09 0.95 32.99
16 47.36 0.95 17.05 0.95 0.84 4.90 4.35 3.79 3.30 2.87 2.50 2.18 1.89 1.65 1.44 1.25 1.09 32.99
Notes By segmentation of 14 pond units which are sequentially harvested one pond every week. there will be obtained a constant feed requirement avter the
week of
T. Chrismadha et al.
An Overview on Managing Minute Duckweed (Lemna Perpusilla Torr) … 189

by the weather so that under extreme weather condition this plant cultivation
might face a failure and it needs some days to a recover.
(6) The institution has also to be capable of collaborations with the govern-
ment agencies in case of relevant regulations and development program
implementation.

8 Conclusion

Involvement of minute duckweed in fisheries aquaculture can potentially become an

alternative way to resolve the current problem of high-cost feed. An appropriate culti-
vation system has to be developed to enable this potential into real implementation.
An integrated culture practice is suggested where the minute duckweed produc-
tion cycle is synchronized with fish farming production target in an optimal way.
Fish culture has to be divided into certain time serial segmentation of stocking and
harvest to let a constant requirement of feed quantity so the minute duckweed target
production can be conveniently set up. Developing a harmonious interaction is very
important in order to make this integrated culture practices become profitable and
sustainable.

Acknowledgements IMTA research was conducted in RCLWR-BRIN under various financial

schemes. including LIPI Competitive Research Program (2012). LIPI Science and Technology
Park (2019) and DIPA of previous RC Limnology-LIPI (2010–2014).

Author Contributionship Tjandra Chrismadha and Awalina Satya. were the main contributors
(data curation. writing original draft); others were supporting contributors: Ika Atman Satya
(review). Azalea Dyah Maysarah Satya (review). Sara Kazemi Yazdi (review). and Efri Mardawati
(review).

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MARPOL.2017.03.012
Structured Lipids Based on Palm Oil

Siti Nurhasanah and S. Joni Munarso

Abstract Palm oil is usually used for the needs of food, chemical industry, and
cosmetic industry. The basic processing of palm fruit can produce two types of oil
namely crude palm oil (CPO) which is produced from the extraction process of the
mesocarp part of the oil palm fruit and palm kernel oil (PKO) as an extract of the
palm kernel part. Naturally, oils and fats have specific characteristics, and the devel-
opment of food processing and technology causes these characteristics to not able to
meet all the expected needs to obtain products with certain functional properties such
as: lipids for sufferers of coronary heart disease, type 2 diabetes sufferers, patients
in the post-operative recovery period, patients who suffer from allergies or diges-
tive problems, and consumers who are controlling their weight low-calorie products.
The dominant fatty acids in palm oil are palmitic, oleic acid, and linoleic acid. CPO
also contains minor components such as squalene, sterols, and carotenoids. Struc-
tured lipids (SLs) are the result of modification or restructuring of triacylglycerols,
which can be obtained by chemical or enzymatic interesterification of triacylglyc-
erols containing short, medium, and/or long chain fatty acids. SLs are the result of
modification or restructuring of triacylglycerols, which can be obtained by chem-
ical or enzymatic interesterification of triacylglycerols. SLs can be sourced from
animal or vegetable fats, or genetic engineering. SLs are synthesized for the purpose
of obtaining functional lipids or nutraceuticals, which can improve or modify the
physical, chemical, and rheological characteristics of oils and fats, and changing or
enhancing nutrition properties of food, giving a certain health benefit. Palm oil has
special fatty acids and other minor components, making it possible to be used as a raw
material for the manufacture of SLs so that their bioavailability increases. Functional
oil and fat production can be catalyzed by lipase. Fats/oils can improve physicochem-
ical and nutritional properties using a lipase catalyst. Palm oil has special fatty acids
and other minor components, making it possible to be used as a raw material for
the manufacture of SLs so that their bioavailability increases. Functional oil and fat

S. Nurhasanah (B)
Department of Food and Technology, Faculty of Agro-Industrial Technology, Padjadjaran
University, Bandung 45363, Indonesia
e-mail: [email protected]
S. J. Munarso
National Research and Innovation Agency, Bogor 16124, Indonesia

© The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2024 193
M. A. R. Lubis et al. (eds.), Biomass Conversion and Sustainable Biorefinery, Green
Energy and Technology, https://doi.org/10.1007/978-981-99-7769-7_9
194 S. Nurhasanah and S. J. Munarso

production can be catalyzed by lipase. Fats/oils can improve physicochemical and

nutritional properties using a lipase catalyst.

Keywords Fatty acid · Interesterification · Palm oil · Structured lipids

1 Introduction

Palm oil is a vegetable oil that can be obtained from the mesocarp of the fruit of
the oil palm tree, generally of the species Elaeis guineensis. Naturally, palm oil is
red in color due to its high beta-carotene content. This oil is a common cooking
ingredient in tropical countries including Africa, Southeast Asia, and parts of Brazil.
The results of the palm oil industry are not only cooking oil but can also be used as a
basic ingredient for other industries such as the food and cosmetics industries. Half
of all fats and vegetable oils consumed come from palm oil (Murgianto et al. 2021).
Palm oil is usually used for the needs of food (popular components in baking,
processed meals, snacks, frozen foods, and chocolate due to their neutral taste,
texture, and practicality), chemical industry, and cosmetic industry. The largest
demand for palm oil is used for food ingredients such as cooking oil, margarine,
shortening, emulsifiers, cocoa butter substitutes, and other derivative products. The
basic processing of palm fruit can produce two types of oil namely crude palm oil
(CPO) which is produced from the extraction process of the mesocarp part of the
oil palm fruit and palm kernel oil (PKO) as an extract of the palm kernel part (Ali
et al. 2014). Commercial cooking oil is made from palm olein, whereas palm stearin
is mostly used for shortening and margarine products. PKO has a sharp melting
profile because high in lauric acid therefore it is suitable for confectioneries and
other specialty fats. PKO is more widely used in a variety of non-edible products,
such as cosmetics, detergents, surfactants, plastics, herbicides, and various other
industrial and agricultural chemicals. The widespread use of palm oil makes this oil
an important commodity that plays a role in food security in the world (Dian et al.
2017; Chew et al. 2021).
Naturally, oils and fats have specific characteristics. The development of food
processing and technology causes these characteristics to be not able to meet all
the expected needs. For example, lipids for sufferers of coronary heart disease, type
2 diabetes sufferers, patients in the post-operative recovery period, patients who
suffer from allergies or digestive problems, and consumers who are controlling their
weight low-calorie products and to obtain products with certain functional properties.
Therefore, it is necessary to modify the fatty acids that make up the triacylglycerol
(TAG) into structured lipids (SLs).
SLs can be carried out by interesterification, either chemically or enzymatically.
The advantages of enzymatic interesterification are that the reaction is more specific,
the reaction conditions (pH, temperature, pressure) are milder and the waste produced
is minimal, thereby reducing environmental pollution. The composition of the TAG
after enzymatic interesterification will depend on the reaction conditions such as
Structured Lipids Based on Palm Oil 195

the type of enzyme, reaction time and temperature, water activity of the enzyme,
substrate ratio, and the type of substrate used. The interesterification process results
in changes in the chemical, physical, and oxidative properties of the substrate of
origin. With the superiority of fatty acids in palm oil, it is possible to serve as SLs
which can be used as raw materials for functional food.

2 Palm Oil

2.1 General

Palm (Elaeis guineensis) is a plant originating from the West African region and
classified as a tropical plant. Palm fruit has an oval shape and is attached to bunches
and can weigh up to 10–40 kg. The palm fruit consists of 2 main parts, namely the
pericarp and the seed. Oil palm fruit bunches are composed of fruit flesh (60%),
bunch skin (29%), and fruit seeds/palm kernel (11%). The pericarp consists of the
exocarp, mesocarp, and endocarp, while the seed portion consists of the endosperm
(palm kernel/kernel), embryo, and seed coat (Ezechi and Muda 2019; Mahlia et al.
2019).
CPO obtained from extraction or from the pressing process of the flesh of the
palm fruit and has not been refined. CPO refinery to make cooking oil always begins
with heating, threshing, boiling, stirring, and pressing then proceed with filtering,
purification, and phase separation (fractionation). Fractional crystallization, which
can be classified into dry and solvent fractionation, is used to separate oils and fats
into two or more components. The first stage of fractionation is crystallization, which
is followed by the separation of solid and liquid fractions (Hasibuan et al. 2021). Palm
oil is very useful for edible oil to make food products, such as frying or cooking oil
including margarine, shortenings, red palm oil, and also specialty fats. Palm olein
has no unpleasant odor, no trans-fatty acid, and has high resistance to oxidation.
Therefore, it is suitable for frying and applications and it is commonly used for
cooking, either with pan frying and deep frying and can be reused several times in
restaurants, fast food restaurants, and also in the manufacture of snack foods and
instant noodles (Purnama et al. 2020).

2.2 Palm Oil Processing

CPO still contains unwanted impurities. Components in the form of phospholipids,

proteins, residues, and carbohydrates in the oil need to be removed through the
degumming process. The principle of degumming is to separate the phosphatides into
196 S. Nurhasanah and S. J. Munarso

the aqueous phase so that they can be separated by precipitation, filtering, or centrifu-
gation. Processing of oil palm bunches begins with heating, threshing, boiling, stir-
ring, and pressing then followed by filtering, purifying, and separating phases (frac-
tionation). Oil refining consists of several stages: gum separation, neutralization,
bleaching, and deodorization. Bleaching process reduces the amount of carotene to
get clear cooking oil (Andarwulan et al. 2018).
The liquid fraction of palm oil that has been refined at the deodorizing stage to
remove free fatty acids and clarification to remove color and deodorize and frac-
tionate is known as refined, bleached, and deodorized palm olein (RBDP Olein).
CPO processing by modifying the refining process, without bleaching, to main-
tain the carotene content in palm oil can produce red palm oil or Red Palm Oil
(RPO) (Sumarna et al. 2022). The content of carotene, especially α-carotene and
β-carotene, is a precursor of vitamin A in the body. Before use, CPO extracted
from fresh fruit bunches needs to be purified because CPO still contains unwanted
impurities. Components in the form of phospholipids, proteins, residues, and carbo-
hydrates in the oil need to be removed through the degumming process. The principle
of degumming is to separate the phosphatides into the aqueous phase so that they
can be separated by precipitation, filtering, or centrifugation.
Degumming process can be carried out in various ways. Wet degumming is able
by adding phosphoric acid, and citric acid. Dry degumming uses the same steps as
wet degumming but without adding water. Of the two methods performed, the wet
degumming method produces better quality than the other method of dry degumming
(Putri et al. 2019; Mayalibit et al. 2020). Neutralization process is carried out to
separate the free fatty acids. Generally neutralization is done by reacting NaOH
with acid free fat contained in the oil to form soap that can be separated (Musyaroh
and Hidayat 2018). The process of deodorization of oil is carried out as one of the
important steps that must be taken in order to reduce unwanted aromas or tastes.
Both of these parameters are usually caused by damage to the oil component in
the oil during processing, such as unsaturated hydrocarbons, terpenes, sterols, and
tocopherols (Ramlah and Sampebarra 2018). Deodorization also affects the reduction
of free fatty acid levels in oil, although not as much as degumming, neutralization,
and bleaching processes (Riyadi et al. 2016).
The fractionation process in oil is carried out to separate the olein fraction (liquid
phase) and the stearin fraction (solid phase) of the oil. The olein fraction is often used
as cooking oil while the stearin fraction is often used as an ingredient raw margarine or
shortening. Fractionation is accomplished by winterization, crystallization (hexane,
acetone, or isopropyl alcohol), and detergent processes. Winterization can result in
oil loss which is abundant whereas organic solvents are flammable and expensive in
the process. The most commonly used is dry fractionation separated by cooling first
so that the stearin freezes and the olein can be filtered.
Palm oil is naturally semi-solid at room temperature, meaning there is no need
for it to be hydrogenated and therefore it contains no trans fats. Replacing trans
fats with palm oil may reduce heart disease risk markers and improve blood lipids.
In the hydrogenation process, unsaturated triglycerides are attacked more quickly
than di-oleo-glycerides, and finally mono-oleo compounds. The concept of fatty
Structured Lipids Based on Palm Oil 197

acid selectivity indicates that the rate of hydrogenation of various unsaturated fatty
acids depends on the concentration of hydrogen. Consequently the reaction rates of
two different unsaturated fatty acids, for example, linoleic acid and oleic acid, are
only a function of the concentrations of these acids and their rate constants, so that
the concentrations of these acids can be determined experimentally, while the ratio
of the hydrogenation rate constant can be calculated (Gunstone and Norris 2013).
During the hydrogenation process, double bonds are not only saturated, but they may
also shift their position along the fatty acid chain (positional isomerization) and/or
undergo cis-trans isomerization. Because trans isomers strongly affect the physical
properties of the triglycerides, the extent of the formation of these isomers has to be
controlled. It is generally expressed as the “trans selectivity” or as the “isomerization
index,” which are defined as the increase in trans content (expressed as % elaidic)
divided by the decrease in iodine value or by the decrease in double bond content,
respectively, as observed in the early stages of the hydrogenation process. Elaidic
acid, C18 H34 O2 , has an essentially linear alkyl chain. The double bond is twisted
across the mean direction of the alkyl chain. In the crystal structure, the molecules
form centrosymmetric O–HO hydrogen-bonded dimers.

2.3 Composition Palm Oil

The RBDPO fractionation will produce two unique fractions, namely the olein frac-
tion which has a high iodine number and low melting point and the stearin fraction
which has a lower iodine number and a high melting point (Tables 1 and 2).
CPO contains unsaturated fatty acids and saturated fatty acids. Saturated fatty
acids have only single bonds between their carbon atoms, whereas unsaturated fatty
acids have at least one double bond between their constituent atoms. Saturated fatty
acids are more stable or less visible than unsaturated fatty acids. Double bonds in
unsaturated fatty acids easily react with oxygen or are easily oxidized. Composition

Table 1 Comparison of fatty acid composition of palm oil

Fatty acids CPO (%) Oleic (%) Stearic (%)
Lauric (C12) 0.10–0.40 (0.24) 0.20–0.40 (0.27) 0.10–0.30 (0.18)
Miristic (C14) 1.00–1.40 (1.11) 0.90–1.20 (1.09) 1.10–1.70 (1.27)
Palmitic (C16) 40.90–47.50 (44.14) 36.80–43.20 (40.93) 49.80–68.10 (56.79)
Stearic (C18) 3.80–4.80 (4.44) 3.70–4.80 (4.18) 3.90–5.60 (4.93)
Oleic (C18:1) 36.40–41.20 (39.04) 39.80–44.60 (41.51) 20.40–34.40 (29.00)
Linoleic (C18:2) 9.20–11.60 (10.57) 10.40–12.90 (11.64) 5.00–8.90 (7.23)
Linolenic (C18:3) 0.05–0.60 (0.37) 0.10–0.60 (0.40) 0.00–0.50 (0.09)
Arachidic (C20:0) 0.20–0.70 (0.38) 0.30–0.50 (0.37) 0.00–0.50 (0.24)
Gee (2007)
198 S. Nurhasanah and S. J. Munarso

Table 2 Physical
Characteristics Typical Range
characteristics and chemical
composition Specific gravity, 30 °C 0.918 0.951–0.920
Index refraction, 25 °C – 1.470–1.474
Iodin value 124.0 118.0–128.0
Saponification – 187–193
Unsaponification – 1.3–2.3
Melting point (°C) – −12 to −10
Solid point (°C) – 1.0–20.0
Stability AOM (h) 19 16–19
α-Tocopherol (ppm) 152 116–172
β-Tocopherol (ppm) 12 0–22
γ-Tocopherol (ppm) 1276 119–1401
δ-Tocopherol (ppm) 61 59–65
O’Brien (2008)

of saturated and unsaturated fatty acids in palm oil is balanced. The dominant fatty
acids consist of palmitic (44–45%), oleic acid (39–40%), and linoleic acid (10–11%).
This allows it to be fractionated into two main fractions: liquid oil (65–70%), palm
olein (mp 18–20 °C), and solid fraction (30–35%), stearin (mp 48–50 °C). CPO also
contains minor components (1%) such as squalene (200–600 ppm), sterols (250–
620 ppm), and carotenoids (500–700 ppm), pigments responsible for the reddish-
orange color and the richest source of tocotrienols in the world (de Almeida et al.
2019).
Beta carotene is a minor component found in palm oil and is a secondary metabo-
lite that belongs to the group of carotenoid compounds synthesized by plants, algae,
and some microorganisms. This type of carotenoid is an organic compound that
is a chromophore and has eleven conjugated double bonds in its structure. The beta
carotene molecule has two beta-ionic rings which theoretically will experience chain
termination at –C15 = C15' – so beta carotene will be converted into two retinol
molecules (Bogacz-Radomska and Harasym 2018).
Beta carotene is a carotenoid compound that has high bioactivity. The bioactivity
properties of beta carotene are useful as a source of provitamin A which can support
the growth of embryo in pregnant women, the growth of children, and influence for
eye health. Apart from that, beta carotene as well has high anticancer and antioxidant
properties so it can ward off cancer, the bad influence of free radicals, increase body
immunity, prevent aging in early childhood, and the risk of cardiovascular disease
(Langi et al. 2018).
Structured Lipids Based on Palm Oil 199

2.4 Deterioration of Palm Oil

The ratio of oil degradation mainly depends on fatty acid composition, type, and
quality of the oil. The selection of oils should be based on the optimization of the
process with regard to culinary aspects as well as nutritional, physiological, and
technological requirements. The application of high temperatures to the oil results
in a change in the composition of the fatty acids and produces monoglycerides,
diglycerides, free fatty acids, primary and secondary oxidation products through
processes such as oxidation, polymerization, and hydrolysis (Jadhav et al. 2022).
Free fatty acid (FFA %) content is the most widely used criterion to determine the
quality of palm oil. Codex Alimentarius standard for maximum concentration of FFA
to 5.0% for CPO and up to 0.3% for RPO in oleic acid. Thus, according to this norm,
all fresh oil (zero month storage) is within the established limits (de Almeida et al.
2019).
Lipid oxidation is an important quality criterion for the food industry. Oxidation
is a reaction between unsaturated fats and oxygen which is accelerated by heat, light,
and metals. Acid value gives an indicator of free fatty acids present in the sample of
oil. Peroxide value is an indicator of primary oxidation products (hydroperoxides)
formed due to oxidation of lipids in food which can be decomposed into aldehydes
and ketones. Formation of secondary oxidation products like aldehyde and ketones by
degradation of peroxide can be measured by p-Anisidine value. Lipid oxidation not
only produces a rancid flavor but can also reduce nutritional quality and safety, namely
the formation of oxidation products which cause toxic products in the presence of
peroxide decomposition to produce secondary reaction products, and provide other
physiological and pathological effects (Nurhasanah et al. 2019; Jadhav et al. 2022).
Thermoxidative changes in heated oil with total polar components (TPC), anisidine
(AV) values, formation of color components, and changes in the composition of fatty
acids and tocopherols.

3 Structured Lipids

3.1 Definition

SLs are the result of modification or restructuring of triacylglycerols, which can be

obtained by chemical or enzymatic interesterification of triacylglycerols containing
short, medium, and/or long chain fatty acids. SLs can be sourced from animal
or vegetable fats, or genetic engineering. SLs are synthesized for the purpose of
obtaining functional lipids or nutraceuticals, which can improve or modify the phys-
ical, chemical, and rheological characteristics of oils and fats, and changing or
enhancing nutrition properties of food, giving a certain health benefit. The properties
of fatty acids based on physical, biological, and nutritional are largely determined
by the position, number, and configuration of their double bonds. These determine
200 S. Nurhasanah and S. J. Munarso

the shape of the molecules, the way molecules can pack together in solid phases,
monolayers, bilayers, and how individual molecules can interact with enzymes and
receptors. Changes in the composition and position of the fatty acids in triacyl-
glycerols are caused by the interesterification reaction causing changes in several
properties such as solid fat content, crystallization behavior, physical properties,
chemical properties, thermal properties, and consistency when compared to native
lipids. The changes are verified through physical, chemical, and functional analysis
due to the fact that SLs can present triacylglycerols. SLs can provide essential fatty
acids such as linoleic (18:2n-6), oleic acid (18:1n-9), and linolenic acid (18:3n-3) as
found in many vegetable oils, such as soybean oil, olive oil, palm oil, coconut oil.
These fatty acids are essential for growth and development throughout the human
life cycle, as well as the promotion of improvement in health, and plays an important
role in reducing risk of metabolic syndrome (Moreira et al. 2017).
One form of lipid structure is specialty fats. Specialty fats are type of fat that has
a special function, so that it has the potential for special applications such as to make
confectionery fat, usually used to replace all or part of cocoa butter and dairy butter.
Among specialty fats, cocoa butter alternatives represent perhaps the most diverse
and widely developed specialty fats. Cocoa butter alternatives are designed to provide
an alternative, both economically and functionally, to a high-value ingredient, cocoa
butter. These fats are formulated or modified from palm oil, since the cocoa butter
and dairy butter are expensive and their supply unreliable. The role of these fats
is to provide specific texture and richness of taste. Furthermore, the confectionery
fats should have a sharp melting behavior to melt easily in the month (Talbot 2015;
Ramadan 2019).
SLs are beneficial for human nutrition because they can be tailor-made to target
specific diseases and metabolic conditions, and reduce calories by specifically posi-
tioning certain fatty acids in the glycerol backbone. Such low-calorie fats are usually
designed to take advantage of the limited absorption of long-chain saturated fatty
acids or the lower caloric density of short-chain saturated fatty acids. SLs also lower
cholesterol, LDL cholesterol, and triglycerides, given a normal diet as well as an
atherogenic diet. In addition, lipid accumulation in the arteries was also significantly
reduced. Thus low-calorie structured fat has the added benefit of reducing serum
and liver lipids which are considered risk factors for cardiovascular disease (Kanjilal
et al. 2016).
Lipase is more promising when certain positional modifications of triacylglyc-
erols are addressed, in addition to resulting in less residue in SL production. In addi-
tion, enzymes can be reused many times, minor lipids, among other compounds with
bioactive functions, are preserved due to milder reactions compared to chemical inter-
esterification. After the interesterification reaction, the number of triacylglycerols is
higher than diacylglycerol and monoacylglycerol content, hydrolysis followed by
esterification leaving a small amount of diacylglycerol and monoacylglycerol, either
by using lipase Lipozyme TL IM or by Rhizopus sp. (Moreira et al. 2017).
Structured Lipids Based on Palm Oil 201

3.2 Structured Lipid Synthesis

3.2.1 Chemical

Chemically catalyzed is the most common interesterification process which is

described as a “reshuffling”. Accidental and limited interesterification can occur
when the oil is heated to above 200 °C, as illustrated by the characteristic change in
crystallization of confectionery fat after deodorization (Gunstone and Norris 2013).
In general, the chemical interesterification process takes place with three kinds of
reactions at once, namely: (1) Alcoholysis, (2) Acidolysis, and (3) Transesterifica-
tion. For the interesterification reaction, use under low pressure in a water bath at
80–85 °C for 30 min. After drying, 1% (m/m) sodium methoxide powder (Oliveira
et al. 2017).
The advantages of chemical interesterification are relatively faster methods, one
of which is for the synthesis of tripalmitin. Palmitic acid is not easily oxidized even
at relatively high temperatures. The final product contained is 97.60% PPP, 1.46%
dipalmitin, 0.08 and 0.26% monopalmitin. The disadvantage of this method is that the
alkaline catalyst in liquid form mixes perfectly with the product so that purification
of the product from the catalyst is relatively difficult. In addition, the use of alkaline
catalysts results in side reactions that are very disturbing, namely the occurrence of
saponification reactions to form unwanted by-products thereby reducing yields (Wei
et al. 2015).

3.2.2 Enzymatic

In addition to chemically catalyzed randomization, there are also directed interester-

ification processes in which the equilibrium associated with complete randomization
is disturbed either by distillation of the most volatile components, such as FAME
(fatty acid methyl esters) (Gunstone and Norris 2013).
Enzymes are well-known as one of the biocatalysts of a wide variety of processes
that are highly effective and efficient catalysts characterized by high activity and
selectivity to accelerate biochemical reactions. The conversion of substrate catalyzed
by lipase into products is carried out by reducing the energy of the activation reaction,
carried out under mild conditions (pH and temperature), very good selectivity, and
substrate activity. Many commercially essential processes, especially in various food,
cosmetic, and pharmaceutical industries use lipases as a natural catalyst. Lipases are
a powerful tool for biotransformation on a broad substrate range (Robinson 2015;
Liu and Dong 2020; Pandey et al. 2020). Catalysts are widely used in the chemical
industry sector: in basic chemistry, in polymerization chemistry, and in refining
such as refining including coconut oil-based products, as well as in liquid reactions
industrial processes (pharmacy, food, cosmetic, etc.) (Bedade et al. 2019).
202 S. Nurhasanah and S. J. Munarso

The enzymes that can be used for the above purposes are Lipozyme TL IM and
Novozyme 435. Lipozyme TL IM is a type of commercial lipase enzyme immobi-
lized using silica gel from Thermomyces lanuginosus and is widely used for various
esterification reactions. This enzyme has positional specificity of the TAG molecule,
namely at the primary position (sn-1,3) with a mild optimal temperature (25–60 °C).
Meanwhile, Novozyme 435 is a type of non-specific immobilized commercial lipase
enzyme using macroporous acrylic resin beads with an optimum temperature of
40–60 °C (Ortiz et al. 2019).
Modification of the simple mixture by these two lipases causes a change in thermal
profile, which causes a delayed crystallization process, as well as the decrease in
enthalpy, indicating that it is interesterified the sample releases less energy during
crystallization. Energy measured in this process refers to the rearrangement of the
liquid phase molecules, which release energy and reformulate in the solid state. In
addition, the crystallization curve also reveals that the higher degree of unsaturation
fatty acids in the TAGs lowers the end temperature and enthalpy of crystallization.
This phenomenon can be explained by the fact that enthalpies are calculated by the
area of each peak and according to the number of crystals formed during cooling indi-
cated crystallization curve of the SLs mixture difference performances presented by
lipases, synthesized SLs with Rhizopus sp. lipase showed three crystallization peaks,
due to incomplete restructuring of the tri- and saturated TAG formed presenting a
higher enthalpy value for each peak compared to samples catalyzed by Lipozyme
TL IM (Moreira et al. 2017).
Bioavailability of fatty acids is not only determined by composition, but also deter-
mined by the position of each type of fatty acid on the glycerol backbone. Unsaturated
long chain fatty acids in position sn-2 can improve bioavailability, because pancre-
atic lipase confers less activity on these fatty acids when esterified to the sn-1 and
sn-3 positions. The absorption of long chain fatty acids and MCFA will be higher
if they are present in the sn-2 position of the TAG. The TAG will be converted into
2 monoglycerides which are more water soluble by pancreatic lipase in the body.
At position sn-1,3 can support low lipid absorption, without compromising the fatty
acids located in sn-2. Based on this perspective, this type of SL synthesis requires
modification at certain positions on the glycerol backbone, which can be obtained
by enzymatic interesterification (Moreira et al. 2017).
Lipases can be produced by plants, animals, and microorganisms, and microbial
lipases are receiving more attention from the industry because of their ability to
remain active at extreme temperatures, organic solvents, pH, exhibit high selectivity,
wide substrate specificity, and do not require cofactors. The advantages of lipases can
reduce the number of hazardous solvents needed; the total reaction steps make the
process cheaper and more environmentally friendly. Other benefits of using lipases
are mild reaction conditions, low energy consumption, biodegradability, and yields of
a pure product. The lipase from Thermomyces lanuginosus expressed in Aspergillus
oryzae is the first commercialized recombinant lipase. Lipase is an enzyme that can
work reversibly, catalyzes the hydrolysis of triacylglycerol to glycerol and free fatty
acids and, or partial hydrolysis to diacylglycerols (DAGs) and monoacylglycerols
(MAGs) (Subroto et al. 2019).
Structured Lipids Based on Palm Oil 203

Enzymatic interesterification process system can be done with the feedbatch

system and system continuous. (Wei et al. 2015) observed Thermomyces lanuginosus
lipase activity during batch interesterification process. Activity enzymes decreased
rapidly after 6 times (equal to 24 h) reaction on batch system.

