Bacterial production of

polyhydroxyalkanoates (PHAs) using

various waste carbon sources
Aansa Naseem1 , Ijaz Rasul1 , Zulfiqar Ali Raza2 , Faizan Muneer1 ,3 ,
Asad ur Rehman4 and Habibullah Nadeem1
1
Department of Bioinformatics and Biotechnology, Government College University, Faisalabad, Punjab,
Pakistan
2
Department of Applied Sciences, National Textile University, Faisalabad, Punjab, Pakistan
3
Biological Oceanography Lab, National Institute of Oceanography, Karachi, Sindh, Pakistan
4
Dr Ikram-ul-Haq Institute of Industrial Biotechnology, Government College University Lahore, Lahore,
Punjab, Pakistan

ABSTRACT
Synthetic plastics are in great demand in society due to their diversified properties, but
they cause environmental pollution due to their non-biodegradable nature. Therefore,
synthetic plastics are in need to be replaced with biodegradable plastics. Polyhy-
droxyalkanoates (PHAs), bacterial biopolymers are natural alternative to synthetic
plastics. These are present inside the bacterial cytoplasm in granular form. Presently,
the production cost of PHA is high due to expensive carbon substrates used in its
biosynthesis. Therefore, this study focuses on the cost-effective production of PHA
using waste carbon sources. Rice bran and sugarcane molasses were used as the carbon
source for PHA production from Bacillus subtilis, Bacillus cereus, Alcaligenes sp. and
Pseudomonas aeruginosa. PHA production from these bacterial strains was confirmed
through Sudan Black-B screening. With rice bran, as carbon source, the highest PHA
yield obtained was for P. aeruginosa, which yielded 93.7% and lowest was 35.5% for
B. cereus. Surprisingly, B. cereus produced the highest cell dry mass (0.045 g/L) but its
extracted PHA contents were lowest being only 0.02 g/L. Alcaligenes sp. with 0.031 g/L
CDM yielded 87.1% PHA. B. subtilis had a CDM 0.029 g/L, 0.02 g/L PHA content and
Submitted 29 January 2024
Accepted 26 July 2024 a yield of 69.10%. In the case of sugarcane molasses, P. aeruginosa produced 95% PHA
Published 24 October 2024 yield, 0.02 g/L CDM, and 0.019 g/L PHA content. Alcaligenes sp. yielded 90.9% PHA,
Corresponding author 0.011 g/L CDM, and 0.01 g/L PHA content. B. subtilis produced 91.6% PHA yield,
Habibullah Nadeem, 0.012 g/L CDM, 0.011 g/L PHA content; B. cereus produced 80% PHA yield, 0.015 g/L
[email protected] CDM, 0.012 g/L PHA content at 37 ◦ C, pH 7. Higher concentrations of carbon sources
Academic editor increased the CDM and decreased the PHA yield. The maximum yield of PHA was
Joseph Gillespie obtained from sugarcane molasses. 24–48 h of incubation was optimal for B. subtilis
Additional Information and and B. cereus, while for Alcaligenes and P. aeruginosa incubation time of 48–96 h was
Declarations can be found on desirable for higher PHA yield. The extracted biopolymers were analyzed by Fourier
page 16
transform infrared spectroscopy (FTIR), which identified the extracted biopolymers as
DOI 10.7717/peerj.17936 poly-3-hydroxybutyrate P(3HB). The thermal properties of the extracted biopolymers,
Copyright such as melting temperatures, were analyzed by differential scanning calorimetry
2024 Naseem et al. (DSC), which confirmed the thermal stability.
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How to cite this article Naseem A, Rasul I, Ali Raza Z, Muneer F, ur Rehman A, Nadeem H. 2024. Bacterial production of polyhydrox-
yalkanoates (PHAs) using various waste carbon sources. PeerJ 12:e17936 http://doi.org/10.7717/peerj.17936
 Subjects Biotechnology, Microbiology
Keywords Polyhydroxyalkanoates, Biopolymers, Plastics, Biodegradation, Bioplastics, FTIR,
Bacteria, DSC

