Journal of Polymers and the Environment (2020) 28:2301–2323

https://doi.org/10.1007/s10924-020-01772-1

REVIEW

Microbial Polyhydroxyalkanoates (PHAs): Efficient Replacement

of Synthetic Polymers
Faizan Muneer1 · Ijaz Rasul1 · Farrukh Azeem1 · Muhammad Hussnain Siddique1 · Muhammad Zubair1 ·
Habibullah Nadeem1

Published online: 1 June 2020

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Abstract
Microbial polyhydroxyalkanoates (PHAs) are biopolyesters produced by microorganisms as intracellular granules under
nutrient stress. Due to non-toxic and biodegradable behavior these polyesters are a sustainable source of wide range of
biomaterials such as bioplastics. As a result of excellent polymeric properties such as melting temperatures ­(Tm), glass
transition temperature (­ Tg), crystallinity, Young’s modulus and stress to break ratio these polyesters are capable of replacing
the synthetic plastics. Bioplastics produced using PHAs as biomass source can be used for packaging material and dispos-
able products on the other hand biofuels can also be generated using PHAs. PHAs find countless applications in industry,
agriculture, pharmaceuticals and health. A large number of bacterial, microalgal and fungal species can produce these poly-
esters. Bacterial species such as Bacillus megaterium, Pseudomonas aeruginosa, P. oleovorans, P. stutzeri and Cupriavidus
necator are some highly studied microorganisms for PHA production. This review summarizes the most important aspects
of PHAs, crystalline and granular structure of PHA biosynthesizing genes and their relevant proteins. Thermal and physical
properties, sources, extraction, purification methods of PHAs are briefly discussed. Applications of PHAs, phylogenetic
analysis of Pseudomonas sp. in term of its PHAs synthesizing genes, future prospects and possible outcomes are discussed.

Keywords  Biopolyesters · Bioplastics · Biodegradation · Biosynthesis · Biomaterials · Biopolymers

Introduction thrown away they accumulate in nature without being dis-

carded [5]. Plastic waste form heaps of debris on land and
Planet earth is losing its natural resources with an ever float on water surface in rivers, lakes and oceans [6]. It is
increasing world population, globalization and industriali- estimated that if the plastic pollution is not replaced with
zation [1]. Uncontrollable and rapid use of non-renewable some environmental friendly and sustainable methods there
energy sources like fossil fuels and minerals have acceler- would be more plastic particles in the ocean than fish by
ated the climate change and environmental pollution [2]. 2050 [7]. Processes like hydrocracking, pyrolysis, thermal
Although there are numerous causes of the growing envi- and catalytic degradation are used to manage waste plastics
ronmental crises, the most common entities directly or by converting into useful products such as fuels and related
indirectly involved are synthetic polymers particularly that byproducts for energy generation purposes [8]. Pyrolysis of
make up plastics [3]. Hydrocarbons are the main ingredients the plastic yields some value added products such as fuel and
of plastics that are actually derived from fossil fuels and important hydrocarbon byproducts but are not sustainable in
are non-renewable in nature [4]. These synthetic polymers terms that these are uneconomical as high temperatures are
(plastics) are non-degradable in natural environment, hence required other than the production of toxic gases during the
once the products made up of these polymers are used and process [9, 10].
Due to severe impacts of plastics on the environment,
economy and health, it is necessary to work on biopolymers
* Habibullah Nadeem
[email protected] that can be used in the manufacturing process of articles,
goods and all other materials that are currently being pro-
1
Department of Bioinformatics and Biotechnology, duced by the synthetic polymers [11]. Biopolymers such
Government College University Faisalabad, Faisalabad, as cellulose, starch, chitosan, chitin, lignin, and microbial
Pakistan

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polyesters such as polyhydroxyalkanoates (PHAs) and poly- and related biomaterials that can help us to diminish the
lactic acid (PLA) can be used to produces bioplastics [12, reliance of modern world on the synthetic plastics thus con-
13]. Bioplastics can be produced from biomass obtained trolling plastic pollution and conserving the natural envi-
from diverse sources such as plants and microbes especially ronment [32]. PHAs depending upon their types and physi-
prokaryotes [14]. PHAs are microbial biopolyesters mainly ochemical conditions find different applications in industry,
produced by prokaryotic organisms such as bacteria in the pharmaceuticals and health [33]. It is therefore, important
form of water insoluble granules of 0.2–0.5 μm inside the to state that biopolymers like PHAs are of great interest in
cell or cytoplasm [15–17]. These granular inclusions are modern world due to their ability to replace the synthetic
produced by microorganisms as a source of energy under plastics from our environment [34]. PHAs as sustainable,
limited supply of nutrients and excess of carbon sources ecofriendly, and non-toxic biomaterials have gained much
[18]. It has been studied that PHAs granules save the bacte- attention in recent years due to their ability in producing
rial cells from the osmotic stress conditions and imbalances biobased and biodegradable plastics that have physiochemi-
[19]. PHAs belong to a diverse class of polymers that have cal properties to become modern day sustainable bioprod-
approximately hundred different types of monomers [20]. ucts [35]. Apart from saving the world from plastic waste,
These monomers are primarily composed of hydrogen and PHAs can ensure the sustainable development of ecofriendly
carbon atoms a reason that lead scientists and researchers to biomaterials that can be used in a wide range of industrial
refer them as “carbonosomes” [21]. In terms of chemistry and domestic applications [36]. Using PHAs for the pro-
these macromolecules or polyesters are synthesized in vivo duction of bioplastics is of great interest in the polymeric
by the polymerization of hydroxyalkanoates (HAs) where science research due to their sustainability and effectiveness
the hydroxyl group is typically located at the β- carbon of to obtain the sustainable development goals as proposed by
the polyester [21]. More than 300 species of microbes such the United Nations [37, 38].
as bacteria, algae and fungi have shown positive results for
the production of PHAs [22]. PHAs are natural alternatives
to modern day synthetic polymers most importantly the pet- Structure and Classification of PHAs
rochemical based plastics [23]. The diverse family of PHAs
provide a large number of polymers that can be used for Depending upon the number of carbon atoms present in the
the synthesis of natural, environmental friendly, biodegrad- monomers, PHAs have various types and structures. The
able plastics called as bioplastics [24]. Some of the PHAs diversity and versatility in PHAs is due to the difference in
that are produced by the microorganisms include poly-3-hy- molecular structure and chain lengths of its members [39].
droxybutyrate (P3HB), poly-4-hydroxybutyrate (P4HB), The most common PHA produced by prokaryotes like bacte-
polyhydroxyvalerate (PHV), and poly(hydroxybutyrate-co- ria is poly-3-hydroxybutyrate (P3HB). This simplest form of
hydroxyvalerate) (PHBV) [25–27]. PHA consists of (R)-3HB repeating unit (monomer) which
PHAs vary in structure and physiochemical properties polymerizes to yield a polymeric chain [17]. Short chain
due to the number of carbon atoms in the monomers, which length PHAs (scl-PHAs), medium chain length PHAs (mcl-
results in different chain lengths and space orientations of PHAs) and long chain length PHAs (lcl-PHAs) are the three
these polyester molecules [24]. Synthetic plastics such as types of PHAs on the basis of carbon chain length [40].
polyvinyl chloride (PVC), polystyrene (PS), polyethylene scl-PHAs have 3-4 carbon atoms such as poly(3-hydroxy-
tetraphalate (PET) and polyethylene (PE) are formed from butyrate) or P(3HB), poly(4-hydroxybutyrate) or P(4HB),
nonrenewable hydrocarbon sources such as fossil fuels poly(3-hydroxyvalerate) or P(3HV) and poly(3-hydroxybu-
which are continuously destroying the environment, living tyrate-co-3-hydroxyvalerate) or PHBV copolymer [41]. scl-
habitats and have severe impacts on health and economy PHAs can be used in food packaging and disposable prod-
[28]. The global production of the synthetic plastics has ucts [22, 42]. Polyhydroxyalkanoates having 6–14 carbon
increased from 150 million metric tons in 2015 to approxi- atoms are termed as medium chain length PHAs these can
mately 395 million metric tons in 2019 [29]. The non-deg- either be homopolymers like poly(3-hydoxyhexanoate), sim-
radability, high production rate, limitations in recycling and plified as P(3HHx) and poly(3-hydroxyoctanoate) or P(3HO)
managing the plastic wastes are involved in changing the [41]. Homopolymers of the scl-PHAs or mcl-PHAs have
scenarios of climate change [30]. It is therefore, the dire and quite different properties [43]. lcl-PHAs are rare in nature
urgent requirement of the 21st century global community to and are particularly of less interest in the developing pro-
tackle plastic pollution and its sociopolitical and economic cess of bioplastics. The general structure of PHAs is shown
challenges that have arisen due to plastics and their non- in Fig. 1. Alkyl side chains of different PHAs are given in
degradable behavior in natural environment [31]. Table 1.
PHAs can be extracted and purified from a number of All forms of PHAs irrespective of the fact of their varying
microbial strains which can be used to produce bioplastics chain lengths i.e. scl-PHAs, mcl-PHAs or lcl-PHAs have the