4 Structured Lipids Based on Palm Oil

Palm oil has special fatty acids and other minor components, making it possible to
be used as a raw material for the manufacture of SLs so that their bioavailability
increases. Functional oil and fat production can be catalyzed by lipase. Fats/oils can
improve physicochemical and nutritional properties using a lipase catalyst. Unsatu-
rated fatty acids in triglycerides are mostly in the sn-2 position. The lipase specificity
of sn-1,3 can be used to catalyze the transesterification reaction while maintaining
sn-2 fatty acids. Strategies for Human Milk Fat Subtitute (HMFS) have been devel-
oped to mimic the fat composition and distribution of human milk. HMFS is used
in infant formula to mimic the fat of breast milk. Breast milk is the main choice for
newborns, infant formula that most closely resembles breast milk is a good substitute
for baby nutrition when breastfeeding is insufficient or cannot be done. Fatty acid
components in human milk fat are oleic acid, palmitic acid, linoleic acid, followed
by stearic acid, myristic acid, and lauric acid. The distribution position of breast milk
fatty acids is 70% palmitic acid is in the sn-2 position and UFA (oleic acid, linoleic
acid linolenic acid, etc.) is in the sn-1.3 position. This characteristic makes breast
milk fat different from most vegetable oils in that most of the UFA is in the sn-2
position and the SFA especially palmitic acid is in the sn-1,3 position (Qin et al.
2014; Hasibuan et al. 2021).
Cocoa butter (CB) is a very important ingredient that contributes to the textural and
sensory properties of confectionery products, particularly chocolate products (up to
32% CB in chocolate formulations). CB is hard and brittle under room temperature,
but when eaten, it melts perfectly in the mouth with a soft creamy texture and a cold
sensation. The CB polymorphism has a major influence on the physical properties of
chocolate products, such as gloss, snap, contraction, heat resistance, fast and sharp
melting in the mouth, and bloom resistance. The special nature of CB is not followed
by supply, price, use in hot climates, and consistency of quality between regions
(Zhang et al. 2020). SL plays a role in the development of bakery products. Based
on the SL1 melting profile, it is suitable for the manufacture of biscuits and cakes
in terms of sensory and organoleptic properties. The organoleptic characteristics
and quality of biscuits and cakes made with SL1 were indistinguishable from those
prepared with traditional bread tallow. This suggests that bakery fats can be fully
replaced by SLs studied to prepare trans-free low-calorie cakes and biscuits. Thus
the use of low-calorie fat can be applied to bakery products, cakes and biscuits
are not only trans-free but also have added value for health such as low calories
and hypocholesterolemic properties (Kanjilal et al. 2016). Binary mixture of PKO
204 S. Nurhasanah and S. J. Munarso

and interesterified fats was dominant in β' crystal. The chocolate showed consistent
texture before and after tempering process (Zhang et al. 2020).
PKO with other vegetable oils can be used as raw material for SL. PKO contains a
wide variety of fatty acids (C6−C20) and has more β' polymorphs than β. Margarine
made with SL from a mixture of PKO, canola oil, and stearin fractions has a hardness,
stickiness, or compactness similar to commercial margarine. Therefore, the SL is
suitable for the formulation of trans-free margarines with low atherogenicity and
desirable textural properties (Kim 2008).

5 Conclusion

Palm oil is produced from the extraction process of the mesocarp part of the palm fruit
(CPO) and palm kernel oil (PKO). Naturally, oils and fats have specific characteris-
tics, developments in food processing and technology have made these characteris-
tics unable to meet all the expected requirements for obtaining products with certain
functional properties by making SLs by modifying or restructuring triacylglycerols,
which can be obtained by chemical or enzymatic interesterification of triacylglyc-
erols. SLs can be sourced from animal or vegetable fats, or genetic engineering. SL
is synthesized with the aim of obtaining functional lipids or nutraceuticals, which
can improve or modify the characteristics of oils and fats, and change or enhance the
nutritional properties of foods, providing certain health benefits. Palm oil has special
fatty acids and other minor components, making it possible to use it as a raw material
for SL so that its bioavailability increases.

Acknowledgements If any.

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Recent Developments
of the Agroindustry Byproducts
Utilization in Bacterial Cellulose
Production and Its Medical Devices
Applications

Khatarina Meldawati Pasaribu, I Putu Mahendra, Novia Amalia Sholeha,

Nanang Masruchin, Efri Mardawati, and Myrtha Karina

Abstract Bacterial cellulose (BC) is a renewable material which is currently playing

a central role in medical device applications due to its biocompatibility and capability
to be structurally, chemically, and morphologically modified at macro, micro, and
nano scales. In addition, BC also has high water content, mechanical strength, and
purity which are also excellent properties for use in biomedical applications. Despite
the numerous advantages of BC properties for biomedical applications, its use for
commercialization is still a challenge due to the high expense of the carbon and
nitrogen sources required for BC synthesis. This study will provide an overview
of numerous alternate sources of carbon and nitrogen from agricultural byproducts
for BC synthesis that have been investigated and the potential of BC to be used for
medical devices.

K. M. Pasaribu (B) · N. A. Sholeha · N. Masruchin · M. Karina

Research Center for Biomass and Bioproducts, National Research and Innovation Agency of
Indonesia (BRIN), Cibinong 16911, Indonesia
e-mail: [email protected]
K. M. Pasaribu · N. A. Sholeha · N. Masruchin · E. Mardawati · M. Karina
Research Collaboration Center for Biomass and Biorefinery, BRIN – UNPAD, Jatinangor 45363,
Indonesia
I. P. Mahendra
Institut Teknologi Sumatera, Jalan Terusan Ryacudu, Way Hui, Jati Agung, Lampung Selatan,
Lampung 35365, Indonesia
K. M. Pasaribu · N. Masruchin · M. Karina
Research Collaboration Center for Nanocellulose, BRIN - UNAND, Padang 25163, Indonesia
E. Mardawati
Faculty of Agroindustrial Technology, Universitas Padjadjaran, Jatinangor, Bandung 45363,
Indonesia
N. A. Sholeha
School of Vocational Studies, Bogor Agricultural University (IPB University), Jalan Kumbang
No. 14, Bogor 16151, Indonesia

© The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2024 207
M. A. R. Lubis et al. (eds.), Biomass Conversion and Sustainable Biorefinery, Green
Energy and Technology, https://doi.org/10.1007/978-981-99-7769-7_10
208 K. M. Pasaribu et al.

Keywords Bacterial cellulose · Production · Substrate · Agroindustry byproduct ·

Medical devices

1 Introduction

In the biomedical field, synthetic or natural polymers are used for different purposes
(Sionkowska 2011; Tian et al. 2012). Although the application of synthetic polymers
in medical devices has advanced significantly, it is known that using these polymer
products in biomedicine poses irritation risk issues for tissue due to their low biocom-
patibility (Gunatillake et al. 2003; Bilgi et al. 2016). The higher biocompatibility
properties of natural polymers make these polymers the most widely recommended
alternatives as synthetic polymers substitutes in medical applications (Aravamudhan
et al. 2014). Currently, plant-derived cellulose is the most abundant natural polymer
produced on earth and is used extensively in the textile and food industries. However,
its use in the medical and cosmetic fields is limited due to its impurities such as lignin,
hemicellulose, and pectin (Klemm et al. 2005; Moran-Mirabal and Cranston 2015).
Apart from being sourced from plants, there is cellulose which is sourced from
microbial synthesis called bacterial cellulose (BC). In contrast to plant-derived cellu-
lose, BC is a cellulose with a high purity, thus it has found extensive use in the
biomedical sector, including as polymer scaffolding for bone and cartilage repair,
wound dressings to restore burned skin, a membrane for skin drug delivery, arti-
ficial blood vessels for microsurgery, and wound dressings to treat burned skin
(Trovatti et al. 2011; Gomes et al. 2013). Bacterial cellulose is a natural extra-
cellular polymer with the molecular formula of (C6 H10 O5 )n which is synthesized
extensively by Gluconacetobacter strains via linear coupling of glucopyranose sugar
monomers (Shoda and Sugano 2005). The strain that was employed to synthesize
this polymer is non-pathogenic, extensively distributed in fruits and their products,
and simple to grow in a lab (Klemm et al. 2009; Moosavi-Nasab and Yousefi 2011).
This polymer is a biomaterial with superior properties due to its nanofibrous network
structure (50–120 nm), 100% purity, high surface area, high crystallinity, high degree
of polymerization, high capacity to absorb and hold water, high wet tensile strength,
biocompatible, and easily degradable (Chen et al. 2013; Dhar et al. 2019).
However, the use of BC for industrial-scale applications for medical devices is
constrained by its high production costs. For industrial-scale application, production
parameters including temperature, pH, surface area to volume ratio of culture medium
air–liquid interface (S/V), inoculum ratio and incubation time should be optimized for
high quality, cost-effective, and high-yield BC production (Bilgi et al. 2016; Gea et al.
2018). Among the factors that affect the production costs, the sources of carbon and
nitrogen used in the BC production are one of the factors that significantly determine
the manufacturing costs, which can amount to 65% of the total cost (Chen et al. 2013;
Sudying et al. 2019). The traditional source of carbon for BC fermentation is sugars
such as glucose, fructose, and sucrose. Coconut water is currently the most affordable
and sustainable raw material utilized in BC industrial production. However, coconuts
Recent Developments of the Agroindustry Byproducts Utilization … 209

are only grown in the tropics countries such as the Philippines, Indonesia, and other
South and Southeast Asian (Cao et al. 2018). In most countries in the world, coconut
production is extremely low and import-dependent. Thus, the use of this alternative
substrate cannot be a solution for reducing BC production costs in many countries.
Meanwhile, the demand for BC, especially in the food and renewable material
industry, continues to increase and it is predicted that demand for BC will eventually
outpace supply in the future (Çakar et al. 2014; Padmanaban et al. 2015; Gea et al.
2022). Adding to this, in 2016, BC market was valued at US$207 million, and it is
anticipated to reach US$700 million in 2026 (Calderón-Toledo et al. 2022).
In recent years, many researchers have focused on efforts to produce BC by
developing cost-effective carbon and nitrogen feedstocks from local agroindustry
product residues (Hong et al. 2011, 2012). Agricultural waste is known to contain
high amounts of lipids, carbohydrates, including mono-, oligo- and polysaccharides,
and proteins which can be converted into renewable energy sources with high added
value (Calderón-Toledo et al. 2022). The advantage of using agricultural residues as
raw materials in BC production is to reduce the cost of raw materials so that they
can be mass-produced and commercialized and can be used as a basis for developing
advanced materials, especially in their applications as biomedical devices (Chen
et al. 2013). In addition, effective utilization of agricultural byproducts will be a
good mode of recycling biomass which will simultaneously reduce the burden of
waste treatment (Cao et al. 2018).

2 Bacterial Cellulose

Bacterial Cellulose is an extracellular polymer produced by bacteria from the genera

Sarcina, Gluconacetobacter, and Agrobacterium through oxidative fermentation in
non-synthetic and synthetic media (Esa et al. 2014; Huang et al. 2014). Among the
various types of bacteria, Gluconacetobacter xylinus is the producer that receives
the most attention for commercial-level production because it can digest a variety
of carbohydrates in liquid media to produce BC in larger quantities (El-Gendi et al.
2022). In the BC synthesis, the bacterial body will releases glucose chains through
the tiny holes in their cell envelopes, and the hydroxyl groups in the polymer chains
form microfibrils, which subsequently group to form random nanofibers, will create
3D nanopore networks (Calderón-Toledo et al. 2022). The main structure of this
fibril is formed of β-1,4-glycosidic linear polysaccharide structure that is connected
by hydrogen bonds (Ul-Islam et al. 2012). Because it is secreted in the form of 2–
4 nm fibrils, which are 100 times smaller than plant cellulose fibrils, this fiber is
categorized as a nanoscale network (Gayathry and Gopalaswamy 2014; Costa et al.
2017).
210 K. M. Pasaribu et al.

In its natural state, BC possesses good hydration and high water holding capacity
which is up to 100 times of its initial weight (Rani and Appaiah 2013). In addi-
tion, BC has a high degree of polymerization, crystallinity, and biological adapt-
ability. BC also offers biocompatibility, biodegradability, and renewable capabili-
ties (Costa et al. 2017). Therefore, BC is a desirable material for future use in a
variety of industries, including electronics, paper, and food as well as the biomedical
industry (bone and cartilage reconstruction, tissue engineering, wound dressings,
implants, corneal restorations, orthodontics, artificial blood vessels), pharmaceuti-
cals, veterinary medicine, and the leather industry (Andritsou et al. 2018; Popa et al.
2022).

3 Nutritional Requirements for Bacterial Cellulose

Production

Currently, the use of bacteria to produce high-purity cellulose is in great demand

because this cellulose has good nanometric, thermal, and mechanical properties.
Moreover, it does not require aggressive treatment for purification and is environ-
mentally friendly (Lin et al. 2013; Shi et al. 2014). Since this sort of cellulose is
produced by bacteria, the availability of nutrient-rich elements in the growth media
is a key factor for the production process as illustrated in Fig. 1 although there are also
several things that need to be considered, such as, culture type (static vs. dynamic,
surface vs. submerged), pH, shear force, and level of oxygenation support (Fernandes
et al. 2020; Popa et al. 2022). Therefore, carbon sources include sugars such as fruc-
tose, glucose, sucrose; polysaccharides such as amylose or starch; nitrogen such as
peptones, yeast extract, and casein hydrolyzates; inorganic salts such as ammonium
sulfate; organic acids such as citric acid; some low molecular weight hydroxylated
compounds such as glycerol, mannitol, and ethanol; are among the most important
in the production of BC.
Currently, synthetic media, such as Hestrin-Schramm (HS) media, are utilized
extensively in laboratory-scale BC production. However, the use of this medium in
a large-scale BC production is not recommended due to its high cost, low yield,
time consumption, and intensive labor required (Calderón-Toledo et al. 2022; Popa
et al. 2022). These facts present a significant barrier and restrict the commercial
use of BC products. Consequently, finding inexpensive substrates becomes crucial
(Cavka et al. 2013; Huang et al. 2014). According to estimations, fermented media
consumes up to 65% of the whole production budget (Chen et al. 2013; Sudying
et al. 2019). Thus, the main challenge in bacterial cellulose production is to find
an abundant, suitable, inexpensive, and non-competitive carbon source with food
production (Dórame-Miranda et al. 2019). In recent years, agroindustrial wastes have
emerged as a potential affordable substitute medium for BC production (El-Gendi
et al. 2022). This method is advantageous because it is able to produce high-quality
Recent Developments of the Agroindustry Byproducts Utilization … 211

Fig. 1 Key factor in BC production process

BC and reduces waste disposal by converting waste water into a cheap fermentation
promoter (Popa et al. 2022).

4 Agroindustry Byproducts that Used as Bacterial

Cellulose Medium Culture

Given that BC has demonstrated excellent potential as a useful biopolymer in many

applications, numerous studies have been conducted to identify an efficient method
for generating BC at a reasonable price. The most notable is the utilization of agri-
cultural byproducts as a replacement of carbon and protein for BC production. Each
of the agroindustry byproducts and the bacterial strain that have been studied in BC
production is shown in Table 1. This table also includes information of BC yields
and highlights of BC produced from each agro-industrial byproduct used.

4.1 Wine Pomace

Pomace grapes are the main solid byproduct of the wine industry consisting of grape
skins and seeds. These byproducts contain lignified fiber and soluble compounds
such as sugars, phenolics, and a number of alcohols (Muhlack et al. 2018; Troncozo
et al. 2019). As a result, grape pomace and its hydrolyzate have the potential to
be used as a low-cost bioactive substrate to produce BC which has antioxidant and
antibacterial activity. The pomace substrate pretreatment process was carried out
by hydrolysis using commercial enzymes, namely pectinase and cellulase (Li et al.
2021).
212 K. M. Pasaribu et al.

Table 1 The utilization of agroindustry byproducts as a carbon and nitrogen source replacement
in BC synthesis
Byproduct Bacterial strain Yield Highlight References
source
Wine pomace Komagataeibacter 4.28 ± 0.21 g/L Possess Li et al. (2021)
rhaeticus in 10 day antioxidant and
antibacterial
activity
Carob-haricot Gluconacetobacter 1.8 g/L in 9 days Predict and Bilgi et al. (2016)
bean extract xylinus optimize BC
production yield
Mango Komagataeibacter 25.34 g/L in Provide a high Calderón-Toledo
(Mangifera rhaeticus 21 days yield with a et al. (2022)
indica) waste reasonable price
extract
Citrus pulp Gluconacetobacter 8.77 g/L in Almost achieve Cao et al. (2018)
hansenii and 10 days industrial level
Gluconacetobacter
xylinum
Corn stalk Acetobacter 2.86 g/L in Matched with the Chen et al. (2013)
xylinum 10 days idea of a
biorefinery
because it is
affordable, green,
and sustainable
Sugarcane Komagataeibacter 2.96 g/L in Provides a Dhar et al. (2019)
straw xylinus 12 days different strategy
to meet the
commercial
demand
Pecan nutshell Gluconacetobacter 2.816 g/L in Chemical Dórame-Miranda
(Carya entanii 28 days functionalization et al. (2019)
illinoinensis) of BC becomes
methylcellulose
and reduces its
crystallinity
Dry olive mill Gluconacetobacter 0.85 g/L in Demonstrate Gomes et al.
residue sacchari 4 days positive outcomes (2013)
to combat high
production costs in
BC
Cashew tree Komagataeibacter 2.8 g L−1 in Up to a 33% of Pacheco et al.
residue rhaeticus 7 days cost reduction (2017)
(continued)
Recent Developments of the Agroindustry Byproducts Utilization … 213

Table 1 (continued)
Byproduct Bacterial strain Yield Highlight References
source
Coffee cherry Gluconacetobacter 8.2 g/L in Provides a more Rani and Appaiah
husk hansenii 14 days affordable (2013)
alternative
substrate and
offers solution for
toxic agricultural
waste disposal
Rice washing Komagataeibacter 0.20 g/L in A potential Sudying et al.
drainage nataicola 13 days alternate carbon (2019)
source that does
not require any
pretreatment
Tobacco waste Acetobacter 5.2 g/L in 7 days Nicotine in TWE, Ye et al. (2019)
extract (TWE) xylinum which is known to
inhibit BC
production, can be
removed by steam
distillation
treatment

4.2 Carob and Haricot Bean Extracts

Carob (Ceratonia siliqua L.) production in Turkey is known to reach 15 × 103 tons/
year with a total production of more than 400 × 103 tons/year in the world. Carob
contains about 50% sugar (75% of sucrose and the rest is fructose, maltose, and
glucose), 8% protein, and a number of important minerals (AYAZ et al. 2007; Bilgi
et al. 2016). Meanwhile, haricot beans (Phaseolus vulgaris) are known to contain
protein of 18.5–22%, a number of minerals (such as magnesium and calcium) and
carbohydrates (de Almeida Costa et al. 2006; Shimelis et al. 2006). Therefore, the
mixture of haricot nuts and carob has the potential to be employed in BC production
as sources of carbon and nitrogen, respectively. According to the studies that have
been published, Plackett-Burman and Central Composite Design techniques were
used to prepare carob and haricot bean extracts as alternative growth media for G.
xylinus in the development of cost-effective BC production methods. While, the
pretreatment process used in this medium is by hydrolyzation process of the extract
with distilled water using an autoclave (Bilgi et al. 2016).

4.3 Mango Variety (Mangifera Indica)

The Food and Agriculture Organization estimates that around 55% of mango
produced globally, equivalent to 1.1 million tons, is wasted due to rotting during
214 K. M. Pasaribu et al.

the transport, packaging, and storage (García-Sánchez et al. 2020). Mango extract
(Mangifera indica) is known to contain 13.7–15.0% of sugar in the form of glucose,
fructose, and sucrose and 1.5–5.5% of protein, making mango waste potentially can
be used as an alternative low-cost substrate source for BC production (Prasanna
et al. 2003; Maldonado-Celis et al. 2019). Moreover, mango fermentation with yeast
and bacteria has been widely used in producing probiotic juice, wine, vinegar, yeast
lipase, etc. (Li et al. 2012; da Pereira et al. 2019). Previous studies stated that it is
necessary to use mangoes as a medium for BC production by first applying a pretreat-
ment in the form of a hydrolysis process followed by a sugars reversion reaction with
acid hydrolysis in 1 M HCl (Calderón-Toledo et al. 2022).

4.4 Citrus Pulp Waste

Oranges are one of the fruits that are produced in very big quantities worldwide,
with an annual production reaching 27.1 million tons where 40–60% of its total
weight is a byproduct in the form of peels and pulp (Fan et al. 2016). Currently,
oranges have been used as vitamins by ingesting their active constituents or as a
beverage ingredient in ethanol, vinegar, liquor, lactic acid beverages, mushrooms,
and high-protein feed (Shan 2016). In China, peel of the orange has been used to
produce traditional medicine by extracting its pectin, refined oil, and flavonoids
content. However, orange pulp manufacturing has not yet been optimized, resulting
in an accumulation of this byproduct. While it is known that citrus pulp is used as a
production medium in BC by first being pretreated with enzymatic hydrolysis using
cellulase and pectinase enzymes to reduce the viscosity of citrus pulp-based medium
and reduce sugar so that mono/disaccharides could be used by the microorganism to
synthesize BC (Cao et al. 2018).

4.5 Corn Stalk

Corn stalks are a byproduct of the annual production of corn agriculture, which
is widely available worldwide (Luo et al. 2017). Thus far, this byproduct has only
been treated inefficiently as compost (Boufi and Chaker 2016). Recently, the utiliza-
tion of this biomass has been developed to produce bioethanol with biorefinery
process (Shen and Wyman 2011; Cheng et al. 2017). Furthermore, according to a
number of research, corn stalk prehydrolyzate contains high sugar content that can be
employed in the fermentation process, offering another option for maximizing the use
of these agricultural byproducts (Boufi and Chaker 2016; Esteves Costa et al. 2016).
However, it is known that the utilization of the prehydrolyzate content in corncobs
does not optimally support the production of BC. Therefore, its utilization as a carbon
source to cultivate Acetobacter xylinum required a pretreatment using acetic acid and
Recent Developments of the Agroindustry Byproducts Utilization … 215

followed by pre-hydrolysis liquor (PHL) detoxification using activated carbon and

ion exchange resin (Cai et al. 2016; Jiang et al. 2016).

4.6 Sugarcane Straw

Sugarcane is an agricultural commodity that is produced in large quantities every

year. Brazil, is one of the largest sugarcane producing countries in the world, esti-
mated to produce 93.3 × 106 tons of sugarcane waste each year in the form of
bagasse and straw (dos Santos Rocha et al. 2017). It is estimated that every 1000 kg
of sugar cane that processed in agroindustrial, will result in residues of 176 kg and
231 kg in the form of straw and dregs respectively. However, this residue is still not
managed adequately, and as a result, the primary method of this waste treatment is
burning. Recently, bagasse has been utilized as a component in bioethanol, biobu-
tanol, or for bioenergy production for power plants, however the utilization of straw
as waste is still rare (Dhar et al. 2019). It is known that sugarcane straw contains cellu-
lose, hemicellulose, and lignin fraction ranging from 35–45%, 25–35%, to 10–25%
respectively, which can be used as a medium for BC synthesis. Several pretreatment
techniques such as enzymatic hydrolysis, acid hydrolysis, or hydrothermal treatment
can be used to optimize the ultilization of this byproduct as a medium for BC produc-
tion. Interestingly, the utilization of biomass as the carbon source was reported to
produce BC with higher yield compared to pure glucose (Costa et al. 2017).

4.7 Pecan Nutshell

Pecan nut (Carya illinoinensis) is an agricultural product in which its seed is the
part that is most widely used. The Food and Agriculture Organization (FAOSTAT)
estimates that 460 × 103 tons of Pecan nut are produced annually while 40–50%
is hazelnut shells which is a byproduct of this agroindustry (do Prado et al. 2014;
Hilbig et al. 2018; Dórame-Miranda et al. 2019). Pecan nut shells are recognized
to have the potential to be used as a source of nutrition for bacteria that specialize
in producing BC because they contain significant levels of crude fiber (particularly
lignin and cellulose) and carbohydrates (~90%) (Flores-Córdova et al. 2016). The
utilization of hazelnut shells as a substrate in the production of BC is also reported
does not require special pretreatment (Dórame-Miranda et al. 2019).

4.8 Dry Olive Mill Residue

Olive oil industry is one of the significant economic activity in several nations,
including Portugal (Trichopoulou and Critselis 2004; Sieri et al. 2004). An estimated
216 K. M. Pasaribu et al.