INTRODUCTION
Synthetic plastics are non-biodegradable and cause environmental pollution due to their
toxic nature (Angra, Sehgal & Gupta, 2023). Therefore, there is an urgency to search for
sustainable and ecofriendly alternatives to modern day plastics. For the purpose, synthetic
plastics are being replaced by natural, eco-friendly, biodegradable polymers such as lignin,
starch, chitin, and microbial polysaccharides and polyesters (Muneer et al., 2021b). These
biopolymers have mechanical and physicochemical similarities to synthetic plastics (Amaro
et al., 2019). The use of biopolymers due to their similarities with synthetic plastics is highly
encouraged for a sustainable environment and biosphere, moreover these can be used in
a wide range of industrial and pharmaceutical applications (Vicente, Proença & Morais,
2023). Bioplastics can be produced from various sources like plants and microorganisms
(Takagi et al., 2004).
Several diverse groups of sustainable polyesters can be used as biomaterials for the
sustainable production of plastic products; one such group of polyester is known as
polyhydroxyalkanoates (PHAs), which is synthesized by microorganisms (Qiang et al.,
2018). PHA is a natural alternative to synthetic plastics that is biodegradable and non-toxic
(Mannina et al., 2019). PHA is the most commonly studied biodegradable polymer. More
than 300 species of bacteria and archaea are used to synthesize PHA naturally (Khatami et
al., 2021). It is a reservoir of carbon and energy accumulated in bacteria which is produced
by microorganisms under nutrient stress of nitrogen, sulfur, zinc, phosphorous, potassium,
magnesium, iron, and oxygen (Masood, Yasin & Hameed, 2014). It is usually present as
granular structure inside the cytoplasm of the microbial cells (Aljuraifani, Berekaa &
Ghazwani, 2019a). These granules are round-shaped and are 0.1–0.2 µm in diameter.
These inclusion bodies perform the function of storage in the vegetative cells of the bacteria
(Marang, Van Loosdrecht & Kleerebezem, 2018). The formation of such granules of PHAs
varies among different microbes, (Zhang et al., 2018). Microbes containing PHA granules
can survive under a scarcity of food and face environmental challenges (Slaninova et al.,
2018; Obruca et al., 2017).
At present, the production cost of PHA is too high, which is the major limitation
of its industrial scale production. Usually, the use of expensive substrates leads to the
higher cost of PHA production (Guerra-Blanco et al., 2018). As a potential solution,
cost-effective carbon sources should be used (Raza, Abid & Banat, 2018). Readily available
and sustainable carbon sources should be used for the sustainable production of PHA
(Koller et al., 2017; Kourmentza et al., 2017). Using waste materials as carbon sources
is another solution to make production cost-effective (Muiruri et al., 2023). A large
amount of waste feedstock is available in the environment, which can be utilized for
PHA production (Sagastume et al., 2016). Pretreated and well-characterized waste material
is used for effective PHA production (Rodriguez et al., 2018). The carbon of pretreated

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 waste material is higher than untreated waste material and supports microbial growth
(Pittmann & Steinmetz, 2016). The pre-treatment methodology increases the dilution of
organic waste, removes solid materials from it, controls temperature and pH, and increases
waste materials’ sterility (Amulya et al., 2016; Basset et al., 2016). The type of carbon source
affects the structure and style of the PHA monomers.
Along with carbon sources, bacterial cell growth and PHA content are also affected
by the carbon and nitrogen (C/N) ratio (Cui, Shi & Gong, 2017). The most common
nitrogen supplements include NH4 Cl, (NH4 )2 SO4 , and NH4 NO3 . Different C:N ratios
have increased the production of microbial biomass and PHA content (Muneer et al.,
2022a). Hence, there is a direct relation between accumulated PHA and C:N ratio, while
there is an inverse relation between PHA accumulation and microbial growth (Ahn, Jho &
Nam, 2015; Cui, Shi & Gong, 2017). Various microorganisms can produce PHA but studies
have shown bacterial PHA production as more economical because of its capacity to
accumulate high contents of PHA (Verlinden et al., 2007). According to various studies, the
best incubation period for PHA production is 24–96 h; the best pH is 7 with a temperature
of 37 ◦ C for different bacterial species. After the growth of microbial biomass under
optimized conditions, extraction and purification of microbial PHA are essential steps
because purified PHA is further used to determine the physical and chemical characteristics
of produced PHA. To cope with the cost and for sustainable production of PHA, waste
biomass can be used as a carbon source for PHA production. Hence, cost-effective PHA
can replace petroleum-based plastics economically (Muneer et al., 2020). Therefore, the
present study aimed to optimize polyhydroxyalkanoates (PHA) production by using rice
bran and sugarcane molasses as the carbon sources under optimized conditions of bacterial
growth and to achieve sustainable development goals (SDGs) by creating a plastic-free
environment to save the global biodiversity and ecosystem from the hazardous effects of
the plastic pollution (Muneer et al., 2021a).

