Received: 27 July 2018 | Revised: 15 September 2018 | Accepted: 18 September 2018

DOI: 10.1002/mbo3.755

ORIGINAL ARTICLE

Bacterial biopolymer (polyhydroxyalkanoate) production from

low‐cost sustainable sources

Amal A. Aljuraifani1 | Mahmoud M. Berekaa2 | Azzah A. Ghazwani1

1
Biology Department, College of
Science, Imam Abdulrahman Bin Faisal Abstract
University, Dammam, Saudi Arabia Twenty‐six different bacterial strains were isolated from samples taken from differ‐
2
Environmental Health Department, College
ent locations Dammam, Saudi Arabia, for screening of their polyhydroxyalkanoate
of Public Health, Imam Abdulrahman Bin
Faisal University, Dammam, Saudi Arabia (PHA) production capability. The initial screening was conducted by staining with
Sudan Black B and Nile Red, followed by examination under fluorescence and elec‐
Correspondence
Amal A. Aljraifani, Biology Department, tron microscopes to characterize PHA granule formation. The PHA‐producing bacte‐
College of Science, Imam Abdulrahman Bin
rial isolates were identified using 16S rRNA gene analyses; the most potent bacterial
Faisal University, Dammam, Saudi Arabia.
Email: [email protected] strain was identified as Pseudomonas sp. strain‐P(16). The PHA production capability
of this strain in the presence of different low‐cost carbon sources, such as rice bran,
dates, and soy molasses, was analyzed. PHA production in the presence of rice bran,
dates, and soy molasses was 90.9%, 82.6%, and 91.6%, respectively.

KEYWORDS
dates, fluorescence microscopy, low‐cost carbon source, PHA yield

1 | I NTRO D U C TI O N been used for PHA production and were found to be cost‐effective
(Raza et al., 2018). In addition to the amount of carbon, the carbon/
Polyhydroxyalkanoates (PHAs) are biodegradable polymers that are nitrogen (C/N) ratio has also been shown to have a profound effect
produced mainly by bacteria in the form of inclusion bodies and act on cell growth and PHA accumulation (Cui, Shi, & Gong, 2017). High
as storage substances inside vegetative cells (Marang, Loosdrecht, & C/N ratios have been reported to promote greater PHA accumula‐
Kleerebezem, 2018; Santiago, Antonio, Alba, & Bernabe, 2018). As tion (Silva et al., 2016). There is great need for carbon sources that
PHAs have properties that are similar to those of synthetic polymers, are sustainable, inexpensive, and readily available (Koller, Marsalek,
they are widely used in the production of biopolymers or bioplastics deSousa, & Braunegg, 2017; Kourmentza et al., 2017). In addition
(Burniol‐Figols et al., 2018, Montiel‐Jarillo, Carrera, & Suarez‐Ojeda, to the choice of substrates, downstream processing is another
2017). They can be produced from many renewable resources under crucial factor in making PHA biosynthesis more efficient (Koller,
varied environmental conditions, which reduces the cost of produc‐ Niebelschutz, & Braunegg, 2013; Madkour, Heinrich, Alghamdi,
tion (Huang et al., 2018; Raza, Abida, & Banat, 2018). The level of PHA Shabbaj, & Steinbuchel, 2013). Although there have been several re‐
granule formation may vary between different producer organisms ports on the use of agro‐industrial wastes in the production of PHA,
(Zhang, Shishatskaya, Volova, Ferreira da Silva, & Chen, 2018). These there are some limitations in their use, as most PHA‐derived medical
differences stem from the variety in substrates, the nature of polym‐ products require high purity to minimize toxicity (Koller, 2018; Peptu
erization, and the diverse metabolic pathways involved in production. & Kowalczuk, 2018). Therefore, the present study was focused on
PHA can be synthesized by a diverse group of microbes, which relies the screening of bacterial strains that could potentially be used for
solely on the types of carbon sources used (Chen & Jiang, 2018). production of PHA in the presence of low‐cost carbon sources, such
The major limitation for PHA production is the cost of production. as rice bran, dates, and soy molasses, that are eco‐friendly, readily
A number of low‐cost carbon sources have been reported to have available sustainable sources.