40 × 103 tons of two-phase olive pomace (OP), which is made up of the pulp, skin,
and stone fragments of the olives, are produced in Portugal each year as a result of
the industrial extraction of olive oil (Fernández-Bolaños et al. 2006). OP has been
used to make OP oil with a yield of 9.2%, therefore this method creates a byproduct
known as dry olive mill residue (DOR), which can reach up to 35% of the mass
of the original dry OP (Vlyssides et al. 2004; Sánchez Moral and Ruiz Méndez
2006). Currently, DOR has been utilized to produce electricity, organic fertilizers,
and additives for animal feed (Martı́n Garcı́a et al. 2003; López-Piñeiro et al. 2007).
The utilization development of DOR continues to be studied and it is reported that
DOR contains a substrate rich in sugars monomer as a source of carbon and nutrients
for BC production after pretreatment in the form of mild acid hydrolysis (Gomes
et al. 2013).

4.9 Cashew Tree Residues

Cashew (Anacardium occidentale) is an agricultural product whose main product

is cashew nuts and cashew juice and has byproducts in the form of cashew pulp
and exudate. Since cashew nuts are the most valuable output of the cashew tree,
the exudate from this tree trunk must regularly be removed in order to stimulate
the production of cashew nuts. Each of these trees produces approximately 700 g
of exudate each year, which becomes a waste (Pacheco et al. 2017). Cashew tree
exudate contains of arabinogalactan proteins, mono- and oligosaccharides, mineral
salts, and 70% of branched heteropolysaccharides, called as cashew gum (Pereira-
Netto et al. 2007; Silva et al. 2010). The polymer chains are mainly composed
of D-galactopyranose units, as the primary building block, which are joined by β-
(1 → 4) glycosidic bonds. Glucose, rhamnose, glucuronic acid, and arabinose are
additional sugars that can incorporate branched cashew gum chemical structures
(Pacheco et al. 2017). At least 68 × 103 and 48 × 103 tons of cashew gum and tree
exudate respectively, are produced annually (Pacheco et al. 2017). Large quantities
of this byproduct made an opportunity to carry out a research in efforts to treat
this waste for the benefit of the economy and environmental sustainability. This
residue has been extensively utilized in the food, biotechnology, and pharmaceutical
industries (Kumar et al. 2012). The usage of cashew tree exudate and cashew gum
as an alternative carbon source in BC production has been examined and claimed
to exhibit good chances to be used in lowering the production cost (Pacheco et al.
2017).

4.10 Coffee Cherry Husk

Coffee cherry husk is one of the most prevalent byproduct that is produced in coffee
cherries agroindustrial. The amount of this byproduct is almost 18% of the total coffee
Recent Developments of the Agroindustry Byproducts Utilization … 217

cherries that are processed (Rani and Appaiah 2013). This husk is rich in polyphenols,
minerals, proteins, and carbohydrates. However, their use in agriculture has been
restricted leading to a significant pollution issue at coffee cherries processing due to
the existence of undesirable compounds like tannins, caffeine, and other polyphenols.
While, according to studies, coffee cherry husk has the potential to be utilized in
bioprocesses as an alternative substrate which is affordable. Coffee cherry husk can
be added straight to the medium without going through any extra processing as a
carbon source (Rani and Appaiah 2013).

4.11 Rice Washing Drainage

The global average of rice consumption per year in 2016 was 478.38 × 106 tons
(Sudying et al. 2019). According to this data, almost all industries that are engaged in
rice processing washed the rice with clean water before being cooked for consump-
tion. In this rice washing drainage, numerous amino acids, saccharides, vitamins,
and other nutrients are contained. Previous study reported the utilization of rice
washing drainage as a source of carbon for BC synthesis can be used as cost reduction
alternative because does not need special pretreatment (Sudying et al. 2019).

4.12 Tobacco Waste Extract

Tobacco (Nicotiana) is a plant with high economic value that is cultivated world-
wide because it is the main ingredient in the cigarette production industry (Wang
et al. 2015). Every year, it is estimated that about half (50%) of the tobacco used
in cigarette production ends up as waste (Liu et al. 2015). This waste is found in
the form of tobacco stems, tobacco leaves, and unwanted waste (Zhong et al. 2010;
Wang et al. 2013). This waste has a high toxic nicotine content so it tends not to
be managed but only thrown away or burned (Zhang et al. 2013; Okunola et al.
2014). This certainly endangers human health and contributes to environmental
pollution. Recently, tobacco waste has been widely studied for its use as a substrate
for producing fertilizers, pectinases, and some drug precursors (Wang et al. 2015;
Zheng et al. 2017). Furthermore, the potential of tobacco waste as a substrate for BC
production was also studied because it has a high sugar content in the form of glucose,
sucrose, fructose, and other polysaccharides. In its utilization as a substrate for BC
pretreatment production in the form of a steam distillation process used to remove
nicotine which can inhibit microorganisms in producing BC. TWE is reported to be
an ideal substrate for lower cost BC production (Ye et al. 2019).
218 K. M. Pasaribu et al.

5 Bacterial Cellulose in Medical Application

The unique physico-mechanical properties of BC, especially its biocompatibility

properties make it widely used for direct applications in biomedical fields such as
tissue engineering, wound healing, and drug delivery (Choi et al. 2022; El-Gendi
et al. 2022). In this section, the superior qualities of BC will be described, along with
the rationale for its use and research development in medical device.

5.1 Wound Dressings

In the biomedical sector to date, BC has played a significant role in the development
of dressings for various types of skin trauma such as chronic skin ulcers, burns,
surgical incisions, and other trauma (Popa et al. 2022). In general, the wound healing
process is a complex process which is divided into four stages, namely: hemostasis,
inflammation, proliferation, and maturation. The type of dressing material used to
treat the wound is known to affect how quickly and effectively each of these stages
progresses (Kushwaha et al. 2022). According to the scientific method of wound
care, a bandage must have a number of qualities in order to support and hasten
the wound healing process. These qualities include the ability to maintain moisture,
absorb exudate, support angiogenesis, enable gas exchange, create thermal insulation
in the wound area, prevent microbial infection, and be non-toxic, non-sticky, and
non-allergenic (Niculescu and Grumezescu 2022).
In this case, BC is one of the greatest materials for creating a good wound dressing
because BC has a high water holding capacity which enables it to absorb wound
exudate, maintain a moist environment at the injury site, and stimulate the acceler-
ation of the re-epithelialization process (Hajmohammadi et al. 2020; Pasaribu et al.
2020a, b). Additionally, the high water content of BC can also prevent pain and
secondary trauma in patients during dressing removal (Weyell et al. 2019). Numerous
hydroxyl groups in BC can form hydrogen bonds with water to produce flexibility,
which makes it flexible enough, especially for contoured skin surface, to act as wound
physical barrier from the outside environment (Swingler et al. 2021). However, from
a biofunctional perspective, BC lacks the antibacterial and antimicrobial proper-
ties which are helpful in preventing infection throughout wound healing phase. In
order to maximize the contribution of BC wound dressings to expedite the wound
healing process, a combination with other substances is required (Choi et al. 2022).
Numerous studies show that BC works well for wound healing when mixed with other
compounds or materials such nanoparticles, benzalkonium chloride, hydroxyapatite,
Aloe vera, and vaccarin (Picheth et al. 2017; Hasibuan et al. 2021). The effectiveness
of BC as a wound dressing is also proven by the availability of several BC commer-
cial wound dressing products such as BioFillTM (Curitiba, Brazil) and DermafillTM
(Londrina, Brazil) which are used to treat burns and boils, Membracel® (Curitiba,
Brazil) for ulcers and leg vein lacerations, xCell® (New York, NY, USA) for venous
Recent Developments of the Agroindustry Byproducts Utilization … 219

leg ulcers, and EpiProtect® (Royal Wootton Bassett, UK) for burns (Cielecka et al.
2019).

5.2 Bone Tissue Engineering

Tissue engineering has recently emerged as a viable solution for the replacement of
damaged tissue (Swetha et al. 2010; Zhou et al. 2014). Therefore, research engaged in
the manufacture of extracellular matrix (ECM) scaffolds that imitate the composition
and architecture of natural ECM of target tissues is widely carried out, especially in
bone tissue engineering (Mano et al. 2007; Stevens 2008; Swetha et al. 2010). Bone,
which is a component of the skeletal system, is produced through a formation process
known as osteogenesis that developed during the prenatal until adulthood phase of
each individual. Thus bone has the capacity for restoration and regeneration to repair
minor injuries and mechanical damage caused by normal trauma as long as it is not
a birth defect. Bone tissue engineering using scaffolds is currently widely recom-
mended in the treatment of bone repair by stimulating bone regeneration through a
complementary combination of cells, biomaterials, and factor therapy (Li et al. 2013;
Vadaye Kheiry et al. 2018).
The selection of biomaterial for scaffolding in bone tissue engineering is crucial
because in this approach the biomaterial acts as a structural and/or functional
supporting template for the cell regeneration process (Sill and von Recum 2008).
Bone tissue engineering requires unique biomaterials with characteristics such as
strong mechanical stress resistance and tunable biodegradability (Atila et al. 2019).
Bacterial cellulose (BC), is one of the biomaterials that have received extensive study
for Bone tissue engineering scaffolding production due to its high compatibility,
although on the other hand, these polymers do not have the appropriate mechan-
ical properties (Sell et al. 2010; Huang et al. 2014). Due to its superior purity, tensile
strength, modulus, and elasticity, BC is used in regenerative medicine more frequently
than plant cellulose. Moreover, BC also has biofunctionality and is biocompatible
(Khan et al. 2015). BC-based scaffolds also have porosity and 3D network struc-
tures that support cell growth (Wan et al. 2007). BC was also reported to have a
structure similar to bone collagen and increase cell proliferation in-vitro (Chen et al.
2009; García-Gareta et al. 2015). When compared to animal-derived biomaterials
like collagen, the usage of BC scaffolds has also been shown to enhance tissue and
bone regeneration and decrease the potential danger of cross-infection (Kong et al.
2004; Popa et al. 2022).

5.3 Dental Implants

In dental clinical practice, dental implants are a common operation, however this
practice is frequently constrained because the maxillary region lacks the necessary
220 K. M. Pasaribu et al.

bone height for the treatment (de Oliveira Barud et al. 2021). The integration of dental
implants into the surrounding tissue is a significant concern. Additionally, for bone
regeneration, osseointegration between the implant and bone must be complete (Choi
et al. 2022). Despite their excellent application in tissue engineering and biomedical
devices, BC-based materials are still under-explored in dentistry. Whereas the use of
BC for commercial purposes in dental applications is very profitable because of its
good absorption capacity, volume retention, and mechanical strength (Mensah et al.
2022). In addition, research suggest that BC can preserve graft space, enhance bone
structure, and be used for dental implant insertion when applied to guided tissue
regeneration techniques for the treatment of periodontal disease (de Oliveira Barud
et al. 2021).

5.4 Vascular Grafts and Artificial Blood Vessels

Vascular Grafts and Artificial Blood Vessels is a method used in replacing blood
vessels by cutting damaged or diseased blood vessels. In this treatment, the devel-
opment of intimal hyperplasia is severely hampered by the material incompati-
bility of artificial blood vessels (Choi et al. 2022). Currently, ePTFE, Dacron, and
polyurethane are the most commonly utilized materials for artificial blood vessels.
Comparative studies have shown that BC is superior to PET and ePTFE products for
usage as a vascular graft material (Picheth et al. 2017). BC is a promising new mate-
rial for application in artificial blood vessels because BC nanofiber may avoid blood
clots by displaying delayed thrombin production on the surface (Fink et al. 2010). In
general, the use of pure BC nanofibrous scaffold for tissue regeneration does have
limitations due to the presence of nanopores which can inhibit cell infiltration and
vascularization of the 3D scaffold. However, several modifications made to the BC
reported the potential for the BC to adapt to mechanical properties similar to those of
small diameter vessels (<5 mm) (Picheth et al. 2017). Development of BC tubes also
shows better resemblance to the human saphenous vein (4.27 × 10–2% per mmHg
for 30–120 mmHg) than commercially available Dacron saphenous vein products
and ePTFE (Choi et al. 2022).

5.5 Delivery of Drug and Bioactive Agents

In recent years, numerous natural biopolymer-based hydrogels have been extensively

researched for drug delivery applications (Dasari et al. 2022). Drugs are manufactured
into drug delivery dosage forms because they are typically administered in numerous
doses, possess fluctuating plasma concentrations, and have shorter half-lives. BC-
based hydrogel scaffolds have recently been employed in drug delivery applications
due to their potential in terms of high reactive surface, fine tissue structure, and high
porosity (Swingler et al. 2021). Moreover, BC can be easily modified, blended, and
Recent Developments of the Agroindustry Byproducts Utilization … 221

impregnated with nanoparticles to change how receptive it is to drug release (Choi

et al. 2022). Lyophilization followed by immersion is the most typical technique used
to load the drug into the membrane of BC (Swingler et al. 2019). The drugs most
commonly incorporated into bacterial cellulose are anti-inflammatory drugs, such
as diclofenac and ibuprofen, and antimicrobial drugs (Ao et al. 2020; Bernardelli de
Mattos et al. 2020; Junka et al. 2020). The effectiveness of BC as a drug delivery
material can be boosted by utilizing the tensile strength and water absorption of BC
to load it with antimicrobial substances such as antibiotics, to provide new features
and functionalities (Gupta et al. 2019, 2020; Swingler et al. 2021). Researchers also
frequently use BC-based as controlled-release drug delivery agents, for instance, the
application of BC powder as paracetamol tablets coat via a spray-coating approach,
demonstrates that the thickness of the BC film is used to affect the in-vitro drug
release rate (Amin et al. 2012). Due to the lack of barrier interference and erythema,
BC has also been widely researched as a transdermal medication delivery agent.
It has been discovered that the skin is well-tolerated and moisturized because BC
porosity can manage hydrophilicity of environment and also alter the release rate of
the drug (Almeida et al. 2014; Ullah et al. 2016).

6 Conclusion and Future Perspective

This study focuses on the review of alternative substrates that sourced from the agroin-
dustry byproducts in order to replace carbon and nitrogen and the advantages of the
basic properties of bacterial cellulose for application as medical devices. Bacterial
cellulose is a polysaccharide synthesized by various non-pathogenic bacteria under
specific cultivation conditions. This appealing biopolymer has a number of physico-
chemical, mechanical, and biological qualities, including: environmental friendli-
ness, biodegradability, biocompatibility, non-toxicity, optimal viscoelasticity, a 3D
porous structure, high tensile strength, easy to modify, sufficient capacity to retain
large amounts of water, and higher crystallinity and purity than plant cellulose. Either
by itself or in combination with other biopolymers and bioactive substances BC has
been reported to have therapeutic effects on various body areas of humans when
used as medical devices. However, the development of BC as the main component
in commercial medical device applications is still constrained due to the high-cost
production of BC. Therefore, further research to find alternative synthesis media to
reduce the BC production costs will continue to develop in the future.

Acknowledgements Khatarina Meldawati Pasaribu expresses her sincere gratitude to the National
Research and Innovation Agency of Indonesia (BRIN) for providing the necessary resources to
conduct her postdoctoral work at the Nanocellulose research group at the Research Center for
Biomass and Bioproducts from July 1, 2022, to June 30, 2023.
222 K. M. Pasaribu et al.

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Extraction, Isolation, Purification,
and Potential Application of Xylose
and Xylooligosaccharides
from Lignocellulosic Biomass

Euis Hermiati, Hans Wijaya, and Dwi Ajias Pramasari

Abstract Xylose and XOS become products of interest and have good markets.
Xylose and XOS are derived from xylans, which are parts of hemicellulose fraction
of lignocellulosic biomass. The demand tends to increase due to depletion of fossil
resources and a new paradigm shift in consumer preferences for healthier and natural
products. There are different extraction methods or fractionation processes to extract
xylose and XOS from lignocellulosic biomass feedstocks, including autohydrol-
ysis and hydrolysis using acid, alkaline, solvent, and inorganic salts. The hydrolysis
usually involves high temperature and pressure. It is important to find the most suit-
able, effective, and affordable method to first fractionate biomass major chemical
components and achieve the practical applications of the method. There are some
unwanted substances and oligosaccharides of various degree of polymerization (DP)
produced during the manufacture of XOS and xylose. These substances should be
removed to obtain xylose and XOS with high purity. Some purification methods
such as solvent extraction, adsorption separation, chromatographic separation, and
membrane filtration, or combinations of those methods could be applied. Xylose can
be utilized for a variety of purposes, either directly as xylose or as a feedstock for the
subsequent production of a variety of products, including furfural, furfuryl alcohol,
xylitol, levulinic acid, ethanol, butanol, and hydrogen through chemical or biological
conversion. XOS can be used as antioxidant, prebiotic, gelling agent, cosmetics, plant

E. Hermiati (B) · D. A. Pramasari

Research Center for Biomass and Bioproducts, National Research and Innovation Agency
(BRIN), Jl. Raya Bogor Km 46, Cibinong, Bogor 16911, Indonesia
e-mail: [email protected]
D. A. Pramasari
e-mail: [email protected]
E. Hermiati
Research Collaboration Center for Biomass and Biorefinery Between BRIN, Universitas
Padjadjaran, Jatinangor, Sumedang 45363, Indonesia
H. Wijaya
Research Center for Applied Microbiology, National Research and Innovation Agency (BRIN), Jl.
Raya Bogor Km 46, Cibinong, Bogor 16911, Indonesia
e-mail: [email protected]

© The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2024 229
M. A. R. Lubis et al. (eds.), Biomass Conversion and Sustainable Biorefinery, Green
Energy and Technology, https://doi.org/10.1007/978-981-99-7769-7_11
230 E. Hermiati et al.

growth regulator, treatment of diabetes, arteriosclerosis, and colon cancer, and are
commercially interesting to be used as animal feed, food, beverage, and pharmaceu-
tical ingredients. The production of xylose and XOS from lignocellulosic biomass
still has some challenges regarding the technology to produce the products that are
feasible commercially, but it has good prospects in the future as the increasing aware-
ness to use renewable resources to produce healthier and environmentally friendlier
products.

Keywords Lignocellulosic biorefinery · Oligosaccharides · Separation ·

Value-added products · Xylan derivatives

1 Introduction

The depletion of fossil resources has triggered research on the use of renewable
resources for producing fuels and other compounds that are now obtained from
fossils. Lignocellulosic biomass is among potential feedstocks for the production
of fuels and chemicals, since it is abundantly available as by-products of agricul-
ture and forestry industries. Lignocellulosic biomass can be used for the production
of cellulosic ethanol, which can substitute the use of gasoline. Unfortunately, the
commercialization of cellulosic ethanol or the second generation of bioethanol is still
hampered by the high production cost, which makes the price of cellulosic ethanol
high and hard to compete with gasoline. Efforts to produce economical cellulosic
ethanol have been conducted through the improvement of technology, starting from
pretreatment up to fermentation. Another effort that could be done is by applying
biorefinery concept. In biorefinery, the transformation of lignocellulosic into energy,
especially cellulosic ethanol, and other chemicals or bioproducts is carried out as
an integrated technology, so that each component of lignocellulosic biomass could
be converted and utilized efficiently. By doing this, the whole process would be
economically more feasible.
Lignocellulosic biomass is made up of three major components: cellulose, hemi-
cellulose, and lignin. In the production of cellulosic ethanol, the focus is to obtain
cellulose that will be converted to glucose, which will further fermented to ethanol.
Lignin is a challenge and becomes a recalcitrance in the production of cellulosic
ethanol, but it is beneficial for producing different kinds of aromatic compounds
or monomers that could substitute fossil-derived monomers in polymer industries.
Hemicellulose is easily hydrolyzed to its oligomers or monomers, for example
through water, acid, alkali, or enzymatic hydrolysis, and then separated from lignin
and cellulose. Unlike cellulose, which has orderly packed structure, hemicellu-
lose has amorphous characteristics with different kinds of branches. After being
recovered and purified from lignocellulosic biomass, hemicellulose monomers can
undergo a variety of transformations that result in compounds with added value,
hence increasing the profit margin for the relevant businesses. Before completely
Extraction, Isolation, Purification, and Potential Application of Xylose … 231

Bioethanol
Glucose Lactic acid
Cellulose
Succinic acid
5-HMF

Pretreatment/ Adhesive
Lignocellulosic Lignin Lignosulfonate
biomass Fractionation
Biosurfactant

Xylitol
Furfural
Hemicellulose XOS Furfuryl alcohol
Xylose Levulinic acid
Biobutanol
Bioethanol

Fig. 1 Schematic diagram of lignocellulosic biomass fractionation

hydrolyzed to its monomers, the hemicellulose could also be hydrolyzed to oligosac-

charides. As an integrated process, it is important to take advantage of every step in the
lignocellulosic biomass conversion to ethanol, for example optimizing the pretreat-
ment process to produce both cellulosic ethanol and sugar monomers or oligosac-
charides. Schematic diagram of lignocellulosic biomass fractionation is shown in
Fig. 1.
Xylose and XOS become products of interest and have good markets. According
to Data Bridge Market Research, during the projection period of 2022–2029, the
global xylose market is projected to increase at a CAGR of 5.4% from its value of
USD 1,797.80 million in 2021 to USD 2,738.21 million by 2029. The price of XOS
is dependent on its purity (70–95%), ranging from USD 25 to 50/kg, and the global
market is anticipated to reach USD 130 million by 2025 (Santibáñez et al. 2021;
Singh et al. 2018; Brenelli et al. 2022). The global XOS market was worth USD
88.09 million in 2016 and is anticipated to reach USD 119.62 million by the end of
2022, expanding at a compound yearly growth rate (CAGR) of 5.23% from 2016 to
2022 (Ahmad 2019).
There are still some challenges in the recovery of xylose or XOS from lignocel-
lulosic biomass, either in the extraction, isolation, or purification step for producing
good quality of xylose and XOS which is economically feasible. However, a new
paradigm shift in consumer preferences for heather and natural products as well as
consumer health consciousness have significantly increased the sales and attraction
for xylose and XOS, especially in the thriving food and beverage industry. In this
chapter highlights will be on the xylan sources, structure, and its derivatives; extrac-
tion and isolation of xylose and XOS, purification of xylose and XOS, and potential
applications of xylose and XOS in food, energy, and pharmaceutical industries.
232 E. Hermiati et al.

2 Sources and Structure of Xylans, XOS, and Xylose

Hemicellulose is the base feedstock for xylose and other xylan-based sugars, particu-
larly xylooligosaccharides (XOS), which are supposed to be utilized in the packaging,
food, pharmaceutical, biomedical, cosmetic, textile, and papermaking industries
(Yohana et al. 2022; Lu et al. 2021; Zhang et al. 2017a). Hemicellulose is a complex
heteropolysaccharide that consists of D-xylose, L-arabinose, D-glucose, L-galactose,
D-mannose, D-glucuronic acid, and D-galacturonic acid. Common composition
of these sugars in hemicellulose is shown in Fig. 2. Hemicelluloses are found in
several polymeric configurations, including xylan, arabinoxylan, xyloglucan, and
glucuronoxylan, and develop in 26% of the dry weight of hardwoods, 22% of soft-
woods, and approximately 25% of agricultural waste (Zhao et al. 2020; Narisetty
et al. 2022).
A dominant component in hemicellulose of lignocellulosic biomass is usually
xylan. Xylan is also the second major component in lignocellulosic biomass after
glucan. The content of glucan and xylan in several lignocellulosic biomass is
presented in Table 1. Terrestrial plants xylans are heteropolymers having a β-
(1 → 4)-d-xylopyranose backbone and branches formed of short carbohydrate
chains, such as D-glucuronic acid or its 4-O-methyl ether, L-arabinose and/or
different oligosaccharides, made up of D-xylose, L-arabinose, D- or L-galactose
and D-glucose (Ebringerová et al. 2005; Scheller and Ulvskov 2010). Based on
its primary structure in plant tissue, xylan is generally divided into homoxy-
lans and heteroxylans, including glucuronoxylan (GX), arabinoglucuronoxylan
(AGX), glucurono-arabinoxylan (GAX), arabinoxylan (AX), and complex heterox-
ylan (CHX) (Ebringerová et al. 2005), with structures shown in Fig. 3a–d. When
hydrolyzed, the xylans are converted to XOS and xylose, depending on the severity
of the process. Production of xylose and xylooligosaccharides (XOS) from hemi-
cellulose fraction of lignocellulosic biomass would be beneficial to support the
commercialization of cellulosic ethanol.

Fig. 2 General composition of lignocellulosic biomass (a), and neutral and acid sugars in
hemicellulose (b) (Narisetty et al. 2022)
Extraction, Isolation, Purification, and Potential Application of Xylose … 233

Table 1 Glucan and xylan content of lignocellulosic biomass

Biomass Glucan (%) Xylan (%) References
Sugarcane straw 38.9 23.9 Brenelli et al. (2022)
Sugarcane bagasse 43.49 22.68 Zhang et al. (2020a)
Corn stover 34.4 22.8 Kumar et al. (2009)
Corn cob 37.9 27.83 Lee and Jeffries (2011)
Sweet sorghum bagasse 37.1 18.2 Wen et al. (2018)
Oil palm empty fruit bunch 36.8 21.5 Ho et al. (2014)
Oil palm frond 40.56 17.40 Goh et al. 2010)
Rice husk 36.83 19.66 Wu et al. (2018)
Rice straw 38.66 22.93 Wu et al. 2018)
Wheat straw 36.3 19.0 Kootstra et al. (2009)
Sweetgum 40.2 15.7 Geng et al. 2019)
Switchgrass 35.5 22.4 Geng et al. (2019)
Maple 43.2 13.0 Geng et al. (2019)
Eucalyptus 44.4 14.3 Geng et al. (2019)
Poplar 43.8 14.8 Kumar et al. (2009)
Aspen 52.4 14.9 Jun et al. (2012)
Loblolly pine 36 7.5 Rana et al. (2012)

XOS (C5n H8n + 2 O4n+1 ) are mixture of oligosaccharides composed of xylose

molecules linked through β-1, 4-xylosidic bonds with a number of xylose residues
vary between 2 and 10 (Aachary and Prapulla 2008), some have branching structures
due to the presence of many side groups (Chen et al. 2021; Yan et al. 2022). As we
know besides xylose, xylan usually contains α-D-glucopyranosyl uronic acid or its
4-O-methyl derivative, acetyl groups, or arabinofuranosyl residues. These structures
result in some branched XOS containing these side groups which have various biolog-
ical properties (Aachary and Prapulla 2011). Figure 3e shows the chemical structure
of XOS. The structural properties of XOS are affected not only by the origin of
the xylan-rich hemicelluloses but also by the manufacturing process (Akpinar et al.
2010; Bian et al. 2013). These variables influence the degree of polymerization (DP),
monomeric units, and linkage types (Aachary and Prapulla 2011).
Xylose or D-xylopyranose (D-Xylp) is an aldopentose sugar, having chemical
formula C5 H10 O5 and molecular weight of 150.13, with a relative density of 1.525
and melting point 145–150 °C. soluble in water, hot ethanol, and pyridine, but not
soluble in ether. It appears as colorless or white crystal or powder, and it has a sweet
taste. Xylose has an aldehyde functional group so that it is classified as reducing
sugar. The chemical structure of xylose can be seen in Fig. 3f.
234 E. Hermiati et al.

a b

Homoxylan (HX):
(a) β-(1→3)-D-xylan; (b) β-(1→3 and 1→4)-D-xylan

Arabinoxylan (AX):
(a) 2, 3-arabino-D-xylan; (b) 3-arabino-D-xylan
c d

Glucuronoxylan
(4-O-methyl-D-glucurono-D-xylan) (MGX) Glucuroarabinoxylan
2, 3-arabino-2-(4-O-methylglucurono acid)-xylan
(GAX)
e f

Fig. 3 Structure of xylans. a Homoxylan. b Arabinoxylan; c Glucuronoxylan; d Glucuroarabi-

noxylan), e XOS and f xylose (Fu et al. 2019; Pinales-Márquez et al. 2021)

Fig. 4 Acid hydrolysis of xylan to xylose (Lu et al. 2021)

Extraction, Isolation, Purification, and Potential Application of Xylose … 235

3 Extraction and Isolation of Xylose

and Xylooligosaccharides

There are different extraction methods or fractionation processes used to extract

xylose and XOS syrup from an abundance of lignocellulosic biomass feedstocks.
In order to produce xylose and XOS efficiently, finding the most suitable, effective,
and affordable method to first fractionate biomass’ major chemical components and
achieve the practical applications of biomass is critical. According to Fernandez
(2019), many existing methods for isolating xylose start with biomass hydrolyzates
or sugar in solution derived from a renewable resource and involve energy-intensive
processes such as evaporation and phase transitions. This is distinct from typical
sugar production, which may be quickly harvested from sugar cane or sugar beets.