MATERIALS AND METHODS

Microorganisms and chemicals
Four bacterial strains Bacillus subtilis (accession number KM282517), Bacillus cereus
(accession number KM282518), Pseudomonas aeruginosa (Maqbool et al., 2016), Alcaligenes
sp. used in this study were collected from Industrial Biotechnology Laboratory Government
College University Faisalabad, Pakistan. These strains were reactivated and fresh cultures
were prepared on nutrient agar plates. The inoculum was prepared by adding the bacterial
cells to the nutrient broth. This inoculum was used for further studies. All the chemicals
used in this study were of analytical grade, purchased from RDH (Germany), CarlRoth
Gmbh (Germany), Sigma-Aldrich (USA), and Europroxima (The Netherlands).

Bacterial screening for PHA production

Sudan Black-B staining was used for the screening of bacterial strains to confirm the
production of PHA. Alcoholic Sudan Black-B (0.3%) was used for the bacterial staining,
with some modifications as described by Mohanrasu et al. (2020). Two methods were used
for the bacterial screening with Sudan Black-B. In the first method, the agar plates with

Naseem et al. (2024), PeerJ, DOI 10.7717/peerj.17936 3/21

 the PHA accumulating strains and control strains were flooded with the prepared Sudan
Black B solution for 30–45 min (Alshehrei, 2019). The second method was the smear slide
method, in which the slides were observed under a microscope after staining with alcoholic
Sudan Black-B solution (Muneer et al., 2022).

Carbon sources
This study used rice bran and sugarcane molasses as cost-effective carbon sources. Rice bran
was collected from the local rice milling point at Sheikhupura Road, Faisalabad, Pakistan.
The sugarcane molasses was arranged from the Tandlianwala and Kanjwani Sugar Mills
Limited, Faisalabad, Pakistan. Raw sugarcane molasses contains variable content of different
sugars like glucose, fructose, and sucrose, which aid in PHA production. Some inhibitory
metal ions are also found in cane molasses, which are unsuitable for PHA production;
therefore, they were pretreated before its use.

Pre-treatment of sugarcane molasses

Raw sugarcane molasses contains variable content of various carbohydrates like glucose,
fructose, and sucrose, which aid in PHA production. Some inhibitory metal ions are also
found in cane molasses, which are unsuitable for PHA production. Deionized distilled
water was added to the concentrated sugarcane molasses to set the required sugar content.
Activated charcoal was added to the diluted molasses, stirred for 1–2 h using a magnetic
stirrer, and centrifuged at 6,000 rpm for 10 min. The pH of the filtered supernatant was set
at 7–7.5 and autoclaved (Jo et al., 2021).

PHA production media & culture conditions

The selected bacterial strains were cultured in shaking flasks containing minimal solution
medium (MSM). Before inoculating into MSM, these bacterial strains were cultured in the
nutrient broth (NB) medium containing: Peptone; 5 g/L, Yeast extract; 1.5 g/L, Beef extract;
1.5 g/L, NaCl; 5 g/L, glucose; 10 g/L, pH; 7, incubated at 37 ◦ C, 150 rpm for 24 h. 10ml of
each inoculum was added in autoclaved MSM. The composition of MSM was derived with
some modifications by (Gomaa, 2014). MSM consisted of different salts in the content ratio
of (g/L): Na2 HPO4 .2H2 O; 2.0, NaHCO3 ; 0.5, MgSO4 .7H2 O; 0.5, NH4 Cl; 1.0, CaCl2 .2H2 O;
0.01, KH2 PO4 ; 2.0, and some trace elements including H3 BO3 ; 0.3, ZnSO4 .7H2 O; 0.08,
CoCl2 .6H2 O; 0.2, NiCl2 .6H2 O; 0.02, MnCl2 .4H2 O; 0.03, CuCl2 .2H2 O; 0.01. Five different
concentrations of rice bran (0.5%, 1.0%, 1.5%, 2.0%, and 2.5%) and pretreated sugarcane
molasses (2.0%, 4.0%, 6.0%, 8.0%, and 10.0%) were added to the MSM medium in
different flasks as a carbon source for PHA production. Three replicates were used during
the experiment using each carbon source and for their various concentrations. The data
obtained is given in Tables 1 and 2.