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Journal of Polymers and the Environment (2020) 28:2301–2323 2303

α‑Form Crystals

P(3HB) crystals usually exits in α-form crystals. This con-

formation of the PHA molecule is produced under different
conditions and environments which includes the melting,
cooling and crystallization conditions of the solution. The
α-form of the PHA crystals is determined using x-ray dif-
fraction which shows that it usually adopts a left-handed ­21
Fig. 1  General structure of PHA. Where ‘n’ varies from 1 to 4 and ‘x’ helix ­(G2T2)2 [47]. In this case two molecules of the poly-
ranges from 100 to 30,000. R denotes alkyl side chain ester pass through the unit cell with the chains being anti-
parallel. Conformational analysis of the polyester crystal
Table 1  Alkyl side chain and their corresponding PHAs was evaluated using data based on energy calculations. The
Alkyl side chain or ‘R’ PHA polyester ester group of the anti-parallel chains of the unit cell have
~ 60° angles at dipoles [48]. P(3HV) like P(3HB) belongs
–CH3 Poly(3-hydroxybutanoate) or PHB to orthorhombic crystal system with a twofold screw left
–CH2–CH3 Poly(3-hydroxyvalerate) or PHV handed symmetry along the molecular axis. Infrared reflec-
–(CH2)2–CH3 Poly(3-hydroxyhexanoate) or PHHx tance, absorption and transmission spectra were used to
–(CH2)4–CH3 Poly(3-hydroxyoctanoate) or PHO study the surface structure of the thin film of P(3HB). It
–(CH2)6–CH3 Poly(3-hydroxydecanoate) or PHD was found that the C–H … O=C hydrogen bonding of the
said polyester exists along the a-axis whereas absent along
b-axis between the C ­ H3 group of one helix to the C=O
same general structure with variations in the main chain of group of the other [49]. On an average, P(3HB) crystals are
their monomer at position –(CH2)n where ‘n’ ranges from around ~5 to 10 μm long ~0.3 to 2 μm wide having lathe-
1 to 4. The second difference comes due to the number of shaped single crystals [47]. These crystals depending upon
repetitions of this monomer to form a polyester chain which molecular weights, solvent and crystallization temperatures
is denoted as ‘X’. The monomer can repeat between 100 have 4–10 nm thickness. PHV crystals solution grown either
to 30,000 times which gives us an idea about the diversity synthetically or from bacterial sources have a square shape
in structure and properties these polyesters. The alkyl side with a thickness of 5–6 nm and 2–4 μm width [50].
chain is denoted as ‘R’. In scl-PHAs such as produced in
Ralstonia eutropha have up to two carbon atoms while in
case of mcl-PHAs the side chain contains at least three car-
bon atoms [41]. Most of the Pseudomonas sp. produce mcl- β‑Form Crystals
PHAs and studied in 1983 for the first time [44]. Structure
of some important PHAs is shown in Fig. 2a, b. P(3HB) exhibits a β-form paracrystalline strain induced
structure with a twisted planner zigzag conformation [51].
The unit cell parameters for this conformation are a =
0.576 nm b = 1.320 nm c (fiber axis) = 0.598 nm. This con-
Crystallographic Structure of PHA formation can be annealed back to α-form at 130 °C. This
shifting of β-form into α-form imparts special characteristics
P(3HB) are usually multi-oriented lamellar crystals. These to the fiber and results in increased crystallinity [45]. It has
crystals grow radially in a twisting manner. The spherulites been studied that the introduction of the β-crystalline forms
structure in which the polymer has banded structure due at least part of the lamellar structures dramatically increases
to the fact that the crystallographic a-axis is radial while the mechanical properties such as Young’s modulus and ten-
b and c axis rotating about it. Molecular weight and the sile strength of the PHA fiber [52].
crystallization temperature impart special characteristics to
the banding pattern of the spherulites. Usually the lamellar
thickness of the spherulites ranges from 4 to 10 nm. The
crystallographic structures of P(3HB) and P(3HV) belong Granular Structure of PHAs
to orthogonal crystal system [45]. The cell parameters of
both these polymers are (a = 0.576 nm, b = 1.320 nm, c = Polyhydroxyalkanoates as we know are granular stor-
0.596 nm) and (a = 0.932 nm, b = 1.002 nm, c = 0.556 nm) age bodies inside the cytoplasm of a number of microbes
respectively [45, 46]. PHA crystal can have two most com- especially bacteria. There can be different granular storage
mon forms i.e.α-form crystals and β-form crystals. bodies inside the bacterial cell, however when a suitable