3.1 Acid Hydrolysis Method

A popular method for hydrolyzing hemicellulose with a significant conversion effi-

ciency is using diluted acid hydrolysis. Hemicellulose breakdown in the solid phase
can be catalyzed by dilute acid and heat to dissolve various oligosaccharides ranging
from xylose to xylotriose and above in the liquid phase. Inorganic acids, including
sulfuric, hydrochloric, and nitric acids, as well as organic acids, such as oxalic,
phosphoric, acetic, maleic, succinic, and citric acids can be used in the conventional
diluted acid hydrolysis. Thus, by introducing acids from outside, the hemicellulose
depolymerization process can be accelerated (Huang et al. 2021; Krishania et al.
2018; Paiva et al. 2009; Rahman et al. 2007; Rocha et al. 2014). In addition, Chen
et al. (2020) described that the activation energy of organic acid degradation is usually
higher than that of inorganic acids, but the degree of glucose and xylose dissolved in
the hydrolysate with organic acids is less than that of inorganic acids, such as sulfuric
acid.
The primary limitation of the acid hydrolysis approach is the production of unfa-
vorable reactions at the same time. The acid hydrolysates usually contain three
groups of undesired chemical components: (a) compounds derived from the hemi-
cellulose structure (organic acids); (b) sugar breakdown derivatives (furfural and
hydroxymethylfurfural (HMF)); and (c) excessive lignin derivatives (aromatic and
polyaromatic compounds). To increase the xylose yield and decrease the creation
of undesired compounds, the precise processing parameters, such as solid-to-liquid
ratio, acid concentration, temperature, and duration of heating, must be followed
(Delgado Arcaño et al. 2020; Harahap 2020; Shahbazi and Zhang 2010).
The two forms of acid employed in the acid-based process are concentrated acid
and diluted acid. Concentrated acids not only dissolve cellulose crystals but also
break down cellulose and hemicellulose into sugars and stimulate the cleavage of
glycosidic bonds. In the United States, sulfuric acid converts hemicellulose and cellu-
lose into sugars at a rate of 80–90%. However, there are risks related to the use of
236 E. Hermiati et al.

concentrated acids, as well as difficulties in acid recovery, which limit the accep-
tance of the technology. Concentrated acid can be produced efficiently at moderate
temperatures, requiring less heating energy. Shahbazi and Zhang (2010) described
that hemicellulose can be efficiently extracted and recovered as dissolved sugars at
medium temperatures (140–190 °C), and the extraction process can be made more
effective by adding an acid solution. On the contrary, dilute acid hydrolysis often
requires greater temperatures with less chemicals. As a result, organic acid hydrol-
ysis has steadily gained prominence and the issue caused by equipment corrosion can
be prevented (Harahap 2020; Cheng et al. 2018; Inamuddin 2021). At temperatures
below 160 °C, acid hydrolysis caused the hemicellulose to become nonhomoge-
neous and dissolve into soluble oligomers, such as XOS and sugar monomers, such
as xylose. Furthermore, the oligomers formed during the hydrolysis of hemicellu-
lose by a random acid attack have different degrees of polymerization (Lee et al.
1999). According to studies on the effects of conventional heating with organic and
inorganic acids on the sugar monomers produced in the hydrolysate and from the
saccharification and fermentation of the solid corncob residues, organic acids such as
maleic acid and oxalic acid could produce more monomer sugars in the hydrolysates
and more ethanol from the solid residuals (Lee and Jeffries 2011).
Some benefits of utilizing dilute acid include a faster reaction rate, lower acid
consumption, lower cost than alkaline pretreatment, and no need for recycling. It is
interesting to note that under particular process circumstances, diluted acid hydrolysis
preferentially extracted the hemicellulose component (Harahap 2020; Cheng et al.
2018; Inamuddin 2021). The acid hydrolysis approach is a conventional method for
dissolving hemicellulose to xylose or XOS. Unfortunately, numerous factors, such
as chemical stability, side reactions, toxic pollution, and proton selectivity, must be
addressed in the near future (Yan et al. 2022; Huang et al. 2021). The summary of
some research regarding the xylose yield after acid hydrolysis method in various
biomass is shown in Table 2.
According to earlier research by Zhang et al. (2017b), concentrated acetic acid
was discovered to perform better than inorganic acid for hydrolysis. Hydronium ions
disrupt the connections in the interiors and/or exteriors of hemicelluloses during
acetic acid hydrolysis, which causes XOS to become soluble. The solid phase of

Table 2 The xylose yield after acid hydrolysis method in various biomass
Solvent (concentration) Biomass Xylose yield (%) References
Oxalic acid (150 mmol/ Corncob 85 Cheng et al. (2018)
L)
Oxalic acid (1.2%) Corncob 96.1 Jin et al. (2018)
Hydrochloric acid (1%) Corn stover 88.8 Feher et al. (2017)
Sulfuric acid (3.1%) Sugarcane bagasse 96 Paiva et al. (2009)
Sulfuric acid (2%) Oil palm empty fruit 91.27 Rahman et al. (2007)
bunch
Sulfuric acid (6%) Kenaf core fiber 86.50 Judiawan et al. (2019)
Extraction, Isolation, Purification, and Potential Application of Xylose … 237

XOS has more exposed cellulose that is ready for cellulolytic hydrolysis due to its
solubility. The majority of the severe inorganic acids have the ability to catalyze and
dissolve the glycosidic linkages that connect xylose units in the skeleton of the xylan
polymer, but their action is random. Consequently, much xylose rather than XOS
is generated (Zhang et al. 2017b). Meanwhile, Yohana et al. (2022) discovered that
the maximum XOS content from sugarcane trash and corncob was 4.74 g/L after
hydrolysis with 2% oxalic acid for 45 min and 1.31 g/L after hydrolysis with 2%
acetic acid for 60 min. Furthermore, according to Zhang et al. (2017a), acetic acid in
corncob hydrolysate generated XOS yields of roughly 45.91%. The XOS yields from
hydrochloric acid and sulfuric acid are 22.54% and 9.38%, respectively. It is also
worth noting that the XOS derived from acetic acid prehydrolyzate was mostly made
up of xylobiose, xylotriose, and xylotetrose, with substantially less xylopentaose and
xylohexaose. This was attributed to the fact that acetic acid pretreatment resulted in a
reduced degree of polymerization (DP) enrichment. Han et al. (2020) discovered that
gluconic acid treated in corncob was effective to produce XOS with a yield 56.2%.
Gluconic acid is a mono-carboxylic acid that could release H+ to depolymerize
hemicelluloses. It is adopted as a green and environmentally friendly solvent for the
extraction of sugars.

3.2 Alkaline Hydrolysis Method

In complement to acid hydrolysis method, the alkaline method is frequently used

to isolate hemicellulose from lignocellulosic biomass by causing cellulose to swell,
rupturing the links between hemicellulose and lignin, and diluting the hemicellulose.
It has been discovered that the yield of hemicellulose is affected by the type and
concentration of alkali, extraction time, and temperature (Huang et al. 2021). Figure 5
illustrates the general mechanism for alkaline hydrolysis, in which the ester link
between lignin’s ferulic acid and the sugar residue of hemicellulose is cleaved (Lu
et al. 2021).
The alkaline hydrolysis was the subject of groundbreaking work by Adams and
Castagne (1951) as stated by Huang et al. (2021). The research was regarding the
various hemicellulose fractions obtained from the holocellulose of wheat straw. The
process also produced the D-xylose and monomethoxyl galacturonic acid complex,
which was resistant to acid. Inorganic alkali solutions are extensively utilized in

Fig. 5 Alkaline hydrolysis of xylan (Lu et al. 2021)

238 E. Hermiati et al.

alkaline hydrolysis, and sodium hydroxide is one of the most prevalent. In addi-
tion, hydrogen peroxide extraction is a frequent alkaline procedure for isolating
hemicellulose from biomass. High extraction rates, purity, and DP are features of
hemicelluloses extracted through alkaline hydrolysis. As a result, alkaline-extracted
hemicellulose is predicted to be used in a variety of industries. The standard alkaline
procedure, on the other hand, involves significant pollution and costs (Lu et al. 2021).
Samanta et al. (2012) reported that sodium hydroxide was superior to potassium
hydroxide in terms of xylan recovery from corncobs. Furthermore, the study discov-
ered that increased alkali concentrations led to higher hemicellulose degradation.
Besides that, a study by Jiang et al. (2019) using sorghum steam discovered that
alkaline hydrolysis using 1.4% NaOH at 37.5 °C and 9.9 h resulted in a maximum
xylose yield (57.7%). The study also stated that increasing alkali loading, reaction
time, and temperature enhanced xylose yield. By using only alkali extraction, the
yield of xylan is relatively low. Combination with other procedures, such as steam
and ultrasound, might be rising the xylan yield. For instance, ultrasound-assisted
alkali extraction could significantly increase XOS yield in corncobs, with a release
level of 174.81 mg/g matrix (Yan et al. 2022; Kawee-ai et al. 2016).

3.3 Autohydrolysis Method

The autohydrolysis process requires specialized equipment at specific temperatures

and pressures. The principle of autohydrolysis is that the hydronium ions are formed
as a result of water autoionization, which results in the catalytic depolymerization of
hemicelluloses to XOS and xylose. The addition of the hydronium ion to the mixture,
which causes cleavage of acetyl groups in acetic acid, allows for the synthesis of
XOS. When the processing severity factor is increased, the DP decreases, and the
degradation of XOS into xylose increases. The downside of this procedure is the
need for specialized equipment as well as the production of undesirable compounds
in the XOS mixture (Samanta et al. 2015; Surek and Buyukkileci 2017).
Nabarlatz et al. (2007a) have used a variety of biomass, consisting of wheat straw,
barley straw, rice husks, corncobs, olive stones, and almond shells, for XOS produc-
tion using autohydrolysis. This study discovered that the XOS yield varied with xylan
content and accessibility and was inversely related to the acetyl concentration in the
biomass feedstock. According to Surek and Buyukkileci (2017), autohydrolysis of
hazelnut shells produced XOS with the highest percentage of low-DP XOS at higher
severity values. To achieve the appropriate DP, temperature and holding time must
be precisely regulated. Additionally, the results indicated that the severity factor
was effective in determining the patterns of oligomers, monomers, and by-products
produced after autohydrolysis.
Extraction, Isolation, Purification, and Potential Application of Xylose … 239

3.4 Combination Hydrolysis (Physical–Chemical Hydrolysis)

Method

Physical hydrolysis includes processes using hydrothermal, steam explosion, ultra-

sonic, and microwave (Yan et al. 2022). A hydrothermal is an instant conversion
of biomass in hot water with controlled high pressure and/or temperature, which
involves the self-expansion of a material. In this process, the moisture in the biomass
is vaporized under the impact of the rapid pressure transfer to a vacuum, which causes
the material to expand and become more texturized. Sometimes the hydrothermal
combines with alkaline hydrolysis or acid hydrolysis to separate hemicellulose to
improve the yield of xylose or XOS (Lu et al. 2021; Yan et al. 2022). The research
conducted by de Sá et al. (2020) found that the hydrothermal-assisted acid hydrolysis
in sugarcane bagasse resulted in 80.13 mmol/L xylose. The XOS produced (35 g/
L) by hydrothermal pretreatment of sugarcane bagasse with sulfuric acid was totally
hydrolyzed by the acid post-hydrolysis, which happened in less than 20 min (Nakasu
et al. 2017).
Harahap (2020) and Dulie et al. (2021) stated that the oligomeric form of hemi-
cellulose predominates in the solution as a result of steam explosion circumstances.
Hemicellulose oligomers need to be post-treated in order to be broken down. To maxi-
mize the performance of the steam explosion, pre-treatment is also required before
the operation. The steam explosion pretreatment method has drawbacks, such as the
destruction of a portion of the pentosan, despite being a cheap pretreatment method.
A xylose yield of 27.58 g/g dry corn cob was obtained by Zhang et al. (2014) from
the pilot-scale production of xylose using a screw-steam explosion extruder and prior
sulfuric acid impregnation. On the other hand, Duangwang et al. (2016) discovered
that a combination between sulfuric acid hydrolysis and superheated steam explo-
sion in a pilot scale gave the highest yield of xylose, up to 87.58 g/kg of dry oil
palm empty fruit bunches. The yield of XOS was 37%, obtained from acetic acid
hydrolysis of wheat straw with steam explosion (Cao et al. 2021).
In the case of alkaline extraction of hemicellulose, the use of ultrasonic has been
shown to be successful. The breaking of the polysaccharide glycoside bond brought
on by ultrasonic pretreatment considerably increases the yield of the extracted hemi-
cellulose at low temperature and short time. Additionally, hemicellulose extracted
with the use of an ultrasonic method has a higher molecular weight, greater thermal
stability, fewer acidic groups, fewer linked lignin molecules, and a lower branching
degree (Lu et al. 2021). Sun and Tomkinson (2002) determined that the yield of
hemicellulose from ultrasonic method in wheat straw hydrolysate was 1.8% higher
than that of conventional alkaline hydrolysis.
Compared to conventional approaches, microwave extraction of hemicellulose has
some benefits, such as energy savings and low toxicity. Additionally, the extraction
of branched hemicellulose with microwave assistance is more successful, resulting
in reduced yield and molecular weight of the hemicellulose recovered through
microwave extraction. It has been challenging to implement in large-scale indus-
trialization thus far (Lu et al. 2021). A study conducted by Lin et al. (2017) stated
240 E. Hermiati et al.

that a practical method for developing XOS production from various hemicellulose
was by utilizing organic acids as catalysts and induced with microwave irradiation.
Using this process, the XOS yield from beechwood xylan, corncob, and waste liquor
pulp is 39.42, 27.46, and 30.89%, respectively. Another research by Hermiati et al.
(2020) demonstrated that the use of maleic acid in combination with microwave
assistance could result in a 24.3% yield of xylose. Meanwhile, microwave-assisted
acid hydrolysis of rice husk could obtain 32.96% of xylose yield (Zhang et al. 2020b).

3.5 Solvent Hydrolysis Method

Organic solvents, such as pure organic solvent and complex organic solvent systems,
can be used to separate hemicellulose. Lu et al. (2021) divided the solvent hydrol-
ysis method into organosolv extraction, ionic liquid extraction, deep-eutectic solvent
extraction, and high-pressure CO2 /H2 O technology. Organosolv extraction has the
benefit of immediate hemicellulose extraction without delignification. The acetyl
functional groups found in lignocellulosic biomass’ cell walls are not transformed
to acetic acid and then expelled. Nevertheless, organic solvents are poisonous,
combustible, volatile, and difficult to decompose, which causes environmental prob-
lems (Lu et al. 2021). Liu et al. (2018) demonstrated that xylose and XOS concen-
tration from ethanol-based auto-catalyzed organosolv method in bamboo can reach
up to 15 g/L and 18 g/L, respectively.
The merits of ionic liquid (IL) extraction include excellent high-recovery effec-
tiveness and toxic-free solvent recycling. Even though ILs are not always costly, the
preparation process is frequently complex and more expensive than using conven-
tional media. Furthermore, hemicellulose recovered by ILs frequently contains
certain contaminants and is only mildly decomposed, which causes some side chains
to partially break. As a result, ILs’ continued commercial application is still chal-
lenging (Lu et al. 2021; Chen et al. 2014). The extraction of xylose by IL is affected by
temperature and time. When temperature raised, the amount of xylan recovered in the
solid fraction of corn stover is reduced, meaning that more xylan was dissolved into
the hydrolysate (Xu et al. 2012). Xylose yield was also increased decreased when the
temperature was increased from 50 to 110 °C, reaching maximum (75.9%) at 110 °C,
but then decreased at higher temperature, implying that at higher temperature some
of the dissolved xylan was degraded to lower molecular weight compound, such as
furfural (Xu et al. 2012). At low temperature (70 °C), the xylose yield is increased
when the duration of corn stover extraction using ionic liquid was increased from 3
to 24 h (Xu et al. 2012).
Deep-eutectic solvent (DES) extraction was an alternative solvent with 100%
atomic economy compared to the previous common solvent for xylose and XOS
extraction. The other advantages of DES are non-toxic, simple preparation, recy-
clable, low purity, and low extraction selectivity of hemicellulose (Lu et al. 2021).
Research by Morais et al. (2018) shows that a yield of 14.81% was obtained when
the optimized aqueous DES solutions were used to extract the hemicelluloses from
Extraction, Isolation, Purification, and Potential Application of Xylose … 241

Eucalyptus globulus wood. These results are higher than the yields resulting from
water or alkaline hydrolysis.
A growing number of studies have focused on high-pressure fluids as potential
solvent substitutes. In light of green chemistry principles, CO2 and H2 O are the
most promising high-pressure fluids since they are renewable and inflammable and
give high xylose yield. As much as 100 mol% of xylose conversion was obtained
using high-pressure CO2 in H2 O/tetrahydrofuran system (Morais and Bogel-Lukasik
2016). Furthermore, the high-pressure CO2 /H2 O method does not require an addi-
tional catalyst, in contrast to conventional hydrothermal processes. Despite its simi-
larity comparable to the pretreatment catalyzed by a mild acid, the depressurization
process for removal of CO2 ensures that the medium acidity does not provide a risk to
the environment. Unfortunately, under conditions of high pressure and temperature,
a significant number of by-products are often produced (Lu et al. 2021; Morais and
Bogel-Lukasik 2016).

3.6 Inorganic Salts Hydrolysis Method

Inorganic salts, such as NaCl, KCl, CaCl2 , MgCl2 , FeSO4 , FeCl3 , and other inorganic
salts, do not include C–H bonds and contain environmentally favorable substances.
The inorganic salt solution is mostly used to dissolve lignocellulosic biomass. The
inclusion of salts can help accelerate lignocellulose hydrolysis and remove more
hemicellulose. As a result, this can lower the reagent distribution cost. The researcher
discovered that the addition of NaCl allowed the reaction to proceed more quickly and
produce the highest amount of xylose (90%) with few secondary reaction products
(Harahap 2020; Jiang et al. 2018).

3.7 Enzymatic Hydrolysis Method

Recent studies have shown that the potential of enzymatic hydrolysis is greater than
that of chemical hydrolysis because of its many benefits. Since managing enzymes
is simpler than handling acids, enzymatic hydrolysis is an eco-friendly process.
Furthermore, it is not necessary to use expensive corrosive equipment. Enzymes
create just one product from substrates because they are highly specific and selective
(Inamuddin 2021).
In order to reduce xylose formation in the hydrolyzates when using enzymatic
hydrolysis for XOS production, it is notable that the enzyme preparations should have
decreased exoxylanase activity. The essential enzyme for generating XOS product
from xylan are β-xylosidase, glycosynthases and endo- xylanases. In addition, the
most popular technique for extracting XOS using xylanase is enzymatic hydrolysis
because of the favorable circumstances and superior results. The extraction of XOS
from xylan varies depending on the xylan source, the activity of the enzyme, and
242 E. Hermiati et al.

the incubation conditions. The compact lignin-hemicellulose structure may have a

significant impact on its extraction from lignocellulose materials. In order to reveal
more hemicelluloses and demonstrate the extraction yield, it is crucial to disrupt the
compact structure. Hence, simultaneous enzymatic hydrolysis in the hemicellulose
could be only used on non-recalcitrant biomass (Chen et al. 2021; Yan et al. 2022;
Samanta et al. 2015).
Currently, Aspergillus xylanases are the most common ones utilized in XOS
manufacturing. Immobilizing xylanase can increase its efficiency and reduce its cost,
making it a viable option for subsequent production. Because of its beneficial impact,
selectivity, and specificity, adjustable degree of hydrolysis, high yield and purity of
XOS, and fewer by-products, enzymatic hydrolysis currently becomes the preferred
method for producing XOS in industrial applications (Yan et al. 2022).

4 Purification of Xylose and Xylooligosaccharides

4.1 Purification of Xylooligosaccharides

Unwanted substances and oligosaccharides from a variety of DP are produced after

the manufacture of XOS (Gullon et al. 2008). This unwanted substances like glucose
and xylose raise the caloric value and alter the sweetening capacity of XOS and also
impurities, such as furfural and HMF, have very little toxicity or adverse effects on
human health in XOS with DP ranges up to 12 (Vazquez et al. 2000). Downstream
processing is necessary to eliminate undesirable components and achieve the desired
product (XOS of DP between 2 and 6) (Aachary and Prapulla 2011). Therefore,
the next primary step in the creation of food-grade XOS is to purify the principal
product by isolating the advantageous high-molecular-weight oligosaccharides from
the unfavorable low-molecular-weight sugars and unwanted compounds. The crude
liquors have been refined using a variety of methods to get rid of unwanted compounds
and concentrate XOS with a yield from 75 to 95% as much as possible to achieve the
required DP (Aachary and Prapulla 2011; Vazquez et al. 2000; Moure et al. 2006).
The purification procedures used and the number of necessary stages have a direct
impact on production costs and, thus, on the Minimum Product Selling Price (MPSP).
For instance, purifying galacto-oligosaccharide (GOS) using activated carbon and
celite for protein and color removal, ion exchange, and simulated moving bed (SMB)
chromatography, known as AO-CH process, increases the MPSP linearly from 2 to 9
USD/kg as the final purity increases from 40% (unpurified) to up to 90% (Illanes et al.
2016). Understanding the above, numerous purification approaches, such as solvent
extraction, adsorption using surface-active compounds, as well as chromatographic
and membrane separation, have been investigated in recent decades.
Extraction, Isolation, Purification, and Potential Application of Xylose … 243

4.1.1 Solvent Extraction Method in XOS Purification

Solvent extraction is often used to extract hemicellulose-based compounds from

pretreatment and treatment steps in the production of XOS (Qing et al. 2013). In the
first stage, vacuum evaporation is used to eliminate volatile components, concen-
trate XOS solutions, and removal of possible explosives (Vazquez et al. 2000; Qing
et al. 2013). The solvent extraction method is useful for removing non-saccharide
parts, resulting in a particularly purified liquid phase and also a solvent component
containing phenolics as well as extractive-derived residues (Moure et al. 2006). The
yield and degree of purification, however, are dependent on the solvent used for
extraction and the kind of lignocellulosic biomass used. Organic solvent precipita-
tion, on the other hand, allows for the extraction of XOS and/or xylose by eliminating
phenolics and extractive-derived chemicals. The most often used solvents for this
process are acetone, ethanol, and 2-propanol (Qing et al. 2013). Other procedures of
varying nature have frequently been used, such as the work of Vegas et al. (2006)
can reach 90.7% XOS purity, by applying a combination of nanofiltration, solvent
extraction, and double ion-exchange chromatography.

4.1.2 Adsorption Separation Method in XOS Purification

Adsorption is a technique that has been used to separate XOS or to eliminate unde-
sirable chemicals. The most often utilized adsorbents, such as diatomaceous earth,
acid clay, titanium, bentonite, activated charcoal, aluminum oxide, silica, and other
synthetic materials, are commonly used in combination with other refining proce-
dures (Vazquez et al. 2000; Xu et al. 2019). Activated charcoal was found to be the
most widely used adsorbent and also has been demonstrated to be a feasible solution
for the elimination of substances derived from extractives, lignin, and carbohydrate-
degradation present in XOS mixtures (Nabarlatz et al. 2007b). The XOS were initially
held by activated charcoal in the study by Pellerin et al. (1991), and then were eluted
with various concentrations of ethanol. A similar technique was used by Reddy and
Krishnan (2014) to extract XOS generated by B. subtilis utilizing activated charcoal.
Activated charcoal (10% w/w) was added to the culture extract, and it was retained
by vacuum filtering. After being adsorbed onto charcoal, XOS was subsequently
eluted with serial percentage of ethanol, resulting in XOS separation depending on
molecular weight. Ion-exchange resins are used in conjunction with other purifica-
tion methods to eliminate heavy metal ions, salts, negatively or positively charged
organic molecules, and pigments from XOS solutions (Vazquez et al. 2000; Chen
et al. 2016). Chen et al. (2016) successfully produce high XOS recovery (91.3%)
and also show that the oligomer composition is not altered by the ion-exchange resin
treatment.
244 E. Hermiati et al.