Extraction and purification of PHA

PHA extraction was done using the previously reported methodology with some
modifications (Mohandas et al., 2018; Aljuraifani, Berekaa & Ghazwani, 2019b). After the
incubation period, the cell culture was collected and harvested. The cells of the culture
containing rice bran and cane molasses were harvested differently. The biomass of the cell

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Naseem et al. (2024), PeerJ, DOI 10.7717/peerj.17936

Table 1 Optimization of rice bran concentration and Incubation time.

Bacterial Incubation Rice Bran %

strains time (h)
0.5 1.0 1.5 2.0 2.5
CDM PHA PHA CDM PHA PHA CDM PHA PHA CDM PHA PHA CDM PHA PHA
(g/L) (g/L) % (g/L) (g/L) % (g/L) (g/L) % (g/L) (g/L) % (g/L) (g/L) %
24 3.8 1.7 44.7 11.0 1.4 12.7 5.5 1.9 12.3 11.2 1.0 8.9 9.3 1.4 15
48 2.9 2.0 69 7.0 0.5 7.1 3.2 1.1 8.3 11.9 1.1 9.2 14.7 1.3 8.8
B. subtilis
72 2.6 0.7 26.9 2.5 0.002 8 6.4 1.6 9.7 11.4 0.5 4.4 10.0 0.5 5
96 1.6 0.5 31.3 11.9 0.8 6.7 3.7 1.2 8.8 11.1 0.9 8.1 12.8 0.8 6.3
24 4.5 1.6 35.5 6.3 1.3 20.6 8.7 1.0 11.5 10.1 1.0 9.9 5.6 0.6 10.7
48 6.7 0.6 8.9 4.8 1.1 23 9.3 0.7 7.5 13.0 0.9 6.9 8.9 1.2 13.5
B. cereus
72 7.6 0.4 5.2 9.8 0.8 8.2 4.6 0.8 5.5 19.8 2.1 10.6 8.5 1.5 17.7
96 4.6 1.1 23.9 11.0 2.2 20 11.5 2.2 19.1 16.6 2.5 15 11.4 1.6 14
24 2.0 0.9 45 3.8 1.3 34.2 6.4 1.5 23.4 7.0 1.1 15.7 4.9 1.6 32.6
48 2.4 1.7 70.8 3.7 1.7 45.9 4.8 3.3 68.7 6.4 2.0 31.2 6.5 2.1 32.3
Alcaligenes 72 3.1 2.7 87.1 5.0 3.2 64 6.4 3.0 46.8 14.5 3.3 22.7 7.3 3.4 46.5
96 2.6 0.3 11.5 4.1 0.8 19.5 6.0 0.2 3.3 8.9 1.0 11.2 8.6 1.0 11.6
120 3.3 0.4 12.1 6.5 0.7 10.8 9.8 0.9 9.2 10.0 0.9 9 10.6 1.8 16.9
24 2.9 1.0 34.4 2.6 1.1 42.3 5.1 1.2 23.5 5.3 1.1 20.7 6.9 1.1 15.9
48 2.5 1.9 76 3.9 2.9 74.3 4.3 1.7 39.5 5.4 1.0 18.5 15.5 2.9 18.7
P. aeruginosa 72 3.2 3.0 93.7 5.2 2.6 50 4.6 3.8 82.6 21.7 3.4 15.6 10.0 3.0 30
96 1.9 0.5 26.3 3.7 0.6 16.2 3.9 0.8 20.5 9.1 1.1 12 6.7 0.7 10.4
120 2.7 0.5 18.5 6.1 0.6 9.8 7.3 0.1 1.4 7.9 0.5 6.3 8.7 0.8 9.2
5/21
Naseem et al. (2024), PeerJ, DOI 10.7717/peerj.17936

Table 2 Optimization of Sugarcane molasses concentration and Incubation time.