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Fig. 2  a Structure of short chain length PHAs. Poly-3-hydroxybu- b Medium chain length PHAs and the combined illustration of scl-
tyrate or P(3HB), Poly-4-hydroxybutyrate or P(4HB), Polyhydroxy- PHAs and mcl-PHAs. Poly-3-hydroxyhexanoate or P(3HHx) and
valerate or (PHV) and Polyhydroxybutyrate-co-valerate or PHBV. Poly-3-hydroxyoctanoate or P(3HO)

growing medium for a bacterial culture is switched to a monolayer with embedded and surface attached proteins as
nutrient deficient medium enriched with a particular carbon can be seen in Fig. 3. Western blot and immunogold label-
source the most widely formed intracellular granules are that ling has also confirmed the presence of high protein content
of polyesters or carbonosomes most importantly referred to in the PHA granule [55]. More accurate data showed that the
as PHAs. Ralstonia eutropha stores PHAs as 0.2–0.5 nm 14 nm thick layer surrounds the PHA granules. Investigation
inclusions inside the cell. Marchessault have given some cal- of the PHA structure is highly dependent on the preparation
culations indicating a single molecule of P(3HB) is made up techniques because the PHAs can undergo denaturation or
of approximately 35,000 units of 3-hydroxybutyrate, having crystallization through the physical stress such as in the case
a length of 24,000 nm [50]. PHA granule has an amorphous of freeze-thaw cycles, highly reactive solvents and strong
polyester core [53]. Early thin section electron microscopy alkalis. The PHA synthase converts the substrate monomers
showed a distinct layer of almost 3–4 nm thick [47, 54]. (mostly from carbon sources such as sugars) into polymers
This suggest that the thin layer is composed of phospholipid that have high molecular weights as compare to the substrate

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Fig. 3  Schematic illustration of
the granular structure of PHA
with the granule associated pro-
teins. The polyester synthase is
a PhaC, while PhaZ and PhaD
are shown together as depoly-
merase due to their depolymer-
izing activity. PhaF or phasin is
shown as structural protein due
to its structural support to the
PHA granule

monomer. The PHA synthase remain covalently attached Pseudomonas putida (Previously called as Pseudomonas
with the growing polyester chain during the PHAs forma- oleovorans) have PHA synthases of Class-I and Class-II
tion and continues to add more and more monomers to the respectively. These both classes of synthases having a sin-
growing chain until all the freely available substrate in the gle subunit with an average size of 60-70 kDa. Class-III
cell is depleted or incorporated for the PHAs biosynthesis. and Class-IV PHA synthases have two subunits. Allochro-
It has been widely accepted that the granular size of the matium vinosum and Bacillus megaterium have PhaC/PhaE
PHA polyester and the number of granules formed by each and PhaC/PhaR subunits, and produce PHA synthases of
microbial cell depend upon a number of factors. Growth class-III and IV respectively [59].
conditions, temperature, pH and carbon sources are highly Different microbial groups have evolved different charac-
responsible factors for the biosynthesis of PHAs and have teristic features in PHAs due to the involvement of enzymes.
direct effects on the form, structure and length of the poly- In most of the bacterial species the genes involved in the
ester. The number of PHAs inclusions formed/produced per biosynthesis of the PHAs are present in form of clusters
cell (from 5 to 10) vary between different microbial strains. in the genome which acts as operon. Ralstonia eutropha
is the most widely studied bacteria with respect to the
bacterial genes governing the synthesis of PHAs. Genes
PHA Biosynthesizing Genes and Enzymes in the R. eutropha constitute a phaCAB operon which in
turn is composed of the genes for PHA synthase i.e. phaC,
PhaA, PhaB, PhaC and PhaE are the key enzymes involved β-ketothiolase or phaA and acetoacetyl- CoA reductase
in the biosynthesis of PHAs. Apart from these essential (phaB) as shown in Fig. 4a. PHA synthase genes cluster-
enzymes for polyhydroxyalkanoates synthesis as energy ing in Ralstonia eutropha, Pseudomonas oleovorans and P.
rich reserves PhaP is an additional PHA related phasin that aeruginosa was determined by using enzymatic analysis,
is responsible for the PHA accumulation and its granular homo and heterologous gene probes and PCR techniques as
morphology [56]. Sugars and fatty acids are converted into shown in Fig. 4b–d [54]. Bacterial species like A. latus and
PHAs by microbes through three different metabolic path- B. cepacia also possess phaCAB operon. A large number of
ways involving acetyl-CoA or acyl-CoA as the intermediate bacterial species such as Pseudomonas sp. genes are clus-
species [57]. All the PHAs producing metabolic pathways tered in a different organizational array. An additional genes
end with the polymerization of the monomers by the PHA cluster phaF1 is present in some microbes downstream of
synthases [58]. phaC was the first PHA synthase that was phaC1 which encodes proteins known as phasins (PhaF) and
discovered almost three decades ago. PHA synthases are Pha1. PhaF is a histone like granule associated protein that
divide into four different classes. PHA synthase that pro- is primarily involved in stabilization of the polyhydroxy-
duces scl-PHA belong to Class-I synthases while Class- alkanoates granular structure. Pha1 is granule associated
II synthases produce mcl-PHAs. Ralstonia eutropha and protein like PhaF with functions of structural support for

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Fig. 4  a phaCAB operon in

Ralstonia eutropha with a
cluster of three genes namely
phaC (encoding PHA syn-
thases); phaA gene encoding
β-ketothiolase; phaB encoding
acetoacetyl-CoA. b–d Organiza-
tion of PHA type-II synthases in
P. oleovorans and P. aeruginosa
which are co-localized with
PHA depolymerize genes i.e.
phaZ. phaC1 and phaC2 genes
encode PHA synthase. phaD
controls the simultaneous
expression of PHAs genes

the stabilization of PHA granules. phaD encode PhaD pro- Microbes Best Suitable for PHA Production
teins with some unknown functions. This protein is not a
granule associated as in case of PhaF or Pha1, however it P(3HB) is the first ever discovered PHA produced by Bacil-
is very essential for the production of PHA in bacteria. It lus magaterium [60]. A French researcher Maurice Lem-
is studied that the deletion of phaD and phaF1 results in oigne first isolated and characterized this polyester some-
the reduction of overproduction of aliphatic PHAs in Pseu- where between 1923 and 1927 and showed that the extract
domonas putida by 70% [14]. Almost all of the genes and can be used to make a transparent film [47]. Microbes such
their relevant proteins that are involved in PHA production as bacteria, fungi and microalgae all have potential for the
have some important role in the production rates, granular biosynthesis of polyesters such as PHAs [61]. From the
structure and monomer incorporation in the polymer chain. discovery of polyhydroxyalkanoates back in 20th century
Some of the major genes that are related to PHA synthesis or to this date hundreds of bacterial species and their differ-
depolymerisation are given in Table 2. Some most important ent strains have been identified as PHA producers [62].
PHA related enzymes corresponding to their functions are Although a number of bacterial species are capable of pro-
enlisted Table 3. ducing a large variety of biopolymers for the production of

Table 2  Different genes and their relevant proteins with UPID numbers involved in PHA synthesis and depolymerisation
Species Domain Gene Protein Enzyme UPID Reference

Haloferax mediterranei Archaea phaA PhaA Beta-ketothiolase I3R3D1 [56]

Haloarcula hispanica Archaea phaB PhaB Acetoacetyl-CoA reductase EIU2R6
Haloarcula hispanica Archaea phaC PhaC PHA synthase subunit C G0HQZ6
Haloarcula hispanica Archaea phaE PhaE PHA synthase Subunit E G0HQZ5
Haloferax mediterranei Archaea phaP PhaP PHA granule-associated protein 13R9Z2
Rhizobium fredii Bacteria phaZ PhaZ Polyhydroalkanoate depolymerase G9A116
Ralstonia eutropha Bacteria phaC PhaC PHA synthase M64341 [54]
Pseudomonas oleovorans, Bacteria phaC1/C2 PhaC1/C2 PHA synthase type-II M58445
phaD PhaD PHA depolymerize
phaZ – –
Pseudomonas aeruginosa Bacteria phaC1/C2 PhaC1/C2 PHA synthase type-II X66592
phaD PhaD PHA depolymerize
phaZ – –