4.1.3 Chromatographic Separation

This method utilizes high-performance liquid chromatography (HPLC), ion-

exchange, affinity, and size-exclusion chromatographic separation techniques to
obtain highly purified XOS at the analytical level (Geetha and Gunasekaran 2017).
In the purification of XOS, chromatographic approaches such as adsorption in acti-
vated charcoal column chromatography and gel filtration chromatography (GFC)
have been studied (Ho et al. 2014). Ho et al. (2014) have purified XOS that produced
by agro-residue autohydrolysis using GFC, which was demonstrated to be effec-
tive in the elimination of oligosaccharides with high DP as well as undesirable
small compounds, such as monosaccharides, acetic acid, and degradation chemicals
(furfural, HMF and phenolics). This method also indicates a competitive advantage
over membrane purification (Moniz et al. 2014). Despite the fact that the reported
final XOS purities were greater than 70%, the product exhibited a wide DP range
(5–40 and 3–23, respectively). As a result, if XOS having DP 2–6 is considered as
the desired product, the purity was greatly overestimated. Chapla et al. (2012), on the
other hand, had employed activated charcoal column chromatography for the purifi-
cation of XOS generated from enzymatic saccharification of pre-extracted corncob
xylan. The refined XOS preparation consists mostly of xylobiose and xylotriose,
and the purity level was not specified. Study by Yang et al. (2007) had successfully
isolated XOS from xylan enzymatic hydrolysis using a packed charcoal column
followed by ethanol elution. The authors used this process to produce XOS mixture
of 71.4% purity, mostly constituted of xylobiose, xylotriose, and xylotetrose, with a
95% XOS recovery.
SMB is a method of chromatographic separation that improves productivity of
the separation and purity of products. Furthermore, this approach lowered solvent
consumption, provided simple operational controls, and increased the performance of
the separation in systems with limited selectivity and resolution (Li et al. 2020). In a
continuous-separation mode, Choi et al. (2016) was able to recover 92.3% xylobiose
from XOS with a purity of 99.5%, which will be beneficial in allowing large-scale,
cost-effective manufacture of high-purity xylobiose.

4.1.4 Purification Through Membrane Filtration

Prebiotic purification to remove proteins and polysaccharides followed by ion

exchange chromatography was employed in the work by Broekaert et al. (2011),
but this process is not economical for industrial application. Membrane technology
is currently regarded as the best downstream approach for industrially producing
high-purity XOS. Membrane technology is a viable technique for concentrating
and refining XOS since it has a high recovery rate, uses little energy, and is a
straightforward process that does not need other compounds as a solvent.
Membrane technology techniques, such as ultrafiltration (UF) and nanofiltration
(NF) seem to be the favored technologies for refining XOS due to their excellent
Extraction, Isolation, Purification, and Potential Application of Xylose … 245

recovery rate with no solvent (Nabarlatz et al. 2007b). The XOS generated by auto-
hydrolysis of almond shells was successfully purified by employing commercial
thin-film polymeric membranes (Nabarlatz et al. 2007b). The findings show that
lignin-related low molar mass compounds are not rejected by low MWCO (1 kDa)
polymeric membranes, enabling continuous diafiltration to remove impurities and
recognize as an appropriate technique to purify XOS solutions produced by the auto-
hydrolysis of lignocellulosic biomass. However, in the case of XOS, UF appears to
be a pre-treatment alternative rather than a method for creating a highly pure product
containing short-chain oligosaccharides (Cordova et al. 2019).
A combination of UF and NF was employed to purify XOS produced from the
sequential autohydrolysis and enzymatic saccharification of almond shells to increase
the elimination of tiny pollutants (Singh et al. 2019a, 2021). In the NF step, the XOS
mixture was concentrated and monosaccharides and acetic acids were removed, while
in the UF step components having high molecular weight, such as enzymes, were
removed, with results similar to those obtained by Akpinar et al. (2007) and Kumar
and Satyanarayana (2015).
Membrane that separates higher-molecular-weight molecules from oligosaccha-
rides requires less energy and is simple to operate and scale-up (Czermak et al. 2004).
However, membrane technology alone cannot perform the best technique to purify
XOS, because membrane has also a drawback due to its poor performance when
small molecules such as monosaccharides should be separated. The hydrolysis of
previously extracted hemicellulose and autohydrolysis from lignocellulosic biomass
produce XOS, which is then purified using UF membrane.
Monosaccharides have the most major impurities that have less difference in
molecular weight (MW) such as glucose (MW = 180 g/mol), which difficult to
separate between xylose (MW = 150 g/mol) and xylobiose (MW = 282 g/mol) (Mah
et al. 2019). The membrane technology mostly does not have a very small cut-off
molecular weight difference. For that reason, the study by Wijaya et al. (Wijaya et al.
2020), has performed a strategy of elimination glucose by the enzymatic reaction that
does not convert cellulose to glucose before the process enters membrane separation
unit. This process is a combination of alkali pretreatment and enzymatic reaction by
xylanase free from cellulase (Wijaya et al. 2020). However, the research for a suitable
way to purify XOS from hemicellulosic hydrolysate has not yet been resolved. Table 3
presents different techniques for recovering XOS using recent purification methods
that have been published.

4.2 Purification of Xylose

The procedures used to convert lignocellulose resources into ethanol include polysac-
charide hydrolysis to xylose and glucose, fermentation, and ethanol purification.
Among these steps, acid hydrolysis is the most important in generating inhibitors,
such as acetic acid, which restrict the fermentation processes in ethanol produc-
tion (Mussatto and Roberto 2004). More particular, the existence of acetate can
246 E. Hermiati et al.

Table 3 Techniques for recovering xylooligosaccharides (XOS) using recent purification methods
have been published
Purifcation Substrate Product Temperature Recovery References
techniques and pH of XOS % (DP*)
production
Anion-exchange Birchwood Acidic XOS 50 °C 85 (NA) Christakopoulos
and size exclusion xylan et al. (2003)
chromatography
Activated carbon Miscanthus X2, X3, X4, Autohydrolysis 91.3 (>2) Chen et al.
and ion exchange giganteus X5 and X6 (2016)
Activated charcoal Corncob X2 and X3 45 °C, pH 5.3 80 (NA) Chapla et al.
column (2012)
chromatography
Activated charcoal Bagasse, XOS 60 °C, pH 7.0 95 (NA) Yang et al.
column corncob, (2007)
chromatography wheat bran,
and peanut
shell
Gel filtration Oil palm XOS NA 83–85 Ho et al. (2014)
chromatography empty fruit (5–40)
bunch
Gel filtration Rice straw XOS Autohydrolysis 80–90 Moniz et al.
chromatography (3–54) (2014)
High-performance Chemical Xylohexose, 50 °C, pH 5 47 & 90.5 Wang et al.
anion exchange pulp Xylobiose (NA) (2018)
and
xylotriose
High-performance Corncob XOS 50 °C, pH 8 32.5 (≤4) Lin et al. (2011)
liquid
chromatography
Nanofiltration, Rice Husks XOS Autohydrolysis 90.7 (NA) Vegas et al.
solvent extraction, (2006)
and double
ion-exchange
chromatography
Nanofiltration Corncob Xylobiose 55 °C, pH 5.5 74.5 (<5) Yuan et al.
meal and (2004)
xylotriose
Nanofiltration Oil palm Xylobiose 50 °C, pH 5 90.1 (NA) Wijaya et al.
empty fruit (2020)
bunch
Nanofiltration Leaves and XOS 190 °C, pH 7 46.0, Oliveira et al.
green tops purity of (2022)
of 20.9 (NA)
sugarcane
plant
(continued)
Extraction, Isolation, Purification, and Potential Application of Xylose … 247

Table 3 (continued)
Purifcation Substrate Product Temperature Recovery References
techniques and pH of XOS % (DP*)
production
Simulated moving Powder Xylobiose NA >92, Choi et al.
bed XOS and XOS purity of (2016)
>99 of X2
form XOS
(NA)
Ultrafiltration Almond XOS 179 °C 58.3 (NA) Nabarlatz et al.
shells (2007b)
Ultrafiltration Wheat bran XOS 60 °C, pH 6.5 44.4 (2–5) Geetha and
Gunasekaran
(2017)
Ultrafiltration, Almond Xylobiose 50 °C, pH5.5 69.6 (<5) Singh et al.
nanofiltration and shells and (2021)
ion-exchange resin xylotriose
DP* Degree of polymerization, NA Not available

inhibit yeast fermentation development and metabolism by lowering intracellular

pH (Lohmeier-vogel et al. 1998). There are several ways for removing acetic acid
from hydrolysate. The detoxification procedures have been thoroughly evaluated in
some studies (Mussatto and Roberto 2004; Parajo et al. 1998; Huang et al. 2008).
Some of the treatments investigated for removing acetic acid include extraction,
neutralization, over-liming, vacuum evaporation, steam stripping, charcoal adsorp-
tion, and ion exchange resins adsorption. There have been many membrane processes
developed recently, including adsorptive membrane and membrane extraction (Han
et al. 2006; Grzenia et al. 2008).
The next step, which is the separation of xylose from glucose, can be chal-
lenging. Desal-5 DK NF membrane with molecular weight cut-off 150–300 Da was
used to remove acetic acid from xylose (Weng et al. 2009). Separation between
xylose and glucose by tailored thin-film composite (TFC) nanofiltration membrane
has been studied as well (Mah et al. 2019). Other strategy involves an enzymatic
reaction as studied by Morthensen et al. (2015). They had done an enzymatic
method for converting glucose to gluconic acid, followed by nanofiltration to sepa-
rate xylose from gluconic acid. The method for separation of xylose from glucose
using concentrated monosaccharide solutions also needs consideration (Sjoman et al.
2007).
Older methods, such as liquid chromatography (LC) by ionic liquid-modified
silica can also be considered (Bi et al. 2010). The chromatographic separation method
can use a cation exchange resin (Chen et al. 2018). In the future, the purification of
xylose can use a combined strategy to achieve a product either ethanol or xylose as
purified sugar.
248 E. Hermiati et al.

5 Potential Applications of Xylose

and Xylooligosaccharides

5.1 Potential Applications of Xylose

Xylose can be used in a variety of applications, either as is or as a feedstock for further

chemical or biological conversion to a variety of products such as xylitol, furfural,
furfuryl alcohol, levulinic acid, levulinic ester, ethanol, butanol, and hydrogen.
Table 4 shows value-added products that could be created from xylose through
chemical and biological catalytic processes and various applications of the prod-
ucts generated in food, healthcare, feed, personal care, cosmetics, pharmaceuticals,
medical, materials, etc. Some important products will be discussed in more detail
in the following paragraphs. The conversion of xylose through chemical process
produces lower yields, and the employment of acidic catalysts and reaction opera-
tion at higher temperatures and pressures renders the process ecologically unfriendly
(Narisetty et al. 2022). Due to the high cost of manufacturing and environmental
incompatibility, the long-term viability of many commercial chemical processes,
including xylitol production, is questionable (Narisetty et al. 2022).

5.1.1 Food and Healthcare

Xylose is usually used as a sweetener that has a high market value (Fernandez
2019). The relative sweetness of xylose is about 70% of sucrose sweetness. This
natural sweetener has a low calorie, is easily absorbed into small intestine, and is not
metabolized by the liver (Fernandez 2019). Therefore, it is beneficial for people with
obesity, diabetes or other illnesses that should limit sugar consumption. Other health
benefits of xylose are its ability to activate and promote the growth of beneficial
bacteria in the human gut, such as Bifidobacterium and Lactobacillus, to prevent
dental caries, to serve as dietary fiber that decreases blood fat, lowers cholesterol,
and prevent colon cancer, and to be compatible with food (Hongzhang et al. 2012).
Xylose is chemically relatively inert and stable, which contributes to the strong
demand for food items, particularly confectionaries, as it is easy to combine into
other food products without altering the recipe. Xylose is a good flavor enhancer
by inducing Maillard reaction compounds, such as amino acids and peptides in
meats, fish, seafood, milk, beans, or peas. The Maillard reaction is a significant
nonenzymatic process that occurs when amino compounds, such as amino acids or
peptides, combine with carbonyl molecules, typically a reducing sugar like xylose or
glucose (Newton et al. 2012; Sun et al. 2019; Martins et al. 2001). Maillard reaction
products (MRPs) containing a variety of volatile compounds have a significant impact
on food flavor and quality. D-xylose is better than glucose or sucrose in remedying
the color, flavor, and fragrant of the food. The addition of 0.05–2% of D-xylose is
enough to enhance the flavor of the food. The usage of xylose provides advantages
Extraction, Isolation, Purification, and Potential Application of Xylose … 249

Table 4 Chemicals from xylose and their applications (Narisetty et al. 2022)
Chemicals Applications
Chemical process
Xylaric acid Sequestering agents
Xylitol Health, food pharmaceutical
Furfural Resins, fuels, adhesives, polymers
Furfuryl alcohol Resins, rocker propellant, adhesives, polymers
2-Methylfuran Gasoline additive, drugs manufacturing
Levulinic acid Precursor for fuels and chemicals
Levulinic ester Fragrance, fuels, fuel additives
Biological process
Ethanol Solvent, automotive gasoline, alcohol beverages, distilled spirits,
hand sanitizers, and medical antiseptics
Acetic acid Polymeric monomers, paints, adhesives, inks, coatings, and food
additives
Acetone Plastics, cosmetics, and solvents
Lactic acid Food, beverages, polyesters, textiles, and pharmaceuticals
Glycerol Pharmaceuticals, food, polymers, humectant, solvents, lubricants,
personal care, and household products
2,3-Butanediol Polymers, solvents, fine chemicals, lactones, fuel additives
Succinic acid Pharmaceutical products, surfactants, detergents, plastics, and
food grade ingredients
Butanol Lubricants, brake fluids, synthetic rubber, polymers, and
automotive fuels
Isobutanol Coatings, chemical derivatives, paints, fuel additive, and solvents
Xylitol Confectionary, chewing gums, syrups, and odontological and
pharmaceutical products
Polyhydroxybutyrate (PHB) Biodegradable plastics

to the flavor industry that uses the Maillard reaction for the creation of thermally
produced flavorings (Gaspar et al. 2012).

5.1.2 Pharmaceutical and Medical

Xylose has been used in an absorption test to evaluate small intestinal absorption
since 1937 because the method is simple and reproducible. The method involves
an oral dosage of xylose and its serum and urine determination after 5 h collection
period. Upon ingestion of xylose, the body is expected to use 75% of its content,
and the 25% remaining is excreted through the urine within 5 h (Gaspar et al. 2012).
Due to the fact that xylose is absorbed unmodified by the duodenum and jejunum,
its inadequate absorption makes it suitable for use as a malabsorption test (Craig
250 E. Hermiati et al.

and Atkinson 1988). The study confirms that the intake of 25 g D-xylose followed
by a 5 h urine collection and a 1 h serum analysis is a very sensitive and specific
diagnostic for malabsorption.
Earlier study by Cook (1975) shows that in healthy Africans, gamma-globulin
serum levels were significantly inversely linked to D-xylose urine excretion. This
indicates that when the D-xylose content in the bloodstream increases or less D-
xylose is excreted in the urine, the concentration of gamma-globulin serum increases
dramatically, hence enhancing the immunological response. Besides that, many other
studies also show that xylose has anti-inflammatory, antiviral, antiglycemic, and
anticancer properties for lung cancer (Cheudjeu 2020). These induce the idea of
using xylose as an alternative therapeutic regimen for a severe case of Covid-19
(Cheudjeu 2020). Nevertheless, this hypothesis should be proved by pre-clinical and
clinical trials. Xylose has a unique and useful texture-enhancing properties, which
are needed in some products, such as cosmetics and personal care products.

5.1.3 Feedstock for Production of Fuels and Chemicals

The major purpose of xylose as an industrial raw material is for producing xylitol.
Xylitol is a platform chemical, having five-carbon sugar alcohol that are widely used
in food, confectionary, personal care, and pharmaceutical industry. The sweetness of
xylitol is the same as that of sucrose, but it has lower calorific value and glycemic
index (2.4 cal/g; 7%) than sucrose (4 cal/g; 60–70%). The metabolism of xylitol
is insulin-independent. Xylitol has a high endothermic heat, thus as it dissolves
in the mouth, a pleasant feeling of cooling and freshness is immediately noticed.
These characteristics mask the unpleasant flavor of numerous pharmacological active
components and excipients. Asia–Pacific, Europe, and the United States account for
the majority of xylitol production. The process of producing xylitol is expensive,
which prevents its production from expanding to other continents.
Currently, the method of producing xylitol from pure D-xylose on an industrial
scale is a chemical process at high temperature and pressure (180 °C, 50 atm) using
aluminum or nickel catalyst for hydrogenation of the xylose (Rafiqul and Sakinah
2013; Carneiro et al. 2019). This process consumes high energy and generates catalyst
residues which causes environmental problem. The amount of xylitol recovered is
around 50–60% of the xylan or 8–15% of the initial feedstock. The following proce-
dures to obtain pure xylitol involve extensive separation and purification processes
which are also time-consuming, energy-intensive, and costly, causing high price of
the product (Saha 2003). Alternative process using bioprocess has been studied and
reported. The bioprocess which usually involves xylose-fermenting yeasts to convert
xylose into xylitol has gained attention, because it is low cost, operates at moderate
reaction conditions, is more sustainable, and environmentally friendlier (Oktaviani
et al. 2021). The yeast could be directly added to the hemicellulose hydrolysate that
contains xylose, followed by the separation of xylitol from the fermentation broth
using different methods, and purification of the xylitol that has been separated.
Extraction, Isolation, Purification, and Potential Application of Xylose … 251

Lactic acid, also known as 2-hydroxyproponoic acid, is an optically active

compound that comes in L and D forms. Lactic acid, as a platform chemical, has
numerous industrial applications in food, cosmetics, polymers, and packaging. The
most valuable application of lactic acid as a monomer is the production of poly-
lactic acid (PLA), a commercial petrochemical polymer substitute (Narisetty et al.
2022). Lactobacillus pentosus and L. brevis naturally can ferment xylose via the
pentose phosphate and phosphoketolase routes, creating lactic acid and a combina-
tion of acetic and lactic acids, respectively (Qiu et al. 2018). Other microbes that
have been reported could produce lactic acid from xylose are Pediococcus acidi-
lactici (Narisetty et al. 2022; Qiu et al. 2018, 2017), engineered Escherichia coli,
Lactobacillus delbrueckii, Lactobacillus lactis, Lactobacillus plantarum, Bacillus
sp., Bacillus coagulans, Corynebacteria glutamicum, and Rhizopus oryzae (Abedi
et al. 2020).
Succinic acid is an aliphatic dicarboxylic acid with four carbon atoms that is used
extensively as a precursor in the pharmaceutical, polymer, and chemical industries.
Similar to lactic acid, succinic acid is a platform chemical that, due to the presence of
two carboxyl acid groups, can be transformed into a range of compounds including
succinic anhydride, succinic esters, 2-pyrrolidine, and polyesters for the synthesis of
biodegradable plastics (Dai et al. 2020).
Ethanol/ethyl-alcohol/bioethanol is the most frequently used biofuel in the trans-
portation industry and offers a number of benefits, including higher octane number,
greater combustion efficiency, and better heat of vaporization. Bioethanol is less
hazardous, rapidly biodegradable, and emits fewer airborne pollutants than petroleum
fuel and other viable fuel options. Due to its hygroscopic nature, however, ethanol
cannot completely replace gasoline because water vapor might damage the engine
(Narisetty et al. 2022). Currently, ethanol is mixed with gasoline at varying percent-
ages (5–20%) over the world. The combined fuels reduce emissions of hydrocarbons
and greenhouse gases significantly. The generation of ethanol from xylose follows
the production of X5P via the pentose phosphate route and continues via the EMP
pathway (Narisetty et al. 2022). The end product of the EMP route, pyruvate, is trans-
formed to ethanol via the intermediate acetaldehyde. Unfortunately, the common
ethanol-fermenting yeast, Saccharomyces cerevisiae, cannot assimilate the xylose.
There are some other yeasts, such as Pachysolen tannophylus and Pichia stipitis,
that can use glucose and xylose to produce ethanol. However, their activities are
restricted due to substrate and product-mediated inhibition. Genetic engineering of
S. cerevisiae is needed to make the yeast assimilate both glucose and xylose (Kwak
and Jin 2017). Even though xylose could be converted to ethanol, it might be more
advantageous if it is converted to other chemicals that have better values than ethanol.
n-Butanol is a four-carbon alcohol with a straight chain and is regarded as a supe-
rior biofuel to ethanol due to its higher heating value, ignition issues, lower volatility,
high octan number, low miscibility with water, and higher viscosity (Vivek et al.
2019). n-Butanol could be produced via chemical or biological route. The chem-
ical process of producing n-butanol is through aldol condensation (oxo process),
which consists of hydroformylation and hydrogenation of propylene. The biological
252 E. Hermiati et al.

process for n-butanol production is a part of acetone–butanol–ethanol (ABE) fermen-

tation using microorganisms, such as Clostridium spp., well-known cell factories
with ABE fermentation (Chacon et al. 2020; Gottumukkala et al. 2013). However,
the manufacture of bio-butanol is hindered by low concentration and yield as well
as product-mediated inhibition. Optimization of solid loading has been performed
to increase the ABE fermentation and reveals that the ideal biomass loading of 10%
combined with the liquid hot water (LHW) pretreatment resulted in a sustainable
route for a successful sugarcane straw ABE fermentation (Pratto et al. 2020). In
addition, the integrated process of hydrolysis and fermentation eliminates problems
of substrate inhibition. Despite the fact that the yield of n-butanol is 20% lower
than that of ethanol, the energy produced by n-butanol is 32% greater than that of
ethanol (Baral and Shah 2016). With the current concentration and yield, the cost of
producing biobutanol is approximately USD 1.8/L. However, further optimization
of the biocatalysts and process conditions could reduce the cost to USD 0.6/L, which
is equivalent to that of gasoline and other fossil fuels (Liu et al. 2016).
Hydrogen is often regarded as one of the most potentially useful alternative ener-
gies because when combined with oxygen, it produces only water and has a 2.75 times
higher energy yield (122 kJ/g) than hydrocarbons (Liang et al. 2012). Fermentation
of sugars to produce hydrogen is usually conducted using thermophilic anaerobic
bacteria, thus, it is usually called dark fermentation. Hydrogen-producing microbes
can utilize both hexoses and pentoses, while some others prefer xylose over glucose
or vice versa. Caldicellulosiruptor members have adapted to xylose and disaccha-
rides (cellobiose) as principal substrates rather than glucose, which may provide
them a competitive advantage in thermophilic lignocellulosic breakdown (Vongkam-
pang et al. 2021). C. saccharolyticus, C. owensensis, and C. kristjanssonii consume
xylose faster than glucose (Zeidan and Niel 2009). In comparison to glucose, C.
kronotskyensis prefers xylose and cellobiose the most (Vongkampang et al. 2021).
Co-culture of microbes has been shown could improve hydrogen production. The
hydrogen yield from the co-culture of C. saccharolyticus and C. kristjanssonii came
close to the theoretical maximum stoichiometry and was much higher than that of the
monoculture of either organism, suggesting the two bacteria synergistic effects on
hydrogen generation (Zeidan and Niel 2009). Genetically engineered microbes could
also increase hydrogen production during fermentation. Higher hydrogen yield and
productivity were observed in the Thermoanaerobacterium aotearoense SCUT27/
nfnAB mutant relative to the original strain (Li et al. 2019a).
Besides microbes, enzyme can also be used in the hydrogen production from
xylose. First, the cellulose and hemicellulose of the biomass were converted to
glucose and xylose, which serve as substrates for phosphorylation and hydrogen
generation using enzyme mixtures. The process is continued with separation and
purification of the hydrogen. The enzymatic production of hydrogen from biomass
hydrolysate in aqueous solution has some benefits over the production of other
biofuels because the process can use lower concentration of sugars and the product
separation is not expensive (Lastname et al. 2015). Hydrogen gas could be easily
separated from an aqueous sugar solution regardless of its concentration. In the
case of ethanol, for energy-efficient distillation of ethanol, its concentration in the
Extraction, Isolation, Purification, and Potential Application of Xylose … 253

fermentation broth should be 4% minimum, which means about 9% sugars in the

hydrolysates.
Furfural is an important bio-based platform chemical derived from C5 sugars,
including xylose. Currently, it is industrially produced from biomass, such as corn
cob or sugarcane bagasse through acid hydrolysis of hemicellulose, especially xylan,
in the biomass, and followed with dehydration of the sugar monomers, especially
xylose (Zhang et al. 2017a, b). The dehydration of xylose to furfural is usually
conducted through chemical processes, without or with different kinds of catalysts,
which is explained in quite detail and comprehensively by Delbecq et al. (2018).
Furfural could be applied in wide variety of applications, such as mentioned in
Table 3. Based on its physical or chemical characteristics, furfural is categorized as a
hazardous substance and listed under numerous globally determined classifications
of hazardous materials (Delbecq et al. 2018).
Furfuryl alcohol (C4 H3 OCH2 OH, 2-furylmethanol, 2 furancarbinol) is produced
through hydrogenation of furfural, which is previously derived from xylose through
dehydration. Both steps usually utilize chemical catalysts. Chemical production of
furfuryl alcohol can give high yields and good selectivity, however, it consumes
high-energy and creates environmental issues that might limit its application. The
conversion of furfural to furfuryl alcohol has been conducted through biological
methods by using some bacteria, such as Bacillus coagulans, Bacillus cereus, and
Escherichia coli that have been genetically engineered. This method has gained
attraction due to its energy-saving and environmentally friendly performance (Liang
et al. 2022). Perez et al. (2017) introduced a one-step conversion of xylose to furfuryl
alcohol via a sulfated zirconia-supported Pt catalyst with balanced acid and metal
sites. In a one-step conversion of xylose to furfuryl alcohol, the reaction takes place
on vicinal acid-metal pare sites formed on single multifunctional catalysts, whereas
in a two-step process furfuryl alcohol is generated through consecutive dehydra-
tion hydrogenation reactions on independent acid and metal sites on dual catalytic
systems. Furfuryl alcohol is widely used in the production of synthetic fiber, rubber
resin, furan resin, vitamin C, lubricant, lysine, and hypergolic rocket fuel (Liang et al.
2022; Millán and Sixta 2020).
Levulinic acid, sometimes referred to as 4-oxopentanoic acid or gamma ketova-
leric acid, is a C-5 fatty acid with the chemical formula C5 H8 O3 and one of the key
platform chemicals (Morone et al. 2015). Levulinic acid has a ketone carbonyl group
(C=O) and an acidic carboxyl group (COOH), which endows it with the capacity to
react with various functional groups to generate a vast array of derivatives, making it a
good platform chemical (Morone et al. 2015; Bozell et al. 2000). Due to the presence
of highly reactive carbonyl and carboxyl groups, levulinic acid can be converted into
a variety of high-value-added chemicals and other bioproducts (Elumalai et al. 2016;
Galletti et al. 2012; Rackemann et al. 2011). Most studies focus on the generation
of levulinic acid from starch or cellulose, or through C6 sugars, and fewer studies
are using C5 sugars, such as xylose. Furfural, which was produced from C5 sugars,
such as xylose, could be transformed into furfuryl alcohol (FA) through a transfer-
hydrogenation (TH), and subsequently, into levulinic acid through a hydrolytic ring
opening reaction (Lange et al. 2009; Chamnankid et al. 2014). While levulinic acid
254 E. Hermiati et al.

has been successfully produced in the lab, its commercialization has been hampered
by a number of factors. These include the high cost of raw materials and equipment,
the low yield of levulinic acid due to undesirable side reactions, the difficulties in
efficient product recovery, the high cost of recovering the catalyst, the inefficiency
of the process economy, and the high cost of energy (Morone et al. 2015).
Polyhydroxybutyrate (PHB), is a biodegradable polymer that is made up of 3-
hydroxybutyrate and can reach a degree of polymerization of 2000. PHB is a member
of the polyhydroxyalkanoate group, that can be used as an alternative to plastics that
are made from petrochemicals (Li et al. 2019b; Lee et al. 2021). The physical and
mechanical properties of PHB such as optical purity, a high melting point, crys-
tallinity, and desirable water and gas barrier properties, are similar and comparable
to those of commercial polymers or plastics derived from fossils (Saratale et al.
2019). Unfortunately, the cost of producing PHB (USD 2.25–2.75/lb) is three to
four times that of conventional plastics (USD 0.60–0.87/lb), limiting its use in the
industrial and commercial sectors (Saratale et al. 2019; Singh et al. 2019b). The
high price of PHB is due to the low productivity, content, and yield as well as the
high price of carbon substrate (Singh et al. 2019c). PHB is produced by numerous
microbes, including Ralstonia, Halomonas, and Bacillus species, that consume sugar
monomers, including glucose, fructose, xylose, arabinose, etc. However, there are
only a small number of strains capable of producing PHB from xylose, the second
abundant and inexpensive carbon source in lignocellulosic biomass (Lee et al. 2021).
R. eutropha, the strain with the highest PHB-producing capacity, cannot readily
absorb and metabolize xylose (Lee et al. 2021; Bhatia et al. 2018; Lopes et al.
2009). Some efforts to increase the yield of PHB have been reported, for example
by screening for xylose-utilizing microbes, co-culturing of the microbes (Lee et al.
2021), or optimizing sugar compositions during fermentation (Li et al. 2019b). Some
other solutions to produce more economical PHB are co-production with other value-
added products, design of an appropriate bioreactor system, and production of PHB
using synthetic biology (Singh et al. 2019c).