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium,
provided the original work is properly cited.
© 2018 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

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2 of 7 | ALJURAIFANI et al.

2 | M E TH O D O LO G Y effects of the incubation period, strain 16 was cultivated on produc‐

tion medium for 12, 24, 36, 48, 60, and 72 hr at 37°C. To determine
the effects of different carbon sources, glucose, fructose, lactose,
2.1 | Isolation and screening of bacterial strains
sucrose, and maltose were used at a concentration of 15 g/L with
Three different soil samples were collected from different loca‐ an incubation time of 72 hr. Similarly, to determine the effects of
tions (a petrol station, a residence, and a garden) in the city of different nitrogen sources, peptone, casein, (NH4)2SO 4,NaNO3, and
Dammam in eastern Saudi Arabia and stored in polyethylene bags. yeast extract were used at a concentration of 2 g/L with an incuba‐
Ten grams of each sample was collected in a sterile poly bag, sealed, tion time of 72 hr.
and transferred to the laboratory or stored at 4°C for later analysis.
Approximately 1 g of each sample was dispersed in 99 ml of sterile
2.3 | Production of PHA biopolymer using low‐cost
distilled water and agitated gently for 2 min. Subsequently, the liq‐
carbon sources
uid portion of the soil suspension was serially diluted and spread on
nutrient agar medium containing peptone (5 g/L), beef (3 g/L), NaCl Three different low‐cost carbon sources, rice bran, date molasses,
(5 g/L), and agar (15 g/L). The inoculated plates were incubated at and soy molasses, were added to PHA production medium as the
28–37°C for 24–48 hr. The isolated bacterial colonies were further sole carbon source at concentrations ranging from 5 to 50 g/L (w/v).
subcultured to obtain pure cultures of each of the respective bac‐ Approximately 2% (v/v) was used as an inoculum in all experiments,
terial strains. Subsequently, the pure cultures were maintained on with the pH maintained at 7.0. The inoculated flasks were incubated
nutrient agar slants at 4°C for use in later analyses (Singh, Kumari, at 37ºC for 72 hr. At the end of the incubation period, the cells were
Mittal, Yadav, & Aggarwal, 2013). harvested by centrifugation at 10,000 rcf for 15 min at 4°C and
All bacterial candidates were qualitatively analyzed for PHA washed aseptically with sterile, distilled water. The cell pellet was
accumulation using the viable colony screening technique, which then dispersed in an equal quantity of sodium hypochlorite (5.5% ac‐
involves the application of Sudan Black B dye. For this purpose, nu‐ tive chlorine) and incubated at 45°C for 2 hours. This extract was
trient agar plates containing 1% glucose and an ethanolic solution centrifuged at 8,000 rcf for 20 min, and the PHA pellet was rinsed
of 0.3% Sudan Black B dye were inoculated with bacterial isolates once with water and twice with a mixture of ethanol and acetone
and incubated for 24 hr at 37°C. All of the Sudan Black B‐positive (2:1). Subsequently, the pellet was dissolved in chloroform and cen‐
isolates were subjected to quantification of their PHA production trifuged briefly at 8,000 rcf to remove the undissolved debris. Finally,