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Table 3  PHA biosynthesis associated enzymes and their relevant functions

Enzyme Function Reference(s)

PhaA β-Ketothiolase or Acetyl-CoA acetyl transferase [56]

PhaB Acetoacetyl-CoA reductase [119]
PhaC PHA synthase [120]
PhaE Class-III PHA synthase subunit [121]
PhaR Class-IV PHA synthase Subunit [122]
PhaZ PHA depolymerase [56]
PhaY PHA oligomer hydrolase [123]
PhaM Activate PHA synthase and functions like PhaF [124]
PhaRt Transcriptional regulator of Phasin. [75]
PhaQ Regulator of phasin transcription [18]
Phasins (family of granule associated proteins)
 PhaP Controls the number and size of PHA granules and promote PHA synthesis by regulating phaC [125]
transcription
 PhaF Regulates phaC transcription and play role in the localization of the PHA granules in the cell [126]
 Phal Works with PhaF and have some unknown functions [127]
 GA14 Controls the size of PHA granules [128]
 ApdA Activate PHB depolymerase [129]
 Mms16 Helps in controlling the size, shape and number of PHA in the cell [130]

bioplastics but only a few are highly studied for their greater number of other polysaccharides can be employed as car-
efficiency and high production rates. Bacillus megaterium, bon source for bacteria and a good quantity of PHA can be
Pseudomonas aeruginosa, Pseudomonas putida, Pseu- harvested [65]. The current production rates of microbial
domonas fluorescens, Pseudomonas oleovorans, Ralstonia PHAs are due to the use of commercially prepared carbon
eutropha and Cupriavidus necator are greatly studied bacte- sources. Using natural carbon sources derived from wheat
ria for the sustainable production of PHAs [63, 64]. Nostoc bran [66], rice bran [67], sugar cane molasses [68], sugar
muscorum, Chlorella minutissima and Botryococcus braunii beet molasses [69], waste cooking oil [70] and crude glyc-
are some microalgal sources that have been studied for the erol [71] can effectively increase the production rates of
production of PHAs. Apart from bacterial and algal sources, PHAs while decreasing the production costs. Researchers
some fungal sources such as Aspergillus fumigatus, Sac- in the field of biomaterials and industrial biotechnology
charomyces cerevisiae, and Yarrowia lipolytica have shown have used and studied a large variety of carbon sources for
positive results for PHAs. Table 4a-c enlists the bacterial, effective and sustainable production of PHAs [72]. Some
fungal and microalgal sources respectively for the produc- of the major carbon sources studied for PHA production
tion of different types of PHA polyesters. using different microbial strains are given in Table  5.
Carbon source effects the type of monomer produced by
the microorganism after the fermentation of the provided
Carbon and Nitrogen Sources sugar or carbon source. Apart from the carbon sources
for the Production of PHAs various other chemical compounds are required such as
nitrogen sources. Some of the most common nitrogen sup-
Carbon is the backbone of almost all of the biomass plements include ­(NH4)2SO4, ­NH4Cl and ­NH4NO3. Carbon
sources. Living organisms either multicellular or unicellu- and nitrogen both have different effects on the overall PHA
lar utilize carbon as an energy source for the normal meta- production rates in different microbial strains. Different
bolic activities. It is evident that carbon is the most essen- C/N (carbon to nitrogen) ratios have shown different rates
tial component for the production of PHAs by bacteria of PHA concentration in bacterial cells [73]. Most of the
and all other microbes. There is a huge carbon feed stock studies shown that limiting nitrogen concentrations while
available in nature which can be used as a sustainable and increasing carbon substrates have positive effects on the
cheap source to feed microorganisms for the production of PHA production rates, however there are exceptions as
PHAs. Glucose is the most common and widely studied well where limiting nitrogen source results in lower pro-
energy source for bacteria. Glucose, sucrose, lactose and a duction rates.

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Table 4  a Different bacterial strains and their produced PHAs polymers. b Fungal sources of PHAs. c Microalgal sources of PHAs
Strain PHA Type Reference(s) Strain PHA type Reference(s)

Acinetobacter junii PB 25 P(3HB-co-3HV) [131] Ferrobacillus sp. mcl-PHA [132]

Actinomycetes sp. PHB [133] Gloeothece sp. PHA [134]
Alcaligenes latus P(3HB-co-4HB) [62] Gloeothece PCC6909 PHB [135]
Aphanocapsa NCCU-542 P(3HB) [136] Haemophilus sp. PHA [137]
Aeromonas hydrophila P(3HB-co-3HHx) [59] Halobacterium PHB, [64]
PHV
Aquaspirillum sp. PHA [138] Haloferax mediterranei P(3HB-co-3HV) [57]
Asticcaulus sp. PHA [139] Hyphomicrobium PHB [140]
ZV620
Azomonas macrocytogenes P(3HB) [141] Lamprocystis roseopersicina PHA [50]
P173 3312
Azospirillum brasilense P(3HB) [142] Leptothrix discophora SS-1 PHA [143]
Azatobacter vinelandii P(3HB) [144] Lampropedia hyalina PHA [145]
Bacillus megaterium P(3HB) [146] Methanomonas sp. P(3HB) [147]
Bacillus licheniformis PHA [148] Methylobacterium sp. GW2 P(3HB) [149]
Burkholderia caryophylli PHB & P(3-hydroxy-4-pen- [53, 120] Methylocystis parvus PHB [150]
tenoate)
Bdellovibrio bacteriovorus mcl-PHA [151] Methylomicrobium sp. V49 P(3HB) [152]
HD100
Botryococcus braunii P(3HB) [133] Methylosinus sp. P(3HB-co-3HV) [153]
Cupriavidus necator P(3HB-co-3HV) [154] Nitrobacter sp. PHA [137]
Caulobacter crescentus PHB [155] Nitrococcus sp. PHA [62]
DSM4727
Chlorofrexeus sp. PHA [62] Nocardia corallina P(3HB-co-3HHx) [156]
Chlorogloea sp. PHA [138] Oceanospirillum scl-PHA [157]
Chromobacterium P(3HV) [50] Paracoccus sp. LL1 P(3HB) [158]
Violaceum
Delftia acidovorans P(3HB-co-4HB) [159] Pseudomonas putida KT2440 mcl-PHA [160]
Ectothiorhodospira shaposh- PHA [161] Pseudomonas oleovorans PHA [162]
nikovii
Escherichia coli P(3HB), [35] Pseudomonas chlororaphis mcl-PHA [75]
P(3HB-co-LA)
Pseudomonas aeruginosa P(3HB) [163, 164] Pseudomonas mendocina P(3HO) [21]
47T2
Pseudomonas fluorescens mcl-PHA [165] Pseudomonas putida PHB [104]
Ralstonia eutropha P(3HB-co-3HV) [166] Rhodobacter spheroids U7 PHB [167]
Rhizobium japonicum P(3HB) [168] Rhodococcus ruber P(3HB-co-3HV) [169]
Rhodopseudomonas sp. S16- P(3HB) [170] Rhodospirillum rubrum PHB [55, 129]
VOGS3
Staphylococcus sp. PHA [66] Streptomyces aureofaciens PHB [171]
Staphylococcus cohni B66, scl-PHA [172] Streptomyces sp. JM3 P(3HB) [105]
Staphylococcus caprae B442
Salmonella enterica P(3HP) [68] Salmonella entrica P(3HB-co-3HV) [84]
Thermus thermophilus HB8 P(3HV-co-3HHx-co-3HO) [166] Thermus thermophilus P(3HB) [173]
JCM10941
Fungi Reference Fungi Reference