5.2 Potential Applications of XOS

XOS has been reported used as antioxidant, prebiotic, plant growth regulator,
cosmetics, gelling agent, and for the treatment of diabetes, arteriosclerosis, and colon
cancer (Ahmad 2019). XOS is commercially appealing for use as animal feed, food,
beverage, and pharmaceutical ingredients (Pinales-Márquez et al. 2021). Further-
more, XOS is utilized in the manufacture of micro or nanoparticles and hydrogels for
medication administration and therapies, particularly for the prevention of gastroin-
testinal problems (Shimoda et al. 2011; Gupta et al. 2016). The relative sweetness
of xylobiose and XOS is 0.34 and 0.26, respectively (Park et al. 2017).
Extraction, Isolation, Purification, and Potential Application of Xylose … 255

5.2.1 Antioxidant

Antioxidant is a compound that can delay or prevent oxidation of a substrate by

scavenging or neutralizing free radicals. The free radical scavenging capability of
XOS is a result of the efficient release of total phenolic compounds and the transfer of
the hydrogen atom from the phenolic compounds (Huang et al. 2005; Gowdhaman
and Ponnusami 2015). The precise mechanism of action of XOS in reducing the
negative effects of oxidative stress has yet to be established by scientific evidence
(Samanta et al. 2015). The scavenging capacity of an antioxidant is determined
using various methods, for example, 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay,
beta carotene bleaching assay, 2,2A-azinobis(3-ethylbenzthiazoline)-6-sulfonic acid
(ABTS) assay, and ferric reducing antioxidant power (FRAP) assay (Antolovich et al.
2002).
The antioxidant activity of XOS depends on several factors, such as types of feed-
stocks or sources of XOS, methods of XOS extraction, concentration and degree
of polymerization of XOS, and total phenolic contents in XOS (Bian et al. 2013;
Samanta et al. 2015; Gowdhaman and Ponnusami 2015). A DPPH assay demon-
strates that the antioxidant activity of XOS derived from sugarcane bagasse is dose-
dependent (Bian et al. 2013). The antioxidant activity of the XOS-rich fraction recov-
ered from wood using ethyl acetate extraction (EC50 = 0.39 g/l) was equivalent to
61% of that presented by butylated hydroxy anisole (BHA) and 12 times higher
than that of butylated hydroxy toluene (BHT), and the fraction contained up to
0.43 g of gallic acid equivalents per 100 g of dry wood (González et al. 2004). The
EC50 (0.23 g/l) was similar to that of BHA when the acid hydrolysis was performed
under circumstances that led to extracts with maximum antioxidant-specific activity
(González et al. 2004). The varied concentrations of XOS from corn cob were shown
to have a range of scavenging abilities between 9.7 and 74.2% (Gowdhaman and
Ponnusami 2015). The antioxidant activity of XOS was shown to have an IC50 value
of 1 mg/ml, which is comparable to earlier results on the antioxidant activity of XOS
extracted from maize and sugarcane bagasse (Aachary and Prapulla 2008; Gowd-
haman and Ponnusami 2015). Study by Rashad et al. (2016) reveals that the XOS
derived from a variety of agricultural wastes demonstrated concentration-dependent
antioxidant activity. This finding is consistent with prior findings regarding XOS
derived from wheat, millet brans, and sugarcane bagasse (Bian et al. 2013; Veenashri
and Muralikrishna 2011; Rivas et al. 2013). The overall antioxidant capacity of XOS
mixes is heavily influenced by both the total phenolic acid concentration and the
type of those phenolic acids (Veenashri and Muralikrishna 2011). The existence of
a higher quantity of total phenolic acid contents in XOS combinations formed from
orange peels and mango peels compared to other XOS mixtures may account for
their considerably stronger antioxidant activity (Rashad et al. 2016).
256 E. Hermiati et al.

5.2.2 Prebiotics

A prebiotic is “a nondigestible food element that promotes the health of the host by
selectively encouraging the growth and/or activity of one or a restricted number of
bacteria in the colon” (Gibson and Roberfroid 1995). A more precise definition of
prebiotic is a fermented element that induces certain changes in the composition and/
or activity of the microbiota of the gastrointestinal tract and offers health benefits
(Aachary and Prapulla 2011; Gibson et al. 2004). The current daily dose of XOS
that is recommended to generate a prebiotic effect is low, which allows them to
compete favorably in terms of pricing. In order to achieve an essential regulatory
status and boost their commercial value, however, additional in vivo experimental
data is required (Amorim et al. 2019).
Several researchers have documented the prebiotic effects of XOS. The XOS
streams utilized as carbon sources for the in vitro fermentability evaluation with
human fecal inoculum exhibited prebiotic effects by stimulating the formation of
lactate, formate, and SCFA (acetate, propionate, and butyrate), as well as alterations
in the selected bacterial populations (Bifidobacterium genus, Lactobacillus, Entero-
coccus group and Bacteroides, Prevotella group) (Buruiana et al. 2017). It appears
that XOS promotes gut health by selectively stimulating the growth of bifidobacteria
and lactobacilli. Additionally, XOS reduces the number of potentially pathogenic
organisms (Chen et al. 2021). In vitro fermentation of the putative probiotic L. brevis
strain validated prebiotic property of XOS-mixture derived from wheat (Faryar et al.
2015).

5.2.3 Food Processing

According to Deutschmann and Dekker ( 2012), XOS is resistance to high tempera-

ture up to 100 °C and has a good adaptability in large range of pH (2.5–8). XOS has a
better resistance to low pH (<4) and high temperature (>90 °C) than other oligosac-
charides and inulin, which is beneficial in food processing point of view and makes
the XOS can be used in carbonated drinks, low-pH juices, and acidic foods (Aachary
and Prapulla 2011). Current study also shows that XOS is stable under high pressure
processing (100–600 MPa) combined with heat treatment (100 °C) (Silva et al. 2022).
In food industry, XOS can also be used as a low-calorie sweetener or feedstock for
producing low-calorie sweeteners, such as xylitol. XOS has been reported as flavor
enhancer in formulating a beverage (Gupta et al. 2016). The addition of XOS in a
non-alcoholic carbonated drink has a positive impact with other sweeteners, such as
mixture of ace sulfame K and aspartame (Gupta et al. 2016). The addition of XOS
in beverages significantly enhances full body character of the beverages without any
drawback of off-flavor perception or mouth feel (Gupta et al. 2016, 2012).
Extraction, Isolation, Purification, and Potential Application of Xylose … 257

5.2.4 Other Biological Functions

XOS has a wide range of biological activities, including anti-inflammatory, antioxi-

dant, antitumor, and antimicrobial properties (Chen et al. 2021). Experiments in rats
show that XOS reduces concentration of sugars and lipids in blood of diabetic rats,
improves calcium absorption in rats, enhances phagocytic activity of neutrophils in
mice, and improves gastrointestinal health of rats (Liang et al. 2012). XOS is non-
cariogenic, preserve pancreatic insulin production, and promote intestinal mineral
absorption (Moure et al. 2006). In human health, XOS has been reported to reduce
risk of colon cancer and have a cytotoxic effect on human leukimia cells (Liang et al.
2012).

6 Concluding Remarks

Lignocellulosic biomass is a potential feedstock because it is widely available, and

relatively inexpensive. It is very beneficial for producing high-value-added products,
including fuels and chemicals, that can be used in various fields and applications. The
production of xylose and XOS from lignocellulosic biomass has become interesting
as a part of lignocellulosic biorefinery, which could promote economic viability of
the biorefinery. Despite significant progress in xylan-based bioproduction over the
last few decades, many challenges remain to be overcome before xylan can be used
as a feedstock at the industrial level. The first challenge comes from the feedstock
itself, which usually varies or not uniform, the scattered existence, and sometimes has
problem with continuous supply. In the process and technology sector, it is important
to select the appropriate pretreatment or fractionation of biomass which could be
applied in a wide variety of biomass, and subsequently facilitate the extraction,
isolation, and purification of xylose and XOS from the biomass using different kinds
of techniques available. The choice of extraction, isolation, and purification should be
feasible technologically and economically. For example, the process should be able
to recover more xylose or XOS, minimize the production of undesirable products,
and retain more cellulose in solid residue. Xylose and XOS could be used as they
are, or to be further transformed to different kinds of chemicals that could be used
for fuels and various applications in food, health care, pharmaceutical, cosmetics,
and personal care. The challenge in this transformation is to find a robust, practical,
inexpensive, efficient, and environmentally friendly process, to generate products of
economic value. Standardized methods of process and analysis of the products are
also important to obtain consistent quality and standardized bioproducts.
258 E. Hermiati et al.

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Carotene Production from Biomass
Waste

Made Tri Ari Penia Kresnowati and Dianika Lestari

Abstract Carotenoids, a group of substances that belongs to the terpenoid group,

are widely used for food colouring to give yellow, orange, or red colour in food
products. In particular, β-carotene can be produced by extraction from biological
substance, such as vegetables, fruits, oils, or microbial fermentation; or produced
synthetically via chemical reactions. This article presents a review of β-carotene
production method, in particular from biomass waste. As a case study, the potential
of β-carotene production from Oil Palm Empty Fruit Bunches (OPEFB), the major
biomass waste from the palm oil industry, is evaluated.

Keywords β-carotene · Extraction · Oil palm empty fruit bunches · Production

1 Introduction

Food colorants are the additive that is widely used by the food industry to improve
the appearance of food products. Carotene is one of the colorants that is widely used
in food industry to give yellow, orange or red color. Carotene also has antioxidant
properties and beta carotene, in particular, is the precursor of vitamin A. Thereby it
is a food additive that also has a nutritional value.
Carotene can be produced synthetically via chemical reactions or obtained from
natural sources. Some of the potential sources for carotene are vegetables such as
carrots, tomatoes, and spinach; vegetable oils such as palm oil and buriti oil, or
microorganisms such as algae, bacteria, and fungi/yeast (Riberio et al. 2011).
Palm oil is one of Indonesian top plantation commodities. Data shows that in
2021 the Indonesian oil palm plantation area was spread at 14.6 million hectares
(Indonesian Statistic Bureau, www.bps.go.id) producing about 46.9 million tonnes
of crude palm oil or CPO (Palm Oil Association, https://gapki.id). Besides producing
CPO, palm oil industries also coproduce a wide array of biomass waste such as oil

M. T. A. P. Kresnowati (B) · D. Lestari

Department of Food Engineering, Faculty of Industrial Technology, Insitut Teknologi Bandung,
Bandung, Indonesia
e-mail: [email protected]

© The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2024 269
M. A. R. Lubis et al. (eds.), Biomass Conversion and Sustainable Biorefinery, Green
Energy and Technology, https://doi.org/10.1007/978-981-99-7769-7_12
270 M. T. A. P. Kresnowati and D. Lestari

palm empty fruit bunches, oil palm fibers, and shells. The oil palm empty fruit
bunches (OPEFB) are the leftover biomass after the pressing or extraction of palm
oil from the fresh fruit bunches. Thereby OPEFB still contains traces of palm oil
and carotene. Palm oil can be one of the potential raw materials for the beta-carotene
industry. Producing carotene from palm oil and its biomass waste can be an additional
value to the palm oil industry.
This paper presents a review of β-carotene production method. Detail evaluation is
presented for the potential of β-carotene production from OPEFB, the major biomass
waste from the palm oil industry.

2 Carotene

Carotenes are hydrophobic organic molecules containing 40 carbon atoms that belong
to the terpenoid group. Carotenes contain 8 isoprene structure and does not contain
any oxygen atom. Carotenes absorb ultraviolet, violet, and blue light, scatter orange,
red and yellow light. The term ‘carotene’ itself was pinned from carrot, from which
it was first extracted. In carrots, carotene is used as the molecule to capture (sun)light
energy for photosynthesis.
The two primary carotene isomers are α-carotene and β-carotene. These molecules
have terminal β-ionone rings on both ends and differ only in the positions of double
bonds (Fig. 1a, b). Another carotene isomer is lycophene, which lacks of β-ionone
ring (Fig. 1c). Another class of molecule sharing the 8-isoprenes-terpenoid group
is called xanthophyll. Some examples of xanthophyll are luthein, fucoxanthin and
astaxanthin. The difference between carotene and xanthophyll is that the first group
does not contain any oxygen atom and thus purely hydrocarbon whereas the second
one contains some oxygen atoms. Both carotene and xanthophyll are grouped as the
carotenoids.

Fig. 1 Molecular structure of α-carotene (a), β-carotene (b), lycophene (c)

Carotene Production from Biomass Waste 271

The presence of β-ring in carotene contributes to the pro-vitamin A activity of

this molecule, which is the main nutritional function of carotene. β-carotene can
be cleaved by a particular enzyme called β-carotene-15,15' -dioxygenase to produce
retinal, which is vitamin A (Grune et al. 2010). On the other hand, the presence
of double bonds in carotene molecules allows the binding of this molecule to
singlet oxygen, providing an antioxidant property. Overall, these valuable proper-
ties of carotene make this compound a desirable food additive and widely applied
as red, orange colorants in food products. Commercially carotenes are applied as
food colorants, nutraceutical supplement, feed additives, and also in cosmetics and
pharmaceutical (Bhosale and Bernstein 2005; Schmidt-Dannert 2000; Mortensen
2009).
β-carotene is sold as capsules, powders, or concentrates either in water-dispersible
form or in oil-based form. A quick survey indicates the price of β-carotene ranges
from 14 to 600 US$/kg, depending on its purity. The global β-carotene market size
was reported to be 432.2 million USD in 2015 and is expected to reach 621.0 million
USD in 2024 (Grand View Research 2024).

3 Commercial Production of β-Carotene

3.1 Synthetic Route of β-Carotene Production

Most of commercial β-carotene is produced via chemical synthetic route. The chem-
ical synthetic route uses β-ionone as the starting chemical precursor. This molecule
can be obtained from natural resources, such as turpentine or lemon grass oil, or
produced from acetone, acetylene, or butadiene (Riberio et al. 2011; Tedeschi 2003;
Vani et al. 2001). The reactions involve the combining of two or more smaller
molecules to give the required carbon skeleton of β-carotene, that is 40 carbon atoms,
either by the Grignard coupling, elimination, and partial hydrogenation reaction or
by the Wittig condensation reaction. The reaction was pioneered by Roche, and now
the two main β-carotene producers are DSM and BASF. These companies have been
producing β-carotene from the early fifties and now supply up to 85% of world
β-carotene demands.

3.2 Natural β-Carotene

B-carotene is naturally present in plants and microorganisms. Some potential sources

of β-carotene are listed in Table 1. Commonly, these organisms contain not only
β-carotene but also other carotenoids, despite in lower quantity. Thereby natural
β-carotene provides more health benefits and can be consumed in larger quantities
272 M. T. A. P. Kresnowati and D. Lestari

(Riberio et al. 2011). The growing health awareness of the usage of chemical additives
in food encourages the growing demand of natural β-carotene.
Palm oil and buriti oil are reported as potential sources for plants-derived carotene.
Their carotene content is much higher than carrots, from where carotene was first
extracted. The processes applied to obtain carotene from vegetable/fruit commonly
include extraction and refinery (i.e. concentration/crystallization) (Fig. 2). A more
complex process is required to obtain carotene from vegetable oil, considering that
carotene is a hydrophobic compound that well dissolves in oil. A review on various
carotene extraction methods from CPO is provided by Othman et al. (2010) and
Sudibyo and Sardjono (2015). The commercial process includes transesterification
process followed by molecular distillation (Ooi et al. 1994).
Wide range of microorganisms can produce carotenoids. Information on strategies
for optimizing the carotenoid content such that it can be applied at commercial
scale, however, is still limited. Nevertheless, using microorganisms as the platform
to produce natural carotene is always an interesting alternative considering the fast
doubling time or fast rate of growth of microbial cells.
An example of commercial microbial source of carotene is Dunaliella salina,
the halotolerant unicellular microalgae. Applying various kinds of stresses during
microalgae cultivation has been reported to increase the carotenoegenesis in this
species such as high salt concentration, light stress, suppression of Nitrogen and Phos-
phorous sources, or addition of organic solvent (Riberio et al. 2011). Other reported
commercial microbial source of carotene is fungus Blakeslea trispora. Similarly,

Table 1 Potential sources of β-carotene

Resources Carotene yield (μg/g) References
Vegetables/plants Carrots (Daucus 85–174 Rodriguez-Amaya
carota) (2001)
Palm oil (Elaeis 470–700 Rodriguez-Amaya
guineensis) (2001)
Sweet potato (Ipomoea 160–226 Rodriguez-Amaya
batatas) (2001)
Buriti oil (Mauritia 1150–3380 Rodriguez-Amaya
vinifera) (2001)
Microorganisms/algae Dunaliella salina 46600 García-González
et al. (2005)
Microorganisms/fungi Blakeslea trispora 67000 Choudhari et al.
(2008)
Phycomyces 32800 Mehta et al (1997)
blakesleeanus
Neurospora sp. 1190 Gmoser et al. (2018),
Priatni (2016)
Microorganism/yeast Rhodotorula sp. 800–900 Buzzini et al. (2005),
Wang et al. (2008)
Carotene Production from Biomass Waste 273

Fig. 2 Block flow diagram of natural carotene production from natural resources: a From oil, fruit,
or vegetable, b from microorganism

some stimuli or inducers should be added to the cultivation/fermentation to accu-

mulate carotene. Some reported inducers are lighting or addition of n-dodecane or
β-ionone to the cultivation (Riberio et al. 2011). Wild type of bacterial species/strains
are rarely reported to produce significant amount of carotene. Some studies explore
the use of genetically modified bacteria using Escherichia coli or Serratia marcensens
as the bacterial platform (Bogasz-Radomska and Harasym 2018). Recently some
studies have been focused on Neurospora sp. for producing carotene (Gmoser et al.
2018; Priatni 2016). This is of particular interest as this fungal species is commonly
used in preparing traditional fermented food in Indonesia such as oncom.
The general scheme of microbial-based carotene production includes the cultiva-
tion of the microorganisms (microalgae/fungi/yeast/bacteria) followed by the down-
stream separation process (Fig. 2). The first step of the downstream separation process
is biomass separation, for example by centrifugation. The products can be sold as
dried biomass containing carotenoids or raw carotene. In this case, the biomass is
dried and pressed. The products can also be sold as concentrate or purified carotene.
In this case, the biomass is disrupted, before carotene is extracted and concentrated.
The concentrated carotene can further be purified by crystallization.
Overall, the extraction process is the critical step in natural carotene production.
Reviews on various carotene extraction methods are given for example by Saini and
Keum (2018) and Rammuni et al. (2019).
Major natural β-carotene producer is Vitatene (Spain) which produces β-carotene
from fungi Blakeslea trispora, and Aquacarotene Limited (Australia) which produces
β-carotene from algae Dunaliella salina. Besides producing β-carotene synthetically,
BASF also has microalgae plants to produce natural β-carotene in Australia. Vitatene
is now part of DSM, another major producer of synthetic β-carotene.
274 M. T. A. P. Kresnowati and D. Lestari

4 Carotene Production from Biomass Waste

Despite the wide distribution of natural carotene in plants and microorganism, they
only comprise of a small portion of the cell which leads to the high cost of natural β-
carotene. The use of low cost substrate, such as biomass waste, as the carotene source
or as the substrate for microbial cultivation for carotene production was suggested
to reduce the production cost of natural β-carotene (Igreja et al. 2021).
Palm oil is the most important plantation commodity in Indonesia. On the other
hands Table 1 shows that the carotenoid content of palm oil is high that palm oil
becomes a potential raw material for carotene production. Most of carotenoid content
in palm oil lost during the refinery, in particular bleaching and deodorization, in order
to produce the light colour oil that is demanded by market. These processes employ
high temperature and pressure such that carotenoid molecules are degraded into
smaller compounds (Ooi et al. 1994). For producing carotene from palm oil, it is
then suggested to use crude palm oil (CPO) as the raw material.
The palm oil industry also coproduces a lot of biomass waste. In the plantation,
there are oil palm fronds and oil palm tree trunks, that need to be regenerated peri-
odically. Within the boundary of the palm oil processing plants there are OPEFB, oil
palm shells, and oil palm fibers. The oil palm fibers are produced as biomass waste
after the oil palm extraction process. The oil palm shells are produced as biomass
waste after the palm kernel extraction process. The empty fruit bunches are produced
as biomass waste after the threshing of oil palm fruits from the fresh fruit bunches.
These biomass waste still contains palm oil residue and therefore it can be expected
to contain materials that dissolve in it.
Some literatures reported the presence of carotene in palm oil residue. Masni
(2004) evaluated the use of palm oil mill wastes, in particular the oil palm pressed
fibers, as the source of carotenoid. The employed processes were extraction followed
by chromatography absorption for purification. The concentration of the produced
carotene concentrate was up to 11580 ppm. Beside the oil palm fiber, Kupan et al.
(2016) also evaluated the use of OPEFB as the source of carotene. The employed
processes were extraction followed by absorption and gave carotene concentrate up
to 1414 and 702 ppm succeedingly for the pressed fibers and empty fruit bunches.
These literatures show that more carotene can be extracted from the fibers, but fibers
are produced much less than the empty fruit bunches in the palm oil mills (Prasertsan
and Prasertsan 1996). Moreover, the oil palm fibers are usually burned to fuel the
boiler in the palm oil mills.

4.1 Empty Fruit Bunch as an Alternative Raw Material

for Carotene Production

As has been discussed previously, empty fruit bunches are produced as biomass waste
after the threshing of oil palm fresh fruit bunches from the fresh fruit bunches and
Carotene Production from Biomass Waste 275

thus still contain palm oil residue and all materials that dissolve in it. Research have
been conducted to develop the carotene production process from OPEFB further.
Direct extraction of OPEFB gave low concentration of carotene extract, in the
order of 1.94–2.54 ppm (Kresnowati et al. 2020) which indicated the need of further
refinery of the carotene extract. Anshori et al. (2022) reported that the type of solvent
used, different parts of OPEFB that was extracted, as well as the cut size of OPEFB
affect the carotene recovery during the extraction. Kresnowati et al. (2020) also
reported the freshness of the OPEFB, or in another word how long the OPEFB has
been dumped nearby the palm oil mill plant, affected the maximum carotene that can
be extracted. Fresh OPEFB still contains a lot of palm oil residue, thus considerable
amount of carotene can be extracted. After just a few days, yellow orange colour
fungi would spontaneously grow on OPEFB, and consistently a significant increase
in the carotene content can be expected. After the growth of yellow orange colour
fungi, the fungal growth would be dominated by the white colour fungi untill the
OPEFB became rotten. Followingly, the carotene content decrease. Although the
results are still limited, the potential is quite promising, the yield of carotene from
empty fruit bunches was estimated up to 180 μg/g which was comparable to that
of carrots and sweet potatoes. Overall, the natural sequential fungal growth during
the storage of OPEFB in the dumpsite nearby the oil palm mill plants indicated
that the presence of fungi increased the carotenoid content of the OPEFB, it also
indicated the potential OPEFB as the substrate for fungal fermentation for carotene
production. In this case, cheap biomass waste materials are used to supply important
precursors that are needed for carotene production, thereby no expensive chemical
precursors need to be added. In addition, the empty fruit bunches themselves also
contain carotene which can increase the overall yield of carotene from the process. In
total, the carotene can be obtained from both the empty fruit bunches and the fungi.
The yellow orange colour fungi that is naturally grown on OPEFB was identified as
Neurospora sp.
OPEFB is lignocellulosic material that is rich in Carbon, but limited Nitrogen
content. Indeed, preliminary research on nutrient addition on the solid state fermen-
tation of Neurospora sp. by using OPEFB as the substrate, such as urea as the
Nitrogen source, and MgSO4 as both the Sulphur source and Magnesium source,
affected fungal growth and carotene production (Tommy and Purba 2020). Other
fermentation parameters that affected the fungal growth and carotene production are
the water activity, lighting, temperature, and fermentation duration (Anshori 2020;
Tambunan and Rucita 2021). Overall, reengineering of the solid state fermentation
of Neurospora sp. on OPEFB substrate may improve carotene production.
The idea of using palm oil wastes as the source of carotene may first sound tedious,
in particular when compared to using the crude palm oil. However, Manurung et al.
(2017) showed that the extraction of carotene or in general the reextraction of palm
oil from oil palm waste is indeed reducing the greenhouse gas emission of the palm
oil industry. Considering that empty fruit bunches are cheap biomass waste that is
available in abundance, the idea of utilizing empty fruit bunches as the source of
carotene offers great potential. The proposed process for carotene production from
OPEFB as well as factors that affecting the process is presented in Fig. 3.
276 M. T. A. P. Kresnowati and D. Lestari

Fig. 3 Carotene production from OPEFB

Using data of Prasertsan and Prasertsan (1996) that about 1 ton of OPEFB are
coproduce during the production of 1 ton of CPO, and potential carotene yield from
empty fruit bunch of 180 μg/g, we estimate 5600 ton of empty fruit bunches will be
required to produce 1 ton of carotene. This means that only 2% of the available empty
fruit bunches will be required to supply the demanded carotene as the provitamin A
for Indonesian population.