(Aswini, Kavitha, Revathy, & Babujanarthanam, 2014; Sunitha & the purified PHA was allowed to dry, and the PHA yield was deter‐
Ujwala, 2013). The positive isolates were further subjected to Nile mined by calculation of the dry weight (g/L). For estimation of the dry
staining for confirmation of PHA accumulation. During this process, cell weight, the whole cell mixture was again centrifuged at 10,000
bacterial smears were stained with Nile Red A stain for 20 min, rcf for 10–15 min and the supernatant was discarded. The cell pellet
cleansed with sterile water and allowed to dry, and then viewed with was washed with water to remove the residual media. Finally, the cell
a fluorescence microscope at a wavelength of 490 nm. PHA‐accu‐ pellet was allowed to dry overnight at 60°C until a constant weight
mulating bacteria exhibited a bright yellowish‐orange color (Bhuwal, (g/L) was obtained. The PHA yield was calculated using the following
Singh, Aggarwal, Goyal, & Yadav, 2013). Twenty‐six different bac‐ formula: % PHA = (weight of PHA/dry cell weight) × 100.
terial strains tested positive during the initial screening procedure;
strain 16 was selected for further analysis on the basis of the amount
2.4 | Identification and characterization of PHA‐
of PHA accumulation, the utilization of carbon sources, and the den‐
producing bacterial strains
sity of cells.
For the molecular identification of PHA‐producing bacteria, the
positive strains were identified using 16S rRNA gene analysis.
2.2 | Optimization of Pseudomonas sp. strain‐P (16)
The strains were sent to the Institute for Research and Medical
for PHA production
Consultation (IRMC) and identified using PCR. Each PCR contained
Pseudomonas sp. strain‐P(16) was optimized for PHA production by Top Taq polymerase (Qiagen, Germany), 10 mM Top Taq PCR buffer,
testing different inoculum concentrations, incubation periods, and 10 mm dNTPs, forward and reverse primers (Applied Biosystems,
carbon and nitrogen sources. The bacterial isolates were inoculated Life Technologies Corporation, USA), and distilled water, to which a
in PHA production media containing (NH4)2SO 4 (2.50 g/L), KH2PO 4 loopful of each bacterial colony was added. Each reaction was sub‐
(1.50 g/L), Na2HPO 4 (3.50 g/L), MgSO 4.7H2O (0.20 g/L), glucose jected to an annealing temperature of 56°C for 75 s in a MyCyclerTM
(20.00 g/L), agar (20.00 g/L), and 1 ml yeast extract trace element (Bio‐Rad, USA). All of the amplified products were purified using
solution (1 mM each of FeSO 4.4H2O, CaCl2.2H2O, MnSO 4.4H2O, a PCR Purification Kit (Qiagen, Germany). The BigDye Terminator
ZnCl2) and incubated at 37°C for 24 hr. To determine the effects Cycle Sequencing Kit (Applied Biosystems, Life Technologies
of strain 16 inoculum concentration on growth and PHA accumula‐ Corporation, USA) was used to sequence the purified amplicons,
tion, inoculum concentrations ranging from 2% to 10% were used which were electrophoresed on a Genetic Analyzer 3500 (Applied
for a 24‐hr incubation period at 37°C. For the determination of the Biosystems, Life Technologies Corporation, USA) using POP 7.
ALJURAIFANI et al. | 3 of 7