Aspergillus fumigatus [174] Saccharomyces cerevisiae [175]

Arxula adeninivorans [176] Wickerhamomyces anomalus [177]
Pichia pastoris [178] Yarrowia lipolytica [179]
Microalgae Reference Microalgae Reference

Aulosira fertilissima, [180, 181] Synechococcus subsalsus, [182, 183]

Botryococcus braunii Synechococcus elongates

13
Journal of Polymers and the Environment (2020) 28:2301–2323 2309

Table 4  (continued)
Microalgae Reference Microalgae Reference

Chlorella minutissima [180] Spirulina platensis [180, 184]

Spirulina sp. LEB-18
Synechocystis salina, [182, 185] Nostoc muscorum [186]
Synechocystis sp. PCC 6803

Table 5  Carbon sources used for different types of PHA production with their yields
Carbon source Microbial strain Cell Dry PHA type Yield (g/L) Reference(s)
weight (g/L)

Glucose Ralstonia eutropha 164 P(3HB) 0.48 [70, 71]

Sucrose Alcaligenes latus 143 P(3HB) 0.43
Xylose Pseudomonas cepacia 2.6 P(3HB) 0.11
Whey Escherichia coli 120 P(3HB) 0.52
Molasses Cupriavidus necator 2.86 P(3HB) 0.78
Cheese whey Bacillus megaterium 51 P(3HB) 1.5 [68]
Crude glycerol Pseudomonas denitrificans DSM 413 38 P(3HB) 15.1
Rice bran Pseudomonas hydrogenovora N.R P(3HB-co-3HV) 1.44 [187]
Octanoate Pseudomonas oleovorans ATCC 29347 10.1 mcl-PHA 1 [40]
Palm oil Pseudomonas aeruginosa IFO 3924 2.7 mcl-PHA 1.06
Sugarcane liquor Pseudomonas fluorescence A2a5 32 scl-PHA 22 [188]
Coconut oil Cupriavidus necator H16 4.7 P(3HB-co-3HV) 3.3 [189]
Soya bean oil Pseudomonas aeruginosa IFO 3924 2.2 mcl-PHA N.R [190]
Styrene Pseudomonas putida CA-3 0.78 (R)-3-HV N.R [191]
Oleic acid Pseudomonas oleovorans NRRL B-14683 1.3 N.R 0.5 [40]
n-alkanoates carbohydrates Bacillus cereus SPV 0.56 scl-PHA 0.45 [192]
Triglycerides Pseudomonas resinovorans NRRLB-2649 3.3 mcl-PHA 2.1 [193]
Corn oil Pseudomonas sp. 12.3 mcl-PHA N.R [194]
Sugar beet molasses Bacillus megaterium 50 P(3HB) 3.7 [195]
Sugar cane molasses Bacillus megaterium BA-019 46 P(3HB) 30.5 [68]
Apple pulp waste Pseudomonas citronellolis NRRL B-2504 4 mcl-PHA 1.2 [196]
Olive oil distillate Pseudomonas citronellolis NRRL B-2504 4.8 mcl-PHA 0.5
Starch Ralstonia eutropha NCIM 5149 179 mcl-PHA 1.14 [40]

N.R not reported

Extraction and Purification of PHAs extraction and purification methods. Different extraction

solvents or surfactants are used to separate these polymeric
Extraction and purification of PHAs macromolecules macromolecules from all other cell debris materials which
produced by microorganisms is crucial step for further ensures its purity and quality. A brief summary, benefits
processing and to determine the quality and quantity of and limitations of various extraction methods are given in
the product. Extreme care and expertise are required for the Table 6.
the extraction purposes so as to avoid the mechanical and
physical harm to the extracted polymer. In order to insure
a polymer of high quality and purity different extraction Thermal and Mechanical Characteristics
methods are adopted with different purification outcomes. of PHAs
Some extraction methods include solvent extraction, flota-
tion method, digestion method, supercritical fluid extrac- PHAs have a wide range of properties but the most impor-
tion and aqueous two phase extraction of PHAs. Cell lysis tant are their thermal and mechanical properties. Thermal
and PHA recovery is almost common step of all these characterization of polyhydroxyalkanoates are highly desir-
able as these properties will be useful to determine the fact

13

2310

Table 6  A brief summary of various methods used for extraction and purification of PHAs

13
Extraction methods Brief summary Benefits Limitations Reference(s)

Solvent extraction Cells are pre-treated to rupture and to make High purity, intact polymer, removes endo- Low temperature (below 40 °C) may affect the [73, 197, 198]
PHA granules accessible. These granules toxins recovery rates
are then made soluble in a suitable solvent
(chloroform, acetone, ethylene carbonate
etc.). Precipitation is done with a non-solvent
solution
Flotation method Modified version of solvent extraction. Cell High purity (98%) with 85% recovery effi- Slow and time consuming [199]
debris floats at the surface of the extrac- ciency, reduction in the polymer wastage
tion solvent when left for 72 h at 30 °C in during extraction
chloroform
Digestion method Non-PHA cellular mass is digested using either Enzymes are target specific which can achieve Digestion with sodium hypochlorite has shown [200, 201]
chemical or enzymatic digestion. Sodium good recovery of PHAs PHA degradation thus effecting the recovery
hypochlorite and surfactants are used for and quality
PHA recovery in chemical digestion while
in enzymatic digestion a series of steps such
as heat treatment, enzymatic hydrolysis and
washing with surfactants is involved
Supercritical fluid extraction Gaseous ­Co2, ammonia and methanol are used Highest purity (99.99%) PHAs obtained for Complex as compare to solvent extraction and [34, 202]
as supercritical fluid extraction. Temperature medical purposes digestion method
and operating pressures are key factors that
change the behavior of cell membrane to
extract PHA
Aqueous two phase extraction Utilizes two phases for PHA extraction. One 12% (w/v) recovery of PHA is reported. Limited recovery rates. Impurities may include [201, 203]
phase is water while the other is non-volatile Effective method for PHA isolation from in final product
phase. The non-organic part settles in water bacteria.
while the organic part moves in the non-
volatile part
Biological extraction method Insects such as Black soldier fly (BSF) can High purity (82.95%) PHB granules obtained N.R [204]
be feed on lyophilized cells of Cupriavidus using this biological method.
necator (grown on rich carbon source for
intracellular PHB production) to increase
their protein contents during early growth
stages. Once the feeding is complete PHB
can be extracted from the fecal pellets by
utilizing the gut of the BSF larvae. 82.95%
pure PHB is extracted using this biological
method