Bacterial Incubation Sugarcane molasses %

strains time (h)
2.0 4.0 6.0 8.0 10.0
CDM PHA PHA CDM PHA PHA CDM PHA PHA CDM PHA PHA CDM PHA PHA
(g/L) (g/L) % (g/L) (g/L) % (g/L) (g/L) % (g/L) (g/L) % (g/L) (g/L) %
24 12.0 1.1 91.6 0.3 0.1 33.3 1.5 0.9 60 1.0 0.2 20 0.3 0.2 66.6
48 0.7 0.2 28.5 0.8 0.4 50 0.5 0.1 20 0.4 0.1 25 0.5 0.3 60
B. subtilis
72 0.6 0.1 16.6 0.7 0.1 14.2 0.4 0.2 50 1.1 0.5 45.4 0.3 0.1 33.3
96 0.2 0.1 50 0.6 0.1 16.6 0.7 0.3 42.8 1.1 0.6 54.5 0.7 0.2 28.5
24 1.5 1.2 80 0.8 0.4 50 0.6 0.1 16.6 0.9 0.3 33.3 0.3 0.2 66
48 1.1 0.8 72.7 0.8 0.6 75 0.9 0.5 55.5 0.2 0.1 50 0.5 0.2 40
B. cereus
72 0.8 0.5 62.5 1.1 0.4 36.3 1.3 0.7 53.8 0.3 0.2 66.6 0.8 0.4 50
96 0.7 0.4 57.1 0.5 0.1 20 0.5 0.2 40 0.4 0.1 25 0.5 0.2 40
24 1.7 1.3 76.4 1.9 1.2 63.1 1.7 1.1 64.7 10.5 1.3 12.3 2.1 0.5 23.8
48 2.0 1.0 50 3.0 2.1 70 2.2 1.6 72.7 2.0 1.1 55 1.5 0.9 60
Alcaligenes
72 1.2 0.2 16.6 1.5 1.0 66.6 1.3 0.7 53.8 1.8 1.4 77.7 2.2 0.4 18.1
96 1.1 0.7 63.6 0.7 0.4 57.1 2.1 1.5 71.4 1.1 1.0 90.9 1.1 0.4 36.3
24 1.0 0.5 50 0.8 0.5 62.5 0.6 0.1 16.6 1.7 0.6 35.2 2.0 0.4 20
48 0.7 0.4 57.1 1.4 1.1 78.5 1.4 1.0 71.4 2.5 1.0 40 1.6 0.5 31.2
P. aeruginosa
72 2.3 2.1 91.3 3.6 2.5 69.4 4.0 1.6 40 4.8 2.8 58.3 4.3 3.3 76.7
96 2.0 1.9 95 3.5 2.3 65.7 3.7 1.7 45.9 4.8 3.0 62.5 5.9 4.3 72.8
6/21
 culture containing the rice bran was collected. An equal volume of sodium hypochlorite
and chloroform was added to the biomass and incubated at 37 ◦ C for 1 h. After incubation,
the mixture was at 8,000 rpm for 15 min, discarding the upper phase containing cellular
digestions. The lower chloroform phase was collected using a sterile syringe to collect
biopolymers by adding 10 volumes of ice-cold methanol. Then, the precipitated PHA was
dried at 65 ◦ C for 24 h. The biomass of the sugarcane molasses cell culture was collected,
washed with distilled water, and lyophilized by adding 40% sodium hypochlorite, vortexed
vigorously, and incubated at 37 ◦ C for 1 h. After incubation, centrifuged the mixture at
8,000 rpm for 15 min, discarded the supernatant, washed twice with distilled water, and
then splashed the pellet with acetone and methanol. Centrifuged at 10,000 rpm for 5 min,
suspended the pellet in boiling chloroform, and dried at 65 ◦ C for 24 h. The formula in
Eq. (1) is used to calculate the PHA content of the extracted biopolymer.
Weight of PHA(g/L)
PHA(%,w/w) = × 100. (1)
CDM(g/L)