(a) (b)

F I G U R E 1 (a) Fluorescence
microscopic view exhibiting bright
yellowish‐orange color and (b)
electron microscopic (EM) view
showing polyhydroxyalkanoate
granules inside the cells of
Pseudomonas sp. strain‐P (16)

The EzTaxon tool was used to determine the similarity of the 16S 3.2 | Optimization of Pseudomonas sp. strain‐P (16)
rRNA sequences obtained from the isolated bacteria with refer‐ for PHA production
ence sequences from various bacterial species (Chun, Vanessa, &
To obtain maximum PHA production, the culture conditions for
Deborah, 2007). Sequencing Analysis Software, version 5.4 (Applied
Pseudomonas sp. strain‐P(16) were optimized in terms of the inoculum
Biosystems, Life Technologies Corporation, USA), was used to verify
concentration, incubation period, and sources of carbon and nitrogen.
the absence of background noise. MAFFT, version 7, was used for
Maximum PHA production (55%–62%) was obtained when 4%–6%
the manual verification of sequence similarity (Katoh & Standley,
(v/v) of inoculum was used (Figure 2a). PHA production increased from
2013). The most potent PHA‐producing bacterial strain that was
10% to 84% with an increase in the incubation period from 12 to 36 hr;
subjected to molecular identification using 16S rRNA gene analysis
beyond 36 hr, there was gradual decline in production (Figure 2b).
is Pseudomonas sp. strain‐P (16).
When the PHA production medium was supplemented with different
carbon sources, such as glucose, fructose, lactose, sucrose, and malt‐
2.5 | Fourier transform infrared (FTIR) analysis ose, maximum PHA production (80%–85%) was obtained when either
glucose or maltose was used as the sole carbon source (Figure 2c).
The extracted PHA was characterized using Fourier transform infra‐
There was no marked variation in PHA production (36%–54%) when
red spectroscopy. Dried PHA polymer from the bacterial candidate
different nitrogen sources were used; an exception was ammonium
was fixed by combining it with KBr powder to form disks. Spectra
−1 sulfate, which leads to a maximum production of 79% (Figure 2d).
were obtained between 400 and 4,000 cm using the SHIMADZU–
IR AFFINITY‐2‐FTIR spectrophotometer (Shimadzu Corporation,
Japan) (Kansiz, Jacobe, & Mc Naughton, 2000). 3.3 | Production of PHA biopolymer using different
low‐cost carbon sources
3 | R E S U LT S In this experiment, PHA production by Pseudomonas sp. strain‐
P(16) was analyzed after growth for 72 hr in the presence of dif‐
3.1 | Isolation and screening of bacterial strains ferent low‐cost carbon sources, such as rice bran, date molasses,
During the present investigation, a total of 26 bacterial strains were and soy molasses (Figure 3a–c). The Results showed that when
isolated from the Eastern Project in Dammam, Saudi Arabia. Of the the medium was supplemented with rice bran at a concentra‐
26 isolates, only eight strains were found to be potent PHA produc‐ tion of 15 g/L (w/v), the maximum production of PHA was 90.9%
ers upon staining with Sudan Black B dye and Nile red stain. Out (R 2 = 0.35), with a CDW of 0.22 g/L. When the medium was sup‐
of these eight potent bacterial strains, Pseudomonas sp. strain‐P(16) plemented with date molasses, the greatest PHA production
was found to be the best potential candidate for PHA production (82.6%, R 2 = 0.53) was obtained at a concentration of 20 g/L,
on the basis of the size of the spherical granules in the vegetative with a CDW of 0.23 g/L. In case of soy molasses, the maximum
cells. Most of these granules were found to occupy 50% of the cellu‐ PHA production was 91.6% (R 2 = 0.0.26) at a concentration of
lar volume and were surrounded by compact membranes (Figure 1). 20 g/L and a CDW of 0.24 g/L. Based on the above findings, it
Transmission electron microscopic studies found that optimal pro‐ can be concluded that all three of the carbon sources gener‐
duction of PHA particles within the bacteria cells occurred in during ate the greatest PHA production at an optimum concentration
the 72‐hr‐long trial. The size distribution of the PHA granules ranged of 15–20 g/L. The present findings revealed an increase in the
from 0.5 to 1.0 μm, with a mean size of 0.5 ± 0.06 μm. The bacte‐ CDW that occurred irrespective of the concentration of the car‐
rial cells contained large, white inclusion particles within the cyto‐ bon sources that was used. It is also pertinent that, with the in‐
plasmic fluid, and were stretched and inflated due to the number of crease in the PHA production, there was a marked decrease in
particles within them. the CDW values.
4 of 7 | ALJURAIFANI et al.

(a) % PHA (w/w) PHA (g/L) CDW (g/L) (a) % PHA (w/w) CDW (g/L)

CDW & PHA ( g/L)

100 y = –2.2542x2 + 21.633x 0.8
80 3
%PHA (w/w)
90 R² = 0.3441 0.7
60 80
2 0.6
40 70

%PHA (w/w)

CDW (g/L)
1 60 0.5
20
50 0.4
0 0 40 0.3
2 4 6 8 10 30
0.2
Inoculum concentraon (%) 20
10 0.1
(b) % PHA (w/w) PHA (g/L) CDW (g/L) 0 0

CDW & PHA ( g/L)

100 2 5 10 15 20 25 30 35 40 45 50
% PHA (w/w)

80 1.5 Rice bran (w/v)

60
1
40 (b) % PHA (w/w) CDW (g/L)
0.5 90 1
20 y = –2.3389x2 + 23.293x 0.9
80
0 0 R² = 0.5343 0.8
70
12 24 36 48 60 72 0.7

%PHA (w/w)
60

CDW (g/L)
Incubaon periods (hr) 0.6
50
0.5
(c) 40
% PHA (w/w) PHA (g/L) CDW (g/L) 0.4
30

CDW & PHA (g/L)

100 2.5 0.3
% PHA (w/w)

80 2 20 0.2
60 1.5 10 0.1
40 1 0 0
20 0.5 5 10 15 20 25 30 35 40 45 50
0 0 Date molasses (w/v)