N.R not reported

Journal of Polymers and the Environment (2020) 28:2301–2323
Journal of Polymers and the Environment (2020) 28:2301–2323 2311

that how these polymers might be used in the daily life these polyesters exhibit a range of properties starting from
production of bioproducts which may include bioplastics, crystallinity like plastics and elasticity of rubbers [17]. A
biomedical implants and plastic replacements. The thermal graphical representation of different physical and thermal
properties are expressed in terms of glass transition tem- properties of homopolymers of mcl-PHAs and scl-PHAs are
perature ­(Tg) and melting temperature ­(Tm) for amorphous given in Fig. 5.
and crystalline phase respectively. Studies have shown that
with an increase in the number of carbons from 1 to 7 in the
side chain ­Tg decreases with an increase in ­Tm. In this case Phylogenetic Analysis of the Different
the melting temperature increases from 45 to 69 °C [22, 74]. Pseudomonas sp. Producing PHAs
Microbial polyesters particularly mcl-PHAs are elastomers
in real sense as these show elastomeric behavior in a very Most of the microbial organisms including bacteria, fungi
narrow range because of their low T ­ m values. These poly- and algae have shown positive results for PHA production
mers are amorphous at temperatures near or above melting however, few of them are more efficient for PHA biosyn-
temperature [75, 76]. PHAs having medium chain length are thesis as compare to others. Pseudomonas have been stud-
crystalline, have tensile strength, high elongation at break ied and investigated for their potential to produce PHAs.
and are therefore show different mechanical properties as Phylogenetic analysis of different Pseudomonas species that
compare to scl-PHA. scl-PHAs are more brittle and stiff due biosynthesize PHAs can shed light on some of the impor-
to which these have a high crystallinity usually 60–80% [77]. tant aspects of the genetic and evolutionary conserved
In order to improve the flexibility and elongation to break regions in PHA synthesizing genes. PHA synthase protein
of the polymer, different monomers are included or blended sequence for at least twenty different Pseudomonas strains
with the PHA [44, 78]. Melting temperature, glass transi- were retrieved in FASTA format from NCBI data base. The
tion temperature (temperature at which the macromolecular retrieved sequences were then used to construct a maximum
mobility of a molecular structure is observed or the polymers likelihood phylogenetic tree as shown in Fig. 6. The phy-
appear viscous or rubbery), crystallinity, tensile strength logenetic tree or the evolutionary history of these strains
and extension to break percentage are some of the physical for the common protein sequence i.e. PHA synthase protein
properties that determines the quality of the PHA polymer (PhaC) was inferred using maximum likelihood method
and its future use applications. Some of these properties are based on the JTT matrix model. MEGA7 software was used
enlisted in Table 7. Physical properties of the polyhydroxy- to construct the tree after carrying out the local and global
alkanoates depends upon their monomers composition and pairwise alignments with ClustalW. The conserved motifs
their purity. Homopolymers and copolymers have varying were then analyzed in the aligned sequences. It was found
physical standards where the previous are soft while the lat- that out of the twenty strains of Pseudomonas that were
ter are brittle in nature. Some polymers such as P(3HB) are used in this analysis some are more closely associated for
stiff and brittle. Depending upon the monomeric properties PHA synthase producing protein sequence due to conserved

Table 7  Thermal and PHA Polymer Melting Glass transi- Crystallinity (%) Tensile Extension Reference(s)
mechanical properties of temp. tion temp. strength to break
different PHAs (°C) (°C) (MPa) (%)

P(3HB) 177 2 60 43 5 [205]

PHBV 145 −1 56 20 50
P(3HB-co-4HA 146 NR 26.7 389 N.R [206]
P(4HB) 60 − 50 N.R 104 1000 [58, 137]
P(3HB-co-3HV) 150 − 7.25 55–70 25 20
PHV 106.2 − 15.7 N.R 6.6 3.5 [132]
P(3HB-co-4HB) 166 N.R N.R 28 45
P(3HO-co-3HH 61 − 35 N.R 9 380
PHA 173.8 23.39 N.R 15.9 3.6 [158]
P(3HB-co-3HO) 133 −8 N.R 17 680 [17]
PHBHx 127 −1 34 21 400 [207]
PHB4B 150 −7 45 26 444
P(3HB-co-3HHx) 122 N.R N.R 10.3 1.1 [208]

N.R not reported

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2312 Journal of Polymers and the Environment (2020) 28:2301–2323

Fig. 5  Graphical representation
of physical and thermal proper-
ties of short and medium chain
length PHA homopolymers

Fig. 6  Phylogenetic analysis of various Pseudomonas strains by drawn to scale, with branch lengths measured in the number of substi-
maximum likelihood method for their conserved motifs of PHA syn- tutions per site. The analysis involved 20 amino acid sequences. Posi-
thase protein i.e. (PhaC). Maximum likelihood method based on JTT tions with gaps and missing data were eliminated
matrix model was used to infer the evolutionary history. The tree is