Optimization of the process parameters

To optimize the different parameters for PHA hyperproduction, other culture conditions
such as concentration of carbon source, incubation period, pH conditions, and temperature
were selected, which helped to determine the optimum conditions for PHA production
by B. subtilis, B. cereus, Alcaligenes sp., and P. aeruginosa respectively. The carbon sources
of different concentrations were used to optimize the production of PHA. In the case of
rice bran as a carbon source, five concentrations of rice bran 0.5%, 1.0%, 1.5%, 2.0%,
and 2.5% were added in five separate Erlenmeyer flasks containing MSM media. In the
case of sugarcane molasses as a carbon source, five concentrations of pretreated sugarcane
molasses 2.0%, 4.0%, 6.0%, 8.0%, and 10.0% were added in five separate Erlenmeyer
flasks containing MSM media. Alcaligenes sp. and P. aeruginosa were incubated for 120 h,
and B. subtilis and B. cereus were incubated for 96 h in the shaking incubator. Cell dry
mass (CDM) and PHA yield were determined at 24, 48, 72, 96, and 120 h along with the
percentage yield of PHA derived from bacterial strains with both carbon sources. The pH
of the bacterial growth medium (MSM) was optimized to determine the effect of pH on
PHA production. The pH of the medium was varied from pH 4.0−9.0 for each bacterial
strain by adding NaOH & HCl.

Analytical characterization
Fourier transform infrared spectrophotometry (FTIR) was used to analyze the chemical
structure of the extracted polyhydroxyalkanoates produced from B. subtilis, B. cereus,
Alcaligenes sp., and P. aeruginosa in the form of granular powder (Morya et al., 2021). FTIR
analysis was done by dissolving the PHA powder in the chloroform and KBr (potassium
bromide) pellet. The spectra were recorded in the 600 to 4,000 cm−1 range using FTIR
spectrophotometer. The extracted PHA samples were further thermally characterized
by a differential scanning calorimeter (TA instruments Trios V4.1) to determine the
extracted PHA’s melting temperature (Tm). The heating temperature was adjusted from

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 25−350 ◦ C, to check the melting temperature (Tm ) of the extracted samples under the
nitrogen atmosphere at the heating rate of 10 ◦ C per minute.

Statistical analysis
These experiments were performed in triplicate and the obtained data was statistically
analyzed with one-way ANOVA. The p-values were calculated. Significant, and non-
significant values were noted.

RESULTS
Bacterial screening for PHA production
B. subtilis, B. cereus, Alcaligenes sp., and P. aeruginosa strains were grown on freshly
prepared nutrient agar plates from the stock cultures. All the strains showed good growth
(Fig. 1A) on the plates and were found satisfactory for further study. These bacterial strains
were stained with Sudan Black-B. The results (Fig. 1B) indicated the production of PHA in
all four bacterial strains (Shah, 2014). Under the compound microscope, the black granules
(Fig. 1C) represented the Sudan Black-B positive isolates for PHA production.

Extraction and purification of PHA biopolymer

PHA polymer was extracted by using sodium hypochlorite and chloroform digestion from
cellular biomass (Fig. 2). PHA production from B. subtilis, B. cereus, Alcaligenes sp., and
P. aeruginosa was analyzed after different time intervals, with rice bran and sugarcane
molasses as carbon source respectively. Under optimized conditions of rice bran, P.
aeruginosa effectively utilized 0.5% rice bran and produced the maximum PHA yield,
which was 93.7%, 3.2 g/L CDM, 3.0 g/L PHA content, after 72 h at 37 ◦ C. According
to previously reported studies, Pseudomonas sp. P (16) produced a 90.9% PHA yield
(Aljuraifani, Berekaa & Ghazwani, 2019b), which has little difference from the research
results presented in PHA production yield obtained from Alcaligenes sp. was 87.1%, 3.1 g/L
CDM, and 2.7 g/L PHA content with 0.5% rice bran, after 72 h at 37 ◦ C. PHA production
yield obtained from B. subtilis was 69%, 2.9 g/L CDM, and 2.0 g/L PHA content with 0.5%
rice bran after 24 h at 37 ◦ C. PHA production yield obtained from B. cereus was 