5 Conclusion

The abundance of oil palm empty fruit bunches (OPEFB), as the waste from palm
oil mills, offers an interesting alternative to utilizing them as the raw material for
carotene. The proposed process for carotene production from empty fruit bunches
consists of fungal fermentation followed by extraction and purification.

Acknowledgements This research was funded by Indonesian Minister of Research Technology

and Higher Education through Penelitian Dasar Unggulan Perguruan Tinggi scheme.

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Effect of Single Clove Black Garlic
Extract on Lipid Accumulation During
Adipocyte Differentiation Using 3T3-L1
Cell Line

Efri Mardawati, Erin Nur Fitriani, Desi Ayu Sundari,

Tenny Putri Wikayani, Nurul Qomarilla, Nova Rachmadona,
Eko Fuji Ariyanto, Roni Kastaman, Totok Pujianto,
and Devi Maulida Rahmah

Abstract Obesity, defined as an excessive adipose tissue mass, is a major factor

in increasing the risk of serious diseases such as heart disease, hypertension, and
diabetes. Obesity is associated with the expansion of adipose tissue by excessive
dietary fat intake, which results in adipocyte hyperplasia and hypertrophy. Thus,
inhibiting adipocyte differentiation and accumulation of lipids are important targets
for preventing obesity. As the mechanism of single clove black garlic (SCBG) extract
affecting lipid metabolism in adipocytes remains unclear, this study aimed to eval-
uate the effect of SCBG extract on lipid metabolism in mature 3T3-L1 adipocytes.
The analysis revealed that SCBG extract contained 23.15 mg/g of polyphenol and
9.75 mg/g of flavonoid compounds. SCBG extract had stronger capacities to scav-
enge α,α-diphenyl-β-picrylhydrazyl (DPPH) than fresh single clove garlic (FSCG)
with half-maximal inhibitory concentration (IC50) values of 0.602 mg/mL. The treat-
ment of SCBG extracts at a concentration of 2.5–7.5 mg/mL had a cytotoxic effect
that reduced cell viability. However, there was no significant difference between the

E. Mardawati (B) · E. N. Fitriani · D. A. Sundari · R. Kastaman · T. Pujianto · D. M. Rahmah

Department of Agroindustrial Technology, Faculty of Agroindustrial Technology, Universitas
Padjadjaran, Jatinangor 45363, Indonesia
e-mail: [email protected]
E. Mardawati · N. Rachmadona · T. Pujianto · D. M. Rahmah
Research Collaboration Center for Biomass and Biorefinery Between BRIN and Universitas
Padjadjaran, Jatinangor 45363, Indonesia
T. P. Wikayani · N. Qomarilla
Cell Culture Laboratory, Faculty of Medicine, Universitas Padjadjaran, Jl. Eckyman No. 38,
Bandung 40161, Indonesia
N. Rachmadona
Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Padjadjaran,
Jatinangor 45363, Indonesia
E. F. Ariyanto
Division of Biochemistry and Molecular Biology, Department of Biomedical Sciences, Faculty of
Medicine, Universitas Padjadjaran, Jatinangor 45363, Indonesia

© The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2024 279
M. A. R. Lubis et al. (eds.), Biomass Conversion and Sustainable Biorefinery, Green
Energy and Technology, https://doi.org/10.1007/978-981-99-7769-7_13
280 E. Mardawati et al.

concentration of extract to the cell viability of adipocytes (p < 0.05). Furthermore,

SCBG extract at 2.5 and 5 significantly reduced lipid accumulation (p < 0.05), and
7.5 mg/mL significantly reduced lipid accumulation (p < 0.01) compared to cell
control indicating potential in anti-obesity effect.

Keywords Black garlic · Phenolic · Flavonoid · Obesity · Lipid accumulation ·

3T3-L1 C

1 Introduction

Obesity is a chronic, systemic disease defined as a pathologically increased fat mass

associated with an increased health risk (Bischoff and Schweinlin 2020). About 35
percent of the adult population worldwide is overweight and obese, according to a
World Health Organization (WHO) scientific survey (WHO 2018). Obesity might
increase the risk of developing chronic diseases such as diabetes, cardiovascular,
musculoskeletal disorders, and behavioral disorders (Chooi et al. 2019). Obesity
is an abnormal or excessive fat accumulation in adipose tissue (Torres-Villarreal
et al. 2019). Fat accumulation in adipose tissue involves two metabolic processes:
an increase in lipid size in adipocytes (hypertrophy) and an increase in the number
of adipose cells (hyperplasia). The process of differentiation of preadipocytes into
mature adipocytes is also called the adipogenesis process (Bahmad et al. 2020). 3T3-
L1 cells are a cell line used widely as a cell model in learning to control molecular
adipogenesis and are associated with obesity (Romao and Guan 2015).
The 3T3-L1 cell line is beneficial in identifying molecular markers, transcrip-
tion factors (TFs), and various interactions that occur in adipogenesis (Zebisch et al.
2012). The amount of differentiation on the 3T3-L1 cell can be seen from the accu-
mulation of lipids after cells are given several pro-differentiating agents. These
agents include insulin, dexamethasone (DEX), and 1-methyl-3-isobutyl xanthine
(IBMX), which can increase intracellular cAMP levels through the presence of fetal
bovine serum (FBS) (Morrison and McGee 2015). 3T3-L1 cells have been used
extensively to evaluate the effects of compounds or nutrients on adipogenesis, to
establish the molecular mechanisms underlying adipogenesis, and to evaluate the
potential applications of various compounds and nutrients in the treatment of obesity
(Kang et al. 2016). In particular, compounds such as quercetin (Eseberri et al. 2015)
and resveratrol (Chang et al. 2016) are known to inhibit adipogenesis in 3T3-L1
adipocytes.
Garlic (Allium Sativum Linn) is a plant considered to have a pharmacological effect
that can be useful in various treatments, including obesity (Batiha et al. 2020). The
organosulfur compounds contained in garlic have been shown to have various biolog-
ical activities. The four most critical organosulfur compounds, which are considered
to be the main biological agents, are diallyl sulfide (DAS), diallyl disulfide (DADS),
diallyl trisulfide (DATS), and allyl-methyl trisulfide (Yang et al. 2018). A study by
Li et al. (2017) evaluated the effects of a garlic compound (DATS) on pre-adipocytes
Effect of Single Clove Black Garlic Extract on Lipid Accumulation … 281

3T3-L1 and the mechanisms involved. The test results indicated that the adminis-
tration of (20–75 μM) DATS could inhibit CCAAT or enhancer-binding protein (C/
EBP α and β). Proliferator-activated receptor (PPAR) c mRNA that causes decreased
expression of fatty acid synthase (FAS) and lipid accumulation in 3T3-L1 cells
suggests that DATS compounds may inhibit the differentiation of preadipocyte cells
3T3-L1 becomes an adipocyte. Garlic is therefore considered to be useful for the
prevention of obesity.
Black garlic is a derivative of garlic products obtained from the fermentation
process for a while (1–2 months) at a controlled high temperature (60–90 °C) with
high humidity (80–90%) (Angeles et al. 2016). Compared to fresh garlic, black garlic
does not have a distinctive taste and a sharp scent that customers are less interested in
due to the reduced content of allicin compounds that are unstable at high temperatures.
Allicin is converted during fermentation into more stable compounds such as S-
allyl cysteine (SAC), bioactive alkaloids, polyphenols, and flavonoids known to be
antioxidants (Kimura et al. 2017).
Currently, research into black garlic products is still based on the form of multi-
clove garlic, and research into the potential of single-clove garlic is still limited. In
research by Chen et al. (2019), a comparison was made with the bioactive content
of 4 varieties of garlic, including multi-clove and single-clove garlic, and the results
of this study showed that the content of bioactive compounds in single-clove garlic
was higher compared to multi-clove garlic. Considering the potential of single-clove
garlic varieties, currently evaluating the potential of black garlic with single-clove
garlic as raw materials against 3T3-L1 anti-obesity is still very limited. In this study,
we examine the effect of single clove black garlic (SCBG) extract on lipid accumu-
lation in the adipocyte differentiation process using 3T3-L1 cell lines as a reference
for the potential use of SCBG in the treatment of obesity.

2 Materials and Method

2.1 Preparation of Single Clove Black Garlic (SCBG) Extract

Single-clove garlic was manufactured for 21 days. SCBG at 1 kg was homogenized

and extracted with 70% methanol (MeOH; 5L) for 24 h. The mixture was then filtered
with Whatman No. 3 filter paper. The total filtrate was concentrated by evaporation,
and the residue of SCBG was dissolved in deionized water.

2.2 Determination of Bioactive Compound

The method described previously by Kim et al. (2013) with slight modification.
Briefly, 3 g of each sample was weighed in an extraction tube, and 10 mL of 70%
282 E. Mardawati et al.

methanol was added. The extract was mixed for 20 min. The extracts were centrifuged
at 6000 rpm for 10 min. The supernatant was decanted in a graduated conical tube.
The extraction step was repeated third times. Both extracts were pooled, and the
volume was adjusted to 10 mL with 70% methanol. One milliliter of the extract was
diluted with water to 50 mL.

2.2.1 Total Phenolic Compound

The total polyphenol compound was determined spectrophotometrically, using gallic

acid as a standard, according to the method described by the International Organiza-
tion for Standardization (ISO) 14502-1. Briefly, 1.0 mL of the diluted sample extract
was transferred in triplicate to separate tubes containing 5.0 mL of a 1/10 dilution
of Folin Ciocalteu’s reagent in water. Then, 4.0 mL of a sodium carbonate solution
(7.5%, w/v) was added. After 1 h at room temperature, the absorbance was read at
(λ) = 756 nm. All values were expressed as mg gallic acid equivalents (GAE)/mg
dry matter of the garlic sample.

2.2.2 Total Flavonoid Compound (TFC)

The total flavonoid compound was determined using a colorimetric method described
previously by Kim et al. (2013). A portion of 0.5 mL extract was taken, and 0.5 mL
of 2% ethanolic solution of AlCl3 was added. After 1 h at room temperature, the
absorbance was read at (λ) = 420 nm. All values were expressed as mg quercetin
equivalents (QE)/mg dry matter of garlic.

2.3 Determination of Antibacterial Activity

Antibacterial activity analysis refers to clinical and international laboratory

committee standards (Riyanti et al. 2020). There are several test procedures, including
the affiliation of tools and materials, the provision of test bacteria, preparation and
standardization of bacterial suspense, and analysis of antibacterial activity.
Petri dishes containing the media are to be prepared at room temperature for 10–
15 min. Vortex bacterial suspense until homogeneous, then insert the swab into the
suspension and apply it to the media evenly. Let stand for 15 min. Then, prepare a
paper disk that has been saturated by the sample and store it on the top layer of the
agar media. Then, incubate at 37 °C for 24 h. After that, observations were made
using the calipers (measuring the diameter/radius of the zone of inhibition).
Effect of Single Clove Black Garlic Extract on Lipid Accumulation … 283

2.4 Determination of Antioxidant Activity

The free radical scavenging activity of SCBG was determined based on the scav-
enging activity of the stable DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical using
the method described by Choi et al. (2014) with slight modification. A sample of
SCBG 5 mg dissolved in 5 mL methanol solution as a stock solution, stock solu-
tions diluted on a range of 0.2, 0.4, 0.6, and 0.8 (mg/mL) and 0.2 mL DPPH was
added dissolved in methanol solution (1 mL). After incubating the solution at room
temperature in the dark for 30 min, the absorbance was measured at (λ) = 517 nm.

2.5 Cell Culture and Cell Viability Assay

3T3-L1 preadipocyte cells were cultured using 12-well plates in DMEM medium
(Stigma, USA) containing 10% FBS (Stigma, USA) in a humidified atmosphere
of 5% CO2 at 37 °C. To induce adipogenesis, the cells were cultured for 2 days
until confluency. This induction was in the presence of the differentiation mixture
containing 0.5 mM methyl-isobutyl-xanthine (Sigma, USA), 1 μM dexamethasone
(Sigma, USA), and 10 μg/ml insulin (Sigma, USA).
Cell viability assay was performed according to the method described by Torres-
Villarreal et al. (2019). Four-well plates were used in this experiment. Each well was
added with different concentrations of C. longa extracts (0, 2.5, 5, and 7.5 mg/mL),
and cells were then incubated for 48 h.
The cells were incubated for 48 h. After 48 h, on day 3, the medium was replaced
with DMEM containing 10 μg/m insulin to optimize glucose uptake into the cells
and lipogenesis during the differentiation process. On day 5, the medium was then
replaced with DMEM. On day 7, the culture medium was replaced again with DMEM.
On day 12, the optimal adipocyte differentiation was obtained in concentration
control (0 mg/mL).

2.6 Determination of Lipid Accumulation by Oil Red O

Staining

Oil Red O staining was performed using Kim’s protocol (Nam et al. 2018) with
minor modifications. The morphology of the cells was examined under an inverted
microscope, and the images were captured. The colored oil droplets were dissolved
in 100% isopropanol, and the relative lipid accumulation content was measured by
reading the absorbance at a wavelength (λ) = 520 nm with an ELISA reader.
284 E. Mardawati et al.

2.7 Statistical Analysis

All experiments were carried out in triplicate, and data were expressed as mean
± standard deviation (SD) using SPSS 25.0 version. One-Way analysis of vari-
ance (ANOVA) and Duncan’s multiple comparison tests were used to determine the
significance of the difference among samples with a significance level of (p < 0.05).

3 Result and Discussion

3.1 Bioactive Compound of Black Garlic

3.1.1 Total Phenolic Compound

Hydroxycinnamic acid derivatives are garlic’s major phenolic acid compounds (Kim
et al. 2013). There is an increase in hydroxycinnamic acid and phenolic acid deriva-
tives during the development phase of black garlic products (Kimura et al. 2017). In
making black garlic, the content of phenolic compounds may be caused by the process
of creating complex compounds bound by esterification and glycolysis reactions,
increasing free phenolic forms (Lu et al. 2017).
The total phenolic compound in SCBG was (23.15 ± 0.15 mg GAE/g dry basis),
which was 2.5 times higher compared to TPC content in FSCG (9.26 ± 0.20 mg GAE/
g dry basis). These results are consistent with those obtained by Choi et al. (2014),
who reported that, compared to fresh garlic (13.91 ± 1.62 mg GAE/g dry basis),
the concentration of polyphenol compounds in black garlic heated for 21 days was
significantly higher (58.33 ± 1.90 mg GAE/g dry basis). In another study, Jang et al.
(2018) showed. an increase in total polyphenol content in samples of black garlic
containing (43.01 ± 2.85 mg GAE/g dry basis), compared to fresh garlic (2.86 ±
0.14 mg GAE/g dry basis). The difference between the value of phenolic compounds
may be related to the different sources of garlic varieties. The increase of TPCs in
SCBG compared to fresh single-clove garlic (FSCG) is shown in Fig. 1.
The antioxidant activity possessed by phenolic compounds was further estab-
lished for treating various diseases. In this case, phenolic compounds are also
considered essential in stabilizing lipids against peroxidation and inhibiting different
forms of oxidizing enzymes to better treat obesity (Anyanwu et al. 2020). The
mitochondrial-targeted antioxidant action of phenolic compounds can be a poten-
tial mechanism to treat obese inflammation. An array of phenolic compounds has
displayed AMPK-activating properties in adipocyte models (Zhang and de Mejia
2020). According to Wang et al. (2014), intake of polyphenol compounds can
help prevent obesity by reducing lipogenesis, increasing lipolysis, stimulating fatty
acid oxidation (FA), inhibiting adipocyte differentiation and growth, weakening the
inflammatory response, and suppressing the occurrence of oxidative stress.
Effect of Single Clove Black Garlic Extract on Lipid Accumulation … 285

Fig. 1 A comparison of 30
total phenolic compound
(mg GAE/g dry basis) of
*
25

Total Phenolic Content (mg/g)

FSCG and SCBG extract.
Data represent mean ± SD,
n = 3; *P < 0.05 significant 20
difference between the two
groups
15

10

0
FSCG SCBG
Samples

3.1.2 Total Flavonoid Compound

Several in vitro studies have shown that flavonoid class compounds affect adipocyte
cells, where flavonoids can reduce cell viability and proliferation, inhibit triglyc-
eride aggregation, promote lipolysis, and reduce inflammation. (Herranz-López et al.
2017). The anti-adipogenic effect of flavonoids is mainly via their effect on the
regulation of several pathways, such as induction of apoptosis, suppression of key
adipogenic transcription factors, activation of AMPK and Wnt pathways, inhibition
of clonal expansion, and cell-cycle arrest (Khalilpourfarshbafi et al. 2019). Quercetin
and structurally similar luteolin are ubiquitous dietary flavones found in a wide range
of herbs, and their anti-obesity effects are well established; this is believed to be medi-
ated by increasing the expression of AMPK, which subsequently reduces the differ-
entiation and proliferation of human preadipocytes 3T3-L1 and induces apoptosis
(Woon and Toh 2014).
Flavonoid compound (TFCs) levels have been expressed as Quercetin equivalents.
The increase of TFCs in SCBG compared to FSCG is shown in Fig. 2. From the
results, the total flavonoids in the SCBG sample were (9.75 ± 0.27 mg QE/g dry
basis). This level increased by 6 times when compared to the total flavonoid content
FSCG (1.35 ± 0.11 mg QE/g dry basis).
Quercetin has been shown to minimize intracellular ROS in the hypertrophic
adipocyte model (Leiherer et al. 2016). Anthocyanins can suppress lipid accumula-
tion in adipocytes due to widespread inhibition of transcription factors that control
lipogenesis, such as active peroxisome receptors and binding proteins to the CCAAT
conjugator (Lee et al. 2014). In studies with animal models, flavonoid compounds
have also shown positive results in preventing and treating obesity. Based on research
from You et al., flavonoids can increase energy expenditure or inhibit food intake
through various processes that suppress oxidative stress and release gastrointestinal
286 E. Mardawati et al.

Fig. 2 A comparison of 12

Total Flavonoid Content

total flavonoid compound *
(mg QE/g dry basis) of 10
FSCG and SCBG extract.
Data represent mean ±SD, 8

(mg/g)
n = 3; *P < 0.05 significant
6
difference between the two
groups 4
2
0
FSCG SCBG
Samples

Table 1 Comparison of total phenolic and total flavonoid compound

Samples Total phenolic compound (mg/g) Total flavonoid compound (mg/g)
FSCG 9.26 ± 0.20a 1.35 ± 0.11a
SCBG 23.15 ± 0.15b 9.75 ± 0.27b
Data represent mean ±SD, n = 3; values by different letters were indicated significantly different
(p < 0.05), according to Duncan’s test
Different symbols indicate significant differences in each aspect/column (consisting of: total
phenolics, total flavonoids, and antioxidant activity) based on the post-hoc test (DMRT, p < 0.05)

hormones. The recapitulation of total phenolic and total flavonoid compounds of

FSCG and SCBG is shown in Table 1.
In addition, black garlic’s total polyphenol and flavonoid contents significantly
increased during the aging period. It shows the potential use of black garlic in various
diseases, including obesity.

3.2 Antibacterial Activity

The antibacterial activity of SCBG can be seen based on the size of the precise zone
diameter (mm) formed around the paper disk against the bacteria tested, namely
Escherichia coli and Staphylococcus aureus. A clear zone is an area not overgrown
with bacteria and looks more apparent than the surrounding area. The greater the
inhibitory zone formed, the greater the ability of antibacterial activity. Based on
the analysis made on samples against the growth of E. coli and S. aureus, a zone
of inhibition formed around the paper disk. The inhibition zone decreased for the
sample with more longer heating duration.
The antibacterial activity of S. aureus looks better when compared to E.coli due
to differences in the cell wall structure in the test bacteria. E.coli is a gram-negative
Effect of Single Clove Black Garlic Extract on Lipid Accumulation … 287

Table 2 Comparison on
Samples Inhibition Zone (mm)
minimum inhibition of
antibacterial activities E. coli S. aureus
FSCG 4.5 6.58
SCBG 2 0.65
Data represent the measurement results of the clear zone average
diameter

bacterium that has a cell wall, higher lipid content, and a multilayer cell wall struc-
ture consisting of lipoprotein, phospholipid outer membrane, and lipopolysaccharide,
which causes gram-negative bacterial cell walls to be difficult to penetrate by antibac-
terial substances while gram-positive bacteria have a layer of peptidoglycan on the
outside and have less role in effective defense permeability. The measurement results
of the apparent zone average of FSCG and SCBG diameter are shown in Table 2.
The results showed that FSCG in this study showed higher antibacterial activity
compared to black garlic. The compound that acts as an antibacterial is an
organosulfur compound, which is allicin (Lawson and Hunsaker 2018). However,
allicin compounds are unstable against high temperatures (Zhang et al. 2015). The
heating carried out in producing black garlic with high temperatures causes the loss
of and damage to the allicin compound. Compared to fresh garlic, black garlic does
not cause a strong taste because of reduced allicin compounds converted into antiox-
idant compounds such as S-ally-cysteine (SAC), bioactive alkaloids, polyphenols,
and flavonoid compounds (Botas et al. 2019).

3.3 Antioxidant Activity

The antioxidant activity of SCBG extracts was investigated using the DPPH scav-
enging assay. The DPPH assay is widely used due to the relatively short time required
for the analysis. The method is based on the scavenging of DPPH by antioxidant
compounds, which includes a reduction reaction that decolorizes the DPPH methanol
solution (Lu et al. 2017). The DPPH radical scavenging activity indicates the ability
of an antioxidant compound to donate electrons or hydrogen, thereby converting
DPPH into a more stable molecule with a reduced absorbance (Wu et al. 2020). The
DPPH radical scavenging activity of black garlic samples is shown in Fig. 3.
Based on the analysis of the percent inhibition value of the sample against DPPH
radicals, it was found that there was an increase in the percent inhibition of SCBG
compared to FSCG. There was an increase in the percent inhibition in the sample
where the FSCG extract had a percent inhibition range (of 9.07–60.6%) while the
SCBG extract was (41.10–95.78%). The DPPH free radical scavenging activity of
SCBG within 21 days of the heating process was significantly higher by approxi-
mately 3-fold from FSCG (p < 0.05). The increase in the percent inhibition of SCBG
extract may be due to an increase in antioxidant compounds in black garlic, such as
288 E. Mardawati et al.

Fig. 3 Radical scavenging 100

activity determined using
DPPH method. The logistic

DPPH (%inhibition)
model in origin was used to 80
calculate half-inhibition
(IC50). “X” represents the 60
different FSCG and SCBG
extract concentrations, and
“Y” represents the radical 40
scavenging activity rate FSCG

20 SCBG

0
0 0.2 0.4 0.6 0.8 1
Extract Concentration (mg/mL)

total phenolic and flavonoid compounds, where there is an increase in the content
of these compounds, which are also known to have antioxidant activity (Bae et al.
2012).
The concept of the half maximal inhibitory concentration (IC50 ) is extensively
used in the pharmaceutical world to measure its effectiveness in inhibiting biolog-
ical or biochemical functions. The IC50 value indicates the inhibitor concentration
required to inhibit a given biological or biochemical function by half. In other words,
large IC50 values denote inhibitors that interact less effectively with an enzyme than
those with small IC50 values (Caldwell et al. 2012). The IC50 value is inversely related
to the percentage value of inhibition or the ability of the compound to inhibit free
radical activity, which is related to the concentration of an extract.
The IC50 value of FSCG and SCBG is shown in Table III. Based on the result,
SCBG has a lower IC50, indicating a more significant free radical scavenging activity
(Table 3).
The abilities to scavenge DPPH radical (%) of SCBG extract (4 different concen-
trations) at the same time were used to calculate the IC50 . According to Duncan’s
test, values by different letters were indicated significantly differently (p < 0.05).
Bae et al. (2012), Choi et al. (2014), and Jang et al. (2018) have described similar
results for DPPH scavenging activity and reducing power using samples of fresh
garlic purchased in local Korean markets and subjected to heat treatment by the
authors.

Table 3 Comparison of IC50

Samples IC50 (mg/mL)
value of single fresh clove
black garlic (FSCG) and FSCG 0.602 ± 0.91a
single clove black garlic SCBG 0.320 ± 0.36b
(SCBG)
Different symbols indicate significant differences in each aspect/
column (consisting of: total phenolics, total flavonoids, and
antioxidant activity) based on the post-hoc test (DMRT, p < 0.05)
Effect of Single Clove Black Garlic Extract on Lipid Accumulation … 289

Cell Control 2.5 mg/mL 5 mg/mL 7.5 mg/mL

Fig. 4 Representative photographs under a 200x magnification microscope for the cell viability
effect of SCBG extract to the 3T3-L1 cells line

3.4 Cell Viability

When a new drug, either derived from natural or synthesized material, is being
developed, it is necessary to check its safety for the host cell or its cytotoxic effect
on cells. This test is known as the cell viability test. Among the viability tests that
depend on converting substrates to chromogenic products by living cells, the MTT
test is one of the most widely used tests (Kumar et al. 2018).
To investigate the effect of SCBG extract on viability in mature 3T3-L1 adipocytes,
the cell was treated with various concentrations of SCBG extract for 72 h. When
viewed from the condition of the photogenic observation cells under the micro-
scope, there was no significant damage to the 3T3-L1 cells affected by the SCBG
extract. Representative photographs under a 200x magnification microscope for the
cell viability effect of SCBG extract are shown in Fig. 4.
Based on cell viability measurement, in preadipocytes, SCBG extract at 2.5, 5,
and 7.5 mg/mL significantly decreased cell viability, resulting in cell viability values
of 84.70 ± 0.80, 79.12 ± 2.92 and 79.37 ± 4.30%. A diagram of the percent cell
viability can be seen in Fig. 5.
There was a decrease in the viability of cells given SCBG extract at 2.5, 5, and
7.5 mg/ mL compared to the control. However, the concentration of the extract did not
significantly decrease cell viability or the number of dead cells. To ensure that SCBG
extract can inhibit lipid accumulation, several tests, such as inhibiting CCAAT or
enhancer-binding protein (C/EBP α and β) and proliferator-activated receptor (PPAR)
c mRNA, should be performed.