% PHA (w/w) CDW (g/L)

(c) 100 y = –2.9695x2 + 30.341x 0.8
Effect of carbon sources (15 g/L) on PHA producon aˆer 72 hr of incubaon
90 R² = 0.2632 0.7
(d) 80
% PHA (w/w) PHA (g/L) CDW (g/L) 0.6
70
CDW & PHA (g/L)

%PHA (w/w)

80 0.6

CDW (g/L)
60 0.5
% PHA (w/w)

60 0.5
0.4 50 0.4
40 0.3 40 0.3
20 0.2 30
0.1 0.2
20
0 0 10 0.1
0 0
5 10 15 20 25 30 35 40 45 50
Soy molasses (w/v)
Effect of nitrogen sources (2 g/L) on PHA producon aˆer 72 hr of incubaon

F I G U R E 3 Production of polyhydroxyalkanoate by Pseudomonas

F I G U R E 2 (a–d) Optimization of Pseudomonas sp. strain‐P(16)
sp. strain‐P(16) supplemented by (a) rice bran (b) date molasses (c)
under various growth conditions

indicated the presence of aliphatic ‐CH3 and ‐CH2 groups. The ab‐
3.4 | Characterization of PHA biopolymer using
sorption bands at 1637.56 and 1261.44 cm−1 in the extracted PHA
FTIR analysis
sample corresponded to the C=O and C–O stretching groups and
Fourier transform infrared spectroscopy was used for the chemical were identical to those produced by PHA that was previously iso‐
characterization of the extracted polymer. Dried PHA polymer from lated from microbes (Hong, Sun, Tian, Chen, & Huang, 1999).
the bacterial candidate was used to prepare KBr disks. Each chemical
complex in the sample has its own unique signature within the ab‐
sorbance spectrum; hence, the IR spectrum obtained from a sample 4 | D I S CU S S I O N
is representative of its total chemical composition. The discreteness
of an independent spectrum that is caused by the chemical struc‐ The current study demonstrated that the selected bacterial strain is
ture of every constituent, and the degree to which each contributes capable of producing large amounts PHA by utilizing low‐cost car‐
to the total spectrum, is directly associated with the concentra‐ bon sources. One of the challenges in the production of biopolymers
tion of each constituent in the sample. Spectra were recorded be‐ is the associated costs (Chen & Jiang, 2017; Chen & Jiang, 2018).
−1
tween 400 and 4,000 cm using a Nicolet 6700 FTIR spectrometer The present findings addressed the possibility of biopolymer pro‐
(Nicolet Instrument Corporation, USA). The biopolymer produced duction by Pseudomonas strain‐P (16) using low‐cost sources, such
by the Pseudomonas sp. strain‐P (16) generated two main absorp‐ as rice bran, dates, and soy molasses. These three easily obtainable
tion peaks within the C–H and carbonyl stretching bands that are raw materials can be successfully exploited for bioconversion into
characteristic of PHA (Figure 4). First, intense hydroxyl stretching high value biopolymer by potent bacterial strains. In the optimiza‐
at 3456.43 cm−1 was observed, which is characteristic of the –OH tion experiment, maximum PHA production was obtained when an
group. Absorption bands occurring at 2986.44 and 2858.50 cm−1 inoculum concentration of 4%–6% was used. When different carbon
ALJURAIFANI et al. | 5 of 7

Transmittance (%)

F I G U R E 4 Fourier transform infrared

spectra analysis of polyhydroxyalkanoate
produced by Pseudomonas sp. strain‐p
(16) Wave number (cm–1)