13
Journal of Polymers and the Environment (2020) 28:2301–2323 2313

motifs as compared to others in the evolutionary tree. A not produce PHAs, however metabolically engineered strain of
total four such groups with different conserved motifs can be Yarrowia lipolytica has shown enhanced PHAs production rate
seen alongside the bacterial strains in Fig. 6. Pseudomonas [40]. Growth parameters, engineering cell size to accumulate
synxantha, P. rhodesiae, P. protegens, P. corrugata, P. more PHAs granules and reprogramming the PHAs synthesis
brassicacearum share more closely in the tree and have a pathways using CRISPR/Cas9 tools leads to hyper produc-
common motif for the protein with amino acid sequence tion of PHAs the production rates are increased from 40% to
of Met-Ser-Asn-Lys-Asn-Asn-Asp-Asp-Leu-Lys. These ten 76% [81]. CRISPRi was used to repress P(4HB) competing
amino acids for a highly conserved motif in these five Pseu- pathways which allowed the glucose generated flux to enhance
domonas strains. The second group in the tree include six P(4HB) synthesis. Different intensities of CRISPRi decided
bacterial strains which are Pseudomonas syringae, P. savas- the P(3HB) and P(4HB) ratios in a copolymer P(3HB-co-4HB)
tanoi, P. putida, P. putida, P. citronellolis, P. mendocina, [82]. PHAs synthesis operon in microbes such as recombinant
P. alcaligenes. These Pseudomonas have a common motif Escherichia coli can be reprogrammed with newly designed
with amino acid sequence of Asn-Pro-Leu-Tyr-Arg-Arg-Tyr- powerful promoters that can enhance PHAs biosynthesis.
Leu-Gln-Thr. The third group contains three bacterial strains When a promoter arabinose ­PBAD was used as a recombi-
which include Psudomonas aeruginosa, P. oleovorans, P. nant in phaCAB operon in E.coli SY-2 the PHAs production
stutzeri. These strains have no well-defined common motif increased from 45% to 93% [83]. The PHA synthesis is a mul-
as there is very small conserved site with a few amino acids tistep enzymatic pathway that is regulated by phbC, phbA and
i.e. Gln-X-X-Lys-X-X-X-Val. The last and fourth group have phbB genes. Adjusting the expression levels of these genes can
six bacterial strains which include Pseudomonas monteilii, improve PHB accumulation in cells [67]. Aldor et al., 2002
P. entomophila, P. veroni, P. chlororaphis, P. fluorescence, engineered a metabolic pathway in recombinant Salmonella
P. koreensis with a conserved motif being Gly-Leu-Arg-Gly- entrica for the production of P(3HB-co-3HV) [84]. prpC strain
X-Asp-Leu-X-Ser-Thr. On the basis of phylogenetic analysis of Salmonella entrica was used with a mutation in its ability to
of the PHA synthase protein sequence it can be inferred that metabolize propionyl coenzyme A. This bacterium was used as
the protein has same function and characteristics but the host for plasmid harboring PHA synthesis operon (phaCAB)
conserved motifs among these strains are vary, which is a having a second plasmid from E. coli with some other PHA
possible indication of the varying amount of PHA produc- activating genes. This system lead to hyper accumulation of
tion in these species. PHBV in the cell with nearly 40% increase in the accumula-
tion of said polyester as compared to the Salmonella entrica
strain having normal metabolic activity for propionyl coen-
Bioengineering Strategies for PHAs zyme A [84]. Cell fission ring location genes such as minC and
Production minD of certain bacterial species such as E. coli can be used
as engineering targets to change the growth parameters of the
Advancements in synthetic biology, biotechnology, genetics cell from binary division to multiple fission resulting in the
and metabolic engineering has enabled scientists to manipulate enhanced PHB production [85]. It was observed that deleting
and advance the molecular and cellular functions. Biochemical minC and minD will lead cells to grow in a multiple fission
pathways to obtain desirable results either in the form of hyper chain process rather than simple binary division which ulti-
production or complete elimination of chemical compounds mately results in the faster growth with more and more PHA
or products on cellular metabolic levels is now possible using accumulation [86]. The three PHA synthesizing pathways
such advanced and sophisticated techniques. Metabolic engi- namely, acetoacetyl-CoA pathway, in situ fatty acid synthesis
neering and synthetic biotechnology has been used to improve and β-oxidation cycles can be engineered to diversify PHA
the bio catalytic activity of PHA synthase with high PHA in terms of its monomers, homo and copolymers [87]. There
biosynthesis rates. Matsumoto et al., 2006 engineered PHA are numerous opportunities in genetics, metabolic engineer-
synthases from Pseudomonas sp. 61-3 with enhanced PHAs ing, synthetic biology and biotechnology for remodeling the
accumulation rates and enabled the regulation of monomer mechanisms and synthesis pathways for the hyper production
composition of PHAs. Multiple mutations in Ser325Cys resi- of PHAs with increased production rates.
due of PHA synthase increases 9-21% PHA production with
alteration of substrate specificity [79]. Low molecular proteins
like “phasins” can be used for the cost effective extraction of Applications
PHAs. Phasins target PHB and phasin bound PHB are secreted
by the cell without disturbing the cellular membranes [80]. Polyhydroxyalkanoates are polyesters that are extremely
Some microbes can be engineered to produce PHAs irrespec- important due to their polymeric properties which enable
tive of the fact that in nature such organisms do not produce them to become a sustainable and ecofriendly replacement
these polyesters. Yarrowia lipolytica is a yeast that usually do for synthetic polymers/plastics [88]. The physiochemical

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2314 Journal of Polymers and the Environment (2020) 28:2301–2323

properties of PHAs bioplastics are quite similar to that of reduce plastic pollution and can help to achieve sustain-
petrochemical based plastics [37]. PHAs have emerged able development goals of the United Nations. Pollution in
as sustainable source for bioplastic production. Microbial the oceans due to the plastic wastes can be managed and
polyhydroxyalkanoates are non-toxic and ecofriendly which solved by using polyhydroxyalkanoates based bioplastics.
means their use as biomaterials have no negative impacts on Biodegradable plastic utensils, which include cups, spoons
the environment. Packaging materials, agriculture mulching and plates can be made using PHAs. Hybrid biomaterials
films, 3D printing materials, textile, medical implants, drug for a large number of sustainable applications can be pro-
delivery carriers, animal nutritional supplements, drugs, fine duced from PHAs by using chemical modification such as
chemicals, as well as new types of biofuels can be produced epoxidation [92]. Some major industrial applications include
using PHA polymers [45, 89]. PHAs as bioplastic materi- packaging, biofuel generation and production of important
als can replace synthetic plastics from the environment thus chemicals.
helping in the conservation of ecosystem [90, 91]. There are
a wide range of applications where PHAs act as biomaterials
for different uses. Figure 7 shows an illustration of various PHAs as Bioplastics for Packaging Materials
uses in different fields of modern era.
Polyhydroxyalkanoates can be used to prepare over 100
different monomers and copolymers. These different types
Industrial Applications of biopolymeric materials thus developed have a variety
of characteristics that enable them to be used in different
PHAs find a wide range of applications in industrial sector. fields of daily life. FDA (Food and Drug Administration) has
Bioplastics made from PHAs can be used to manufacture approved a PHA copolymer called “Metabolix” which is a
and produce packaging materials. Using these packaging blend of PHB and P(3HO) i.e. P(3HB-co-3HO). Metabolix
materials rather than the synthetic ones can effectively is used for the production of food additives and packaging

Fig. 7  Applications of PHAs in various industrial sectors such as agriculture, health and pharmaceuticals