3.5 Effects of Single Clove Black Garlic on Lipid

Accumulation in 3T3-L1 Preadipocytes Differentiation

The inhibitory effect of SCBG extract on lipid accumulation was evaluated by Oil Red
O (ORO) staining. ORO staining is widely used to detect lipids in cells and tissues.
The increase in adipocytes is known to be closely related to the accumulation of lipid
290 E. Mardawati et al.

Fig. 5 Cell viability. Cell *

viability was determined by 120 *
CCK-8 assay. 3T3-L1 *
preadipocytes were treated 100

Cell Viability (%)

with 0, 2.5, 5, and 7.5 mg/
mL SCBG extract for 72 h.
Data represent mean ±SD,
80
n = 9; *P < 0.05 significant
difference between the two 60
groups
40

20

0
Cell Control 2.5 5 7.5
Extract Concentration (mg/mL)

content (Kang et al. 2021). Therefore, ORO-stained cells indicate the degree of lipid
accumulation in 3T3-L1 adipocytes.
The differentiation of 3T3-L1 preadipocytes was initiated with an inducer after two
days of contact inhibition (when preadipocytes exited from the growth cycle). 3T3-
L1 preadipocytes differentiated with the treatment of SCBG extract at the indicated
concentration for 18 days. After the differentiation of preadipocytes along with the
treatment of SCBG for 18 days, ORO staining and subsequent quantification were
performed to examine intracellular lipid accumulation.
As seen in Fig. 6, microscopic observations show that the treatment of black
garlic extract can reduce lipid data in the 3T3-L1 cell line. The lipid droplets in
differentiation media-treated cells became larger with a deeper red color; however,
the treatment decreased these phenomena, which is indicated by the reduction of
red cells. It could be related to the synthesis of triglycerides in mature adipocytes
and the increased hydrolysis of intracellular triglycerides, which in turn prevents the
accumulation of lipids in adipocytes, thereby inhibiting fat cell hypertrophy.
After Oil-Red O staining, the stained oil droplets were dissolved in 100%
isopropanol, and the relative triglyceride content was measured by reading the
absorbance at the wavelength (λ) = 520 nm with an ELISA reader. Based on the
results of the calculation of the percentage of accumulated lipids, black garlic extract
at concentrations of 2.5, 5, and 7.5 mg/mL resulted in lipid accumulation values of
76.15 ± 6.50, 71.93 ± 8.49 and 57.84 ± 4.10%, diagram of the accumulation of
lipid content can be seen in Fig. 7.
The measurement results of cell lipid accumulation added with black garlic, and
the value was smaller when compared to control cells (p < 0.05), (p < 0.01), the
value of lipid accumulation in the sample significantly decreased with the higher
concentration of the extract added.
Effect of Single Clove Black Garlic Extract on Lipid Accumulation … 291

(a)

(b)

Cell Control 2.5 mg/mL 5 mg/mL 7.5 mg/mL

Fig. 6 Lipid accumulation. 3T3-L1 preadipocytes were treated with SCBG extract with 0, 2.5, 5,
and 7.5 mg/mL. Mature 3T3-L1 adipocytes were stained with oil red O after 18 days. a Lipid
accumulation was observed in cell culture media. b Microscopic pictures were taken at 200x
magnification

Fig. 7 Lipid accumulation. **

*
Values were measured from 120 ** *
isopropanol elution of the oil *

red O stained cells. Data 100 **

represent mean ±SD, n = 9;
Lipid Accumulation (%)

*
*P < 0.05 significant *
80
difference between the two
groups. **P < 0.01
significant difference 60
between the two groups
40

20

0
Cell Control 2.5 5 7.5
Extract Concentration (mg/mL)

Wu et al. specifically tested melanoidin compounds in black garlic to determine

their anti-obesity effect, based on the results of research on the effect of melanoidin on
C57BL/6 J mice with obesity induced by a high-fat diet (HFD) that the administration
of melanoidin orally, has a significant effect in reducing weight gain and white
adipose tissue weight and reversing glucose tolerance, especially at high doses. Nam
et al. reported that aged black garlic (ABG) extract at 2.5 and 5 mg/mL significantly
reduced protein expression of proliferator-activated receptor c (PPARc) and perilipin
292 E. Mardawati et al.

in mature 3T3-L1 adipocytes. The hormone-sensitive lipase (HSL) and Ser563-pHSL

levels were significantly reduced by treatment with 5 mg/mL of ABG extract.

4 Conclusion

Single-clove black garlic (SCBG), produced by aging single-clove garlic for 21 days,
has shown higher antioxidant properties when compared to fresh single-clove garlic
(FSCG). Measurement results of lipid accumulation added with SCBG. The value
was lower compared to control cells (p < 0,05), (p < 0.01), and the value of lipid
accumulation in the sample decreased significantly with a higher concentration of
the added extract. These results suggest that SCBG extract may have anti-lipogenic
effects on adult 3T3-L1 adipocytes that could be associated with potential. These
results suggest that black garlic extract may have anti-lipogenic effects on mature
3T3-L1 adipocytes, which could be associated with the potential for black garlic in
treating obesity.

Acknowledgements This research was funded by Directorate of Higher Education and Universitas
Padjadjaran, Indonesian Ministry of National Education (DIKTI) under the scheme of Science and
Technology Application Research. We would also like to express our gratitude to Dr. Afiat Berbudi
for the kind gift of 3T3-L1 cell line and to Universitas Padjadjaran for the research fund. This study
was conducted at Cell Culture Laboratory, Faculty of Medicine, Universitas Padjadjaran, Indonesia

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Physical and Organoleptic Characteristic
of Bread Substituted with Spirulina
Platensis

Souvia Rahimah, Muhammad Fadly Sonjaya, Robi Andoyo, Awalina Satya,

Siti Nurhasanah, and Tjandra Chrismadha

Abstract Spirulina is a microalgae that can be used as an alternative protein source

to improve the physical and sensory properties of baked products. This article outlines
the effects of Spirulina platensis substitution on the physical, i.e., specific volume,
porosity, texture, and color index, and the organoleptic characteristics of the bread.
This study allowed us to identify the optimal concentration of spirulina addition
which ranges from 2 to 4% (dry matter). At this concentration, the bread has a
reduced specific volume, crumb hardness, and lightness degree. Contrarily, there is
a significant increase in bread porosity and gumminess. In terms of the organoleptic
parameters, spirulina substitution is acceptable to be added within 2–4%, though the
hedonic trend decreases as the concentration increases.

Keywords Spirulina platensis · Bread · Organoleptic · Protein alternative ·

Substitution

1 Introduction

Spirulina platensis is a microalgae that contains a protein content of 60–70% (dry

weight) followed by levels of carbohydrates, fats, and water ((Danesi et al. 2004;
Rosa et al. 2015). The protein is composed of essential and non-essential amino
acids which are supported by other components such as γ linoleic acid, carotenoids,
linoleic acid, arachidonic acid, vitamins, iron, calcium, phosphorus, RNA and DNA
nucleic acids, chlorophyll, and phycocyanin (Singh et al. 2015).
Spirulina has been added to various food products and processed products (Rabelo
et al. 2013; Saharan and Jood 2021). Apart from its high protein content, spirulina

S. Rahimah (B) · M. F. Sonjaya · R. Andoyo · S. Nurhasanah

Department of Food Industrial Technology, Faculty of Agro-Industrial Technology, Universitas
Padjadjaran, Jatinangor, Indonesia
e-mail: [email protected]
A. Satya · T. Chrismadha
Research Centre for Limnology and Water Resources, National Research and Innovation Agency,
KST Soekarno, Cibinong, Indonesia

© The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2024 295
M. A. R. Lubis et al. (eds.), Biomass Conversion and Sustainable Biorefinery, Green
Energy and Technology, https://doi.org/10.1007/978-981-99-7769-7_14
296 S. Rahimah et al.

also has a group of bioactive compounds in the form of pigments which are useful as
antioxidants, anti-inflammatories, anti-microbials, and preventive effects for certain
diseases (Borowitzka 2009; Lafarga et al. 2020). Substitution of spirulina into food
products is one way to increase the functional value of food products.
The trend to consume functional foods is increasing globally. As a result, the
food industry has gained around 30% more profits than the previous year (2019)
(BPS 2020). Functional food products are categorized into several product groups,
i.e. fermented products (tape, yogurt, kefir), plant-based analogue products (meat
analogue, cheese analogue, and rice analogue), and fortified products (vitamin D
fortified milk and vitamin E palm oil) (Stübler et al. 2020). This trend was also
followed by an increase in the consumption of bakery products. Therefore, the devel-
opment of functional food in bread products by utilizing spirulina has great potential
for the food industry.
The development of bread as a bakery product into functional food has been carried
out by many national and international researchers. The development was carried
out to improve the quality of bread from the nutritional aspect, physicochemical,
and organoleptic characteristics into functional food (Sadeghi et al. 2019; Pinto et al.
2014; Pan 2017).
Gluten-free bakery products are one of the most developed product by replacing
wheat flour with other flours such as soy flour and sweet potato flour (Pătraşcu et al.
2017; Lafarga 2018). Gluten-free bread is intended for consumers who have certain
medical conditions such as gluten intolerance, allergies, and celiac disease so they
cannot consume gluten-based products (Quiñones et al. 2015; Skendi et al. 2021).
Gluten-free bakery products are also currently in great demand by consumers who
are concerned about environmental issues on the basis of the negative impact of land
clearing for wheat fields (Recchia et al. 2019). The high market demand for gluten-
free bread products has encouraged researchers to develop other functional breads,
especially gluten-free bread.
Gluten-free bread dough, which uses composite or non-wheat flour has rheological
characteristics that have lower elasticity and cohesive values than flour-based bread
dough which has a high gluten content (Cappelli et al. 2020). Gluten-free bread
also has a crumbly texture and color image that is less attractive to consumers.
This deficiency can be overcome by using several strategies, one of which is to use
alternative materials that can mimic the viscoelastic characteristics of the gluten
network. These active ingredients include hydrocolloids, enzymes, emulsifiers, and
alternative protein sources (Cappelli et al. 2020). However, until now no technology
has been found that can exactly match the characteristics of wheat flour-based bread
with gluten-free bread.
One method that can be used to improve the characteristics of gluten-free bread is
to add alternative protein sources such as Spirulina. Spirulina has a protein content
that reaches 50–70% (dry weight). According to Cauvain and Young in (Selmo and
Salas-Mellado 2014), the addition of alternative protein sources can help regulate
the characteristics of gluten-free dough, preventing the product from having a brittle
texture so that it can maintain the volume of bread.
Physical and Organoleptic Characteristic of Bread Substituted … 297

The addition of spirulina to bread products has been shown to significantly

increase protein, mineral, and active compounds such as phenolics which have antiox-
idant capacity content (Matos et al. 2017). The concentration of spirulina added to the
dough needs to be considered because the high protein content in spirulina will affect
the final properties of a product (de Nogueira and Steel 2018; Khemiri et al. 2020).
One of the characteristics that need attention is the functional characteristics of the
dough which includes the rheological properties of the dough (viscosity, extensibility
and elasticity), dough development time, and dough stability (Singh et al. 2015).

2 Conventional and Alternative Bread Formulations

and Methods

Bread is a bakery product that is composed of wheat flour, water, and yeast as the main
ingredients. In addition, salt, sugar, and emulsifiers are used which will strengthen
the crumb structure and give a certain flavor (Cauvain 2012). Bread is produced by
going through three stages, namely: kneading, fermentation (proofing), and heating
(baking/steaming) where each stage has its own role in forming the desired structure,
texture, color, and characteristics (Manano et al. 2021; Yang et al. 2020).
Not only the ingredients used, but also each stage that the dough goes through
each has its own critical point that determines the final quality of the bakery product
(Cappelli et al. 2020). Therefore, bread making is good experimentally and industrial
scale, must know the changes in the parameters of each stage. The final quality
in question includes texture, pore uniformity, volume, color, and taste which is a
parameter of consumer acceptance (Ekafitri and Isworo 2014).
According to Table 1, the formulations and methods used in bread making affect
the physical quality of bread products, in this case the specific volume and the
resulting color index. Research conducted by Amoriello et al. (2021) is a control
parameter for making normal bread dough using basic ingredients without any addi-
tions. This research uses the no time dough method where the fermentation time
is only around 20–30 min. The specific volume produced is 2.83 ml/g. Unlike the
research conducted by (Babajide et al. 2014), the specific volume produced ranges
from 3.95 to 5.40 ml/g depending on the concentration of sugar or honey added.
Sugar added in bread making is in the range of 5–20% of the total flour mass (Baba-
jide et al. 2014). Both studies show that the difference in methods affects the specific
volume of bread produced This is in line with the statement put forward (Zhou et al.
2014) that the straight dough method will produce a higher specific volume because
the dough has increased water absorption capacity during the fermentation process.
Pătraşcu et al. (2017) and Manano et al. (2021) conducted a study using two
different methods with specific volumes which showed the same value (1.57 ml/g).
This contrasts with other studies where the method influenced the specific volume
results. The figure shows that the method is not one of the factors that determine the
specific volume of bread. There is interaction involvement between the ingredients
298 S. Rahimah et al.

Table 1 Bread characteristics based on dough formulation and method

Method Ingredients Specific Color index References
volume
spesifik
(mL/g)
No-time Flour, yeast, salt, sugar, 2,83 Amoriello et al.
dough water (2021)
Straight Flour, yeast, oil, salt, water 3,00 Saharan and Jood
dough (2021)
Straight Composite flour (cassava: 3,95–5,40 Crust: L* = Babajide et al. (2014)
dough wheat), yeast, honey 45,25–39,18; a* =
(10–50%), water, butter 11,58–12,25; b* =
23,99–27,88
Crumb: L* = 64,
23–50,8
a* = 10,00–14,05;
b* = 18,14–33,65
No-time Composite flour (rice, 1,57 Crumb: L* = 76,94 Pătraşcu et al. (2017)
dough cornstach, potato), psyllium a* = 3,2
husk, yeast, salt, sugar, b* = 27,5
protein extract, soybean,
gum guar
No-time Composite flour (rice, 2,26 Warna crumb: L* = Pătraşcu et al. (2017)
dough cornstach, potato), psyllium 66,6
husk, yeast, salt, sugar, a* = 2,57
protein extract, egg flour, b* = 26,61
gum guar
Straight Composite flour (cassava 1,57–2,1 – Pătraşcu et al. (2017)
dough 10–60%), yeast, salt, sugar,
margarine

used which will affect the physical characteristics of the bread. Pătraşcu et al. (2017)
used composite flour, hydrocolloids, and protein extracts as additives to improve the
characteristics of gluten-free bread. Meanwhile, Manano et al. (2021) does not use
any additional ingredients other than the main ingredient. Thus, although the no-time
dough method cannot produce optimal volume, if the right additional ingredients are
used, the expected characteristics can be achieved.
Physical and Organoleptic Characteristic of Bread Substituted … 299

3 Comparison of Spirulina Platensis Components

and Their Interaction on Bread Characteristics

3.1 Effect of Protein on Bread Characteristics

Spirulina is a microalgae that belongs to Cyanobacteria (blue-green) group with a

cylindrical trichome shape and lives naturally in lakes, rivers, swamps, and oceans
with different concentrations depending on the influence of growth factors (Morais
Junior et al. 2020). Spirulina has been known as a food additive and labeled as a
superfood because it contains nutrients that have a positive effect on the human body
when consumed in sufficient levels. Components such as protein, essential fatty acids,
vitamins, minerals, chlorophyll, and phycocyanin are the components contained in
spirulina which can have a positive effect on the body. In addition, spirulina is able to
provide linoleic acid (ALA), linolenic acid, stearidonic acid, eicosapentaenoic acid,
docosahexanoic acid, and arachidonic acid (Vo et al. 2015) (Table 2).
Spirulina type algae have the highest protein group compared to other types of
algae. This is confirmed by comparing the protein levels of spirulina (46–71%) in
various alternative protein sources such as soybeans (36–42%), chickpeas (17–22%),
mushrooms (19–35%), and peanuts. green (23–25%) The data shows that microalgae
belonging to the Spirulina group have the highest protein content and great potential
to improve the quality of gluten-free bread (Christwardana et al. 2013) (Table 3).
The use of Chlorella algae with composite flour significantly reduced the bright-
ness level of the bread and intensified the green and yellow colors of the bread (Diprat
et al. 2020). This is in line with the research by Selmo and Salas-Mellado (2014) who
used Spirulina where the brightness level of the mixture decreased significantly and
the green color became darker (L*: 34.59). This color change is caused by pigments
that are owned by algae where the Chlorella and spirulina pigments that contribute
to giving the color are chlorophyll and phycocyanin, respectively (Fradinho et al.
2020; Ścieszka and Klewicka 2020).

Table 2 Nutritional content

Parameter Content (%)
of Spirulina platensis
Protein 56–62
Fat 4–6
Carbohidrate 17–25
Linoleic acid (Gamma) 0.8
Chlorofil 0.8
Phycoyanin 6.7–11.7
Caroten 0.43
Zeaxanthin 0.1
Water 3–6
Christwardana et al. (2013)
300 S. Rahimah et al.

Table 3 Alternative protein applications on gluten-free bread

Protein source Concentration (%) Flour control References
Chlorella powder 2.1, 4.2 25% rice flour, 58.3% Diprat et al. (2020)
(Chlorella sorokiniana) corn flour, 16.7% pea
flour
Spirulina platensis 1–4 100% rice flour Selmo and
powder (strain LEB18) Salas-Mellado (2014)
Acheta domesticus flour 2, 6, 10 80% corn flour Kowalczewski et al.
20% potato flour (2019)
Eggs white powder 5, 10, 15 Commercial gluten-free Aprodu and Banu
flour (potato starch, (2021)
cassava starch, sorghum)
Eggs white powder 2, 4 100% Tepung Beras Phongthai et al.
(2016)
Soybean powder 2, 4, 6 100% rice flour Srikanlaya et al.
(2018)
Potato protein extract 2, 6, 10 80% corn flour Witczak et al. (2017)
20% potato flour
Pea powder 5, 10 50% rice flour Pico et al. (2019)
50% corn flour
Canola protein extract 3, 6, 9 100% wheat flour Salah et al. (2019)
100% rice flour

Chlorella and spirulina both have a relatively high protein composition. As a

result, when given chlorella algae (4.2%), the porosity of the crumb was much higher
compared to the control (Diprat et al. 2020). Selmo and Salas-Mellado (2014) stated
that increasing the concentration of spirulina would decrease its specific volume. The
presence of high protein content in the dough causes competition between protein
in algae and starch in the dough for hydration. If these two components are not
optimally hydrated, it will be difficult to maintain the texture and structure of the
gluten-free bread (Bhattarai et al. 2016).
Changes in bread volume also occur when adding animal protein such as egg
whites and milk to the gluten-free dough. Phongthai et al. (2016) explained that
there was an increase in the volume of bread up to an addition of 2%, then the
volume of bread experienced a downward trend up to the addition of 4, but there was
an increase in the specific volume of bread up to the addition of 10% albumin.
The application of vegetable protein to gluten-free bread dough, on the other hand,
decreases the volume of the dough on bread. Vegetable proteins such as soy and pea
protein isolate only affect the porosity of the crumb, where the crumb has larger pores
than the control (Srikanlaya et al. 2018; Pico et al. 2019). This is because gluten-
based dough has a high proline content, compared to gluten-free dough. Proline is
a type of amino acid that plays a role in volume development during the proofing
period. Gluten-free dough has high levels of lysine and arginine and low levels of
proline (Skendi et al. 2021).
Physical and Organoleptic Characteristic of Bread Substituted … 301

3.2 The Effect of Spirulina on the Physical Characteristics

of Bread

The physical characteristics of bread are one of the important parameters of bread
quality that can affect consumer acceptance. These characteristics include specific
volume, porosity, texture profile, and color index. The following table shows the
effect of spirulina on the physical characteristics of bakery products (Table 4).
Based on research by Amoriello et al. (2021), the addition of spirulina caused
the dough development time to decrease from 9.1 to 1 min. According to him, the
interaction between fat, fiber, and protein components is the cause of reduced dough
development time due to the weakening of the gluten network so that it can increase
the diffusion of water into the dough. This causes the required mixing time to be
significantly reduced.
The addition of spirulina also increased the specific volume of the dough by 5%
from the control. In contrast to research conducted by Selmo and Salas-Mellado
(2014) where the addition of spirulina actually reduced the specific volume of the
dough like the majority of other algae. The decrease in specific volume can be
caused by the interaction between fiber and non-gluten protein, and the dilution of
starch in wheat flour. These causes have implications for decreasing extensibility and
weakening of the gluten network which of course affects the volume of bread.

Table 4 Physical characteristics of bread on the addition of Spirulina platensis

Product type Flour Spirulina Results References
concentration With Spirulina Control
(%)
Bread Soft wheat 1, 2.5, 4 Volume: –Volume: Amoriello
3,02–2,93 mL/ 2,83 mL/g et al. (2021)
g –Porositas:
Porosity: 18,9%
38,4–56,5% –L* crumb:
–L* crumb: 74,68
54,55–37,40
Baguette Hard wheat 10 Gumminess: Gumminess: Sanjari et al.
2,23 N 0,84 N (2018)
Springiness: Springiness:
10,56 mm 8,43 mm
Cohesiveness: Cohesiveness:
0,43 0,46
Hardness: 20 g Hardness: 55 g
Bread Rice flour 1–4 Volume: Selmo and
2,93–3,10 mL/ Salas-Mellado
g (2014)
Firmness:
436,39–316,25
L* crumb:
34,59–53,21
302 S. Rahimah et al.

Based on several studies, the addition of algae significantly affects the color of the
crumb and crust on bread. The decrease in L*, a*, and b* values was affected by the
high chlorophyll content in the algae. In other words, the higher the concentration
added, the lower the brightness level (L*). In spirulina, the b* value decreases with
increasing spirulina concentration, due to the high phycocyanin content (Bhattarai
et al. 2016; Sanjari et al. 2018; Skendi et al. 2021).
Spirulina affects the texture profile of bakery products (Saharan and Jood 2021).
There was an increase in the elasticity parameter from the research that has been
done. This can occur due to the interaction between the protein in spirulina and the
protein in the dough. The interaction is a hydrogen bond between amidehydroxyl
and hydroxyl-carbonyl groups with polar compound groups. This bond can increase
the hardness and resistance of the dough.
Nevertheless, the study stated that there was a decrease in the hardness value
in the sample with the addition of spirulina. According to Nikoozade et al. (2011)
in Sanjari et al. (2018), this can occur due to the fiber content which prevents the
release of some water into the air because it is bound by the fiber. In addition, fiber
also reacts with molecules in wheat flour starch which has implications for inhibiting
the retrogradation process in the final bread product.

3.3 The Effect of Spirulina on the Organoleptic

Characteristics of Bread

Spirulina added to flour-based food products ranges from 1 to 8% with the best
acceptance in the 2–4% concentration range. The significant change that occurs due
to the addition of spirulina is the change in the intensity of the green color which
is directly proportional to the concentration added. The application of spirulina also
makes bakery products have a distinctive spirulina taste that can be accepted by
consumers up to a concentration of 4%. If viewed from the shelf life, bread products
with the addition of spirulina can last up to two days at room temperature and four
days at refrigerator temperature.
Saharan and Jood (2021) conducted organoleptic testing of bread products with
the addition of spirulina concentrations of 2, 4, and 6% on 10 semi-trained panelists.
Panelists assessed the color, appearance, aroma, texture, taste, and overall acceptance
using the hedonic method with 9 scales (9 point hedonic rating scale) on bread
stored at room temperature and refrigerator temperature. Overall, in two different
conditions, bread with the addition of 2% spirulina was the most preferred with a
score of 8.10 (very like), then 7.60 and 7.16 (quite like) at concentrations of 4 and
6%. While the control value is at 8.00 (very favorable).
Research conducted by Saharan and Jood (2021) states that the shelf life of bread
stored at room temperature in terms of its organoleptic acceptability is 2 days of
shelf life. The organoleptic quality of bread stored at refrigator temperature can be
maintained for up to 4 days of shelf life.
Physical and Organoleptic Characteristic of Bread Substituted … 303

Batista et al. (2019) conducted a study on the evaluation of the organoleptic cracker
added to four types of microalgae at a concentration of 2% which included Arthospira
platensis, Chlorella vulgaris, Tetraselmis suecica, and Phaeodactylum tricornutum.
This research was conducted by 30 untrained panelists with an age range of 19–
38 years. Parameters that are then assessed are color, aroma, taste, texture, and
overall acceptance. In his research, it was known that the control crackers had a
higher score (>4) compared to the treatment sample. However, the samples with the
addition of microalgae had a texture that resembled the control sample. Spirulina
platensis in this study had advantages in color acceptance, aroma, taste, and overall
acceptance (3–4) compared to other types of microalgae. Research conducted by
Fradinho et al. (2020) on the addition of 2% spirulina to gluten-free pasta products
was tested on 31 untrained panelists. The panelists assessed the parameters of texture,
color, flavor, extentability, and overall acceptability. Fradinho et al. (2020) stated that
panelists preferred control samples without the addition of microalgae, even though
the quantitative differences were not that significant. However, the level of consumer
preference for products with the addition of 2% spirulina is satisfactory (>3.7).
Evaluation of hedonic quality on a scale of 10 in biscuit products was carried
out by Singh et al. (2015). The test involved 10 semi-trained panelists consisting of
students and academic staff of Indian universities. Parameters tested include color
intensity, taste, sweetness, graininess, and crispiness. Of the five parameters tested,
spirulina had a significant impact only on the color intensity and taste parameters.
The color intensity in this study specifically assesses the intensity of the green
color. In this study, it was stated that the increase in the concentration of spirulina
platensis was directly proportional to the color intensity value. This is caused by
the pigments contained in spirulina in the form of chlorophyll-a, chlorophyll b,
carotenoids, and phycocyanins which contribute to giving certain colors to food
products (Nuhu 2013).
The taste score on the organoleptic test for this biscuit product ranged from 7.38
to 9.02. Based on the data obtained, the addition of spirulina powder concentration
lowered the taste assessment of this product. This is in line with research conducted
by Saharan and Jood (2021) where the addition of spirulina concentration in bakery
products actually reduces the taste value of a product. This is because spirulina
has a strong enough aroma to change the original aroma of a product. One of the
characteristics of spirulina itself is that it has a fairly fishy smell.

4 Conclusion

The addition of spirulina to bread products can affect the specific volume of bread,
porosity, color intensity, and organoleptic properties on parameters of color, aroma,
taste, and overall acceptability. The addition of spirulina to bread products is generally
at a concentration of 2–4% where there is a decrease in specific volume and brightness
level (L*), as well as an increase in crumb porosity. In terms of its organoleptic
304 S. Rahimah et al.

properties, the addition of spirulina to bakery products was acceptable to the panelists
at a concentration of 2–4%.

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