sources were tested, glucose and maltose supported better PHA pro‐ 5 | CO N C LU S I O N
duction. Different nitrogen sources were also tested, among which
ammonium sulfate promoted maximum PHA production. In the pre‐ Though extensive research has been conducted on microbial
sent study, the optimum concentration for maximum PHA produc‐ biopolymer production, its cost compared with that of petro‐
tion using rice bran (90.9%) was 15 g/L (w/v); for dates (82.6%) and leum‐based plastics is still a major concern in regard to large‐
soy molasses (91.6%), it was 20 g/L (w/v). Huang, Duan, Huang, and scale production of this natural polymer. Thus, the present study
Chen (2006) obtained a maximum PHA production of 55.6% using revealed that alternative, low‐cost carbon sources that can be
Haloferax mediterranei in the presence of extruded rice bran and corn‐ utilized for large‐scale production of microbial biopolymer. The
starch at a ratio of 1:8 g/g. Omar, Rayes, Eqaab, Vob, and Steinbtlchel present findings also confirmed that Pseudomonas strain‐P(16) is
(2001) obtained a PHB content of 50% (w/w) using Bacillus mega‐ a potent PHA‐accumulating microbe that may be useful for the
terium with date syrup at a concentration of 5% (w/v). Similarly, economical production of biopolymer. The greatest challenge is
Ataei, Vasheghani‐Farahani, Shojaosadati, and Tehrani (2008) ob‐ the effective recovery of the accumulated PHA and its conversion
tained a maximum PHA production of 71% using date syrup waste. into the finished product.
Solaiman, Ashby, Hotchkiss, and Foglia (2006) produced crude PHA
at 5%–17% using 2%–5% (w/v) of soy molasses. Similarly, Full, Jung,
AC K N OW L E D G M E N T S
and Madigan (2006) produced 25.4% PHA using soy molasses at a
concentration of 0%–2%. In addition to optimizing PHA production The researchers would like to thank the research units at the Faculty
using low‐cost resources, improvement of the overall yield is a major of Science, Imam Abdulrahman Bin Faisal University, for their help in
concern (Huang, Chen, Wen, & Lee, 2017). In the present study, the performing the experiments.
maximum PHA yield recorded was in the range of 73%–92%, which is
much greater than that achieved during previous studies (Abid, Raza,
C O N FL I C T O F I N T E R E S T
& Hussain, 2016; Huang et al., 2006). PHA production by bacterial
strains also depends on the carbon source used (Raza et al., 2018). The authors declare no conflict of interests.
During the optimization experiments, glucose and maltose were
found to be suitable carbon sources for Pseudomonas strain‐P(16)
AU T H O R S C O N T R I B U T I O N
that improved the PHA yield. Of the three carbon sources used with
the P(16) strain, the maximum yield was obtained using soy molasses Mrs. Azzah participated in the laboratory work (Isolation and ex‐
(92%), followed by rice bran (91%) and date molasses (83%). Thus, the traction of materials). Dr. Mahmoud contributed to Results and
present study revealed that the PHA yield depends on the type of Discussion. Dr. Amal contributed to Results and Discussion and is a
substrate used (Venkateswar‐Reddy et al., 2016). corresponding author.
6 of 7 | ALJURAIFANI et al.

E T H I C S S TAT E M E N T cultivation strategy. Bioresource Technology, 241, 802–811. https://

doi.org/10.1016/j.biortech.2017.05.192
The research was carried out under the rules of ethics approved by Huang, L., Chen, Z., Wen, Q., Zhao, L., Lee, D. J., Yang, L., & Wang,
the Imam Abdulrahman Bin Faisal University. Y. (2018). Insights into Feast‐Famine polyhydroxyalkanoate
(PHA)‐producer selection: Microbial community succession,
relationships with system function and underlying driving
DATA ACC E S S I B I L I T Y forces. Water Research, 131, 167–176. https://doi.org/10.1016/j.
watres.2017.12.033
The data presented in this manuscript are available upon request Huang, T. Y., Duan, K. J., Huang, S. Y., & Chen, C. W. (2006). Production
from the institutional archives of the corresponding author. of polyhydroxyalkanoates from inexpensive extruded rice bran and
starch by Haloferax mediterranei. Journal of Industrial Microbiology
and Biotechnology, 33, 701–706. https://doi.org/10.1007/
s10295-006- 0098-z
ORCID
Kansiz, M., Jacobe, H. B., & Mc Naughton, D. (2000). Quantitative deter‐
Amal A. Aljuraifani http://orcid.org/0000-0003-2863-7620 mination of the biodegradable polymer poly (β‐hydroxybutyrate) in a
recombinant Escherichia coli Strain by use of mid‐infrared spectros‐
copy and multivariative statistics. Applied Environmental Microbiology,
66, 3415–3420.
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