13
Journal of Polymers and the Environment (2020) 28:2301–2323 2315

materials which have the characteristic properties like pet- Health and Pharmaceuticals
rochemical derived plastics [93]. PHAs and its copolymers
can effectively be employed for the formation of food grade PHA based biocompatible suture have been approved by FDA
packaging materials. Bioplastics formed by PHB and PHBV for medical use. PHA based fibers, nanoparticles and blends
shown higher barrier to aromatic compounds which helps are used for a number of medical applications including regen-
the packed food to maintain its aroma and taste quality for erative medicine and tissue engineering [104]. Catabolic activ-
a long time [94]. PHBV can be used to manufacture blow ity of PHAs results in 3-hydroxyacids which can be used and
molded bottles for beverages and as a coating on papers. modified to synthesize some important anti-microbial com-
These polymers (PHAs) in blended form can resist tempera- pounds [58]. mcl-3HAs produced by some bacterial strains
ture up to 200 °C and are UV protectant which make them such as Streptomyces sp. have anti-bacterial activity against
a good candidate for food and daily life grocery packaging Salmonella typhimurium and Listeria monocytogenes patho-
[95]. PHA can also be used to make diapers for example US gens [105]. 3HB monomers from PHA sources can help fight
patents No. 4826493 and 4880592 have described the pro- Parkinson and Alzheimer diseases by preventing the neural
duction of P(3HB) and P(3HB-co-3HV) as biofilms which cell death [106]. Hydroxyalkanoic acid can be used as precur-
can be used as diaper back sheets [96, 97]. sors for the production of antibiotics, pheromones, vitamins
and aromatic compounds [102]. 3-hydroxyalkanoic acids are
­ a2+ channel activation
studied for potential applications in C
Biofuels and Fine Chemicals and memory enhancement [107, 108].
Poly-3-hydroxybutyrate act as anti-osteoporosis agent
An important research area which employs PHA polyesters by helping serum alkaline phosphatize activity and calcium
is the production of biofuels. Used bioplastic products can deposition in bones [109]. Osteogenic initiation in bone mar-
be methyl esterified to produce biofuels which are sustain- row derived mesenchymal stem cell was observed due to the
able fuels for energy sectors. The first study for biofuel pro- grooved P(3HB-co-3HHx) film. This finding confirms the use
duction from PHA based materials was reported in 2009. of PHAs in treating osteoporosis [110]. Lysozyme embedded
Short and medium chain length PHAs can be esterified with PHA films are useful in wound dressing because these inhibits
methanol to produce hydroxyalkanoate methyl esters [98]. the formation of bacterial biofilms which can cause inflam-
Butanol (an important biofuel) was produced from glucose mation of wounds [111]. Some medium chain length PHAs
through PHB synthesis pathway [99]. Using PHAs as a like P(3HB) and P(4HB) have been investigated for wound
source of biofuel production is highly sustainable because healing and skin infections [112]. Poly-3-hydroxybutyrate
PHAs can be produced from activated sludge and nutrient composites films containing glass nano-particles were used for
rich waste water from different sources [100]. Biofuel pro- wound healing process (Francis et al. [113]). Anti-cancerous
duced through PHA generated from activated sludge should activity of PHA can be observed by combining 3HAs with
be around US$ 1200 per ton [101]. Apart from a source of D-peptide [114]. Poly-3-hydroxybutyrate as food supplement
biofuel generation PHAs can be used to derive industrially in giant tiger prawn has shown anti-pathogenic activity in its
important chemicals like hydroxyalkanoates (HAs) which intestine [115]. Pseudomonas fluorescens GK13 produces a
are the precursors for the synthesis of chemical compounds PHA depolymerase enzyme encoded by a gene phaZGK13
such as antibiotics, aromatics and pheromones [102]. which depolymerizes PHAs to monomers. These monomers
effectively reduced the infections caused by Staphylococcus
aureus [116]. A number of medical devices used during sur-
Environment Remediation by Removal gical operations are made from biomaterials based on PHAs,
of Heavy Metals some of the important devices include cardiovascular patch
grafting, stents, surgical mesh and orthopedic pins [107].
PHAs can be employed for the environmental remediation P(3HB-co-3HV) has been studied for its suitability in blood
purposes apart from its other sustainable applications. Hun- coagulation, complement reaction and hemostasis tests [24].
gund et al., 2018 developed and used PHAs composites such
as PHB-starch and PHB-polyethylene glycol (PEG) com-
posites for the removal of heavy metals from the aqueous PHAs as Drug Delivery Agents
solution. These composites have shown successful adsorp-
tion of ­Pb2+ ions. It was observed that the lead adsorption Treating a disease, at the right time and at the right target
capacity of PHB was 20 µg/ml which was higher than its location is essential for positive treatment results. Drugs are
composites i.e. PHB-PEG and PHB-starch. The adsorption normally delivered through oral, intravenous and subcuta-
capacity of PHB-PEG and PHB-starch was 13 µg/ml and neous routes. PHAs as biocompatible and biodegradable
19 µg/ml respectively [103].

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2316 Journal of Polymers and the Environment (2020) 28:2301–2323

materials can be good source of drug carriers to the target of microspheres have been studied in nanotechnology to act
cells [40]. Poly(4-hydroxybutyrate)-mPEG nanocarriers as nano-carriers of the herbicides with less genotoxicity and
were used to deliver cisplatin which is a known anti-carci- higher biodegradability rates thus increasing the herbicide
nogenic drug to mouse hippocampal HT22 cells with a noted activity [33].
suppression of its growth [117]. Rifampicin in combination
with PHAs form microspheres that behave as drug carriers Future Prospects and Challenges
and are very effective in releasing the antibiotic drug at the
target site [107]. Bioactive compounds can be encapsulated Polyhydroxyalkanoates are the marvelous gift of nature that
in PHB carriers to target the specific sites for more accurate are stored inside the microbial cells. As microbial polyesters
and efficient treatment [21]. Nanotechnology can be used to these amazing granular inclusions can help us to fight the envi-
bind PHAs on lignocellulosic fibers which enables them to ronmental pollution by tackling the current plastic debris and
become potential drug delivery agents [40]. waste mismanagement scenarios. PHAs are not just helpful as
biomass for bioplastic production but these are equally impor-
tant in health and pharmaceuticals. Tissue engineering and
PHAs in Tissue Engineering drug delivery can be revolutionized in future by using PHAs
and their blends. Researchers working in the fields like cancer,
Chemically modified PHAs are very important in tissue Parkinson disease, Alzheimer’s disease, nerve regeneration,
engineering. These engineered products can be applied bioplastics and biofuels can find new gate ways by focusing
for the manufacture of tissue graphs, cardiovascular valves on the important characteristic properties of these microbial
and nerve tissues [118]. P(3HB) and P(3HB-co-3HV) are polyesters. Industry can employ PHAs to find sustainable solu-
extensively studied for biomedical applications, however tions to fight plastic waste by manufacturing and producing
their brittleness and stiffness have made them limited, as bioplastics, biofuels and other economically important chemi-
biomedical process need more elastomeric properties. cals. Despite all these unique and unambiguous applications of
P(3HB-co-3HHx) and P(3HO) are important polymers in PHAs, there are certain limitations which include the selection
biomedical applications as these mcl-PHAs have more elas- of a host microorganisms that can express all the PHA synthe-
ticity as compare to scl-PHAs [44]. In tissue engineering sizing genes. Studies are also required for the hyper production
cells are usually grown on scaffold that support the growing of these polyester from cheap nutrients and biomass sources.
cells. PHAs have been investigated as scaffolds to seed cells Biotechnologically and genetically improved and engineered
and induce the growth of new tissues. PHAs biopolymers bacterial sources are required that can produce PHAs on larger
are also studied for soft tissue engineering such as vascular scales. In conclusion, PHAs have the potential to change the
grafting where malfunctioned blood vessels are replaced or material science, medicine and industry if the challenges that
repaired with biocompatible materials [44]. The vast uses of limits its production rates, economic feasibility are defeated.
PHA monomers, copolymers and their blends as biocompat-
ible materials have placed them at top of the list priority for
future research in tissue engineering particularly cardiovas- Author Contributions  FM and HN designed and organized the data for
the manuscript. IR, MHS and FA contributed in bioinformatics analysis
cular grafting, wound healing and artificial skin engineering of genes involved in PHA production. MZ provided important data and
[118]. contributed in Figure designing. FM wrote the manuscript. HN proof
read the manuscript. All authors approved the final manuscript.

PHAs in Agriculture Sector Compliance with Ethical Standards 

PHAs can be used to make agriculture nets, mulch films and Conflict of interest  All the authors declare that there is no conflict of
interest in this work.
agriculture grow bags [88]. PHAs made biofilms or bioplas-
tics for mulching is used not only to protect the crops but also Ethical Approval  This article does not contain any studies with human
increase its yield. Copolymer such as P(3HB-co-3HHx) are participants or animals performed by any of the authors.
used to manufacture agriculture mulching [33]. Commer-
cial examples of agriculture mulch produced from PHA is
Nodax™ and Mirel™. The increased rate of synthetic mulch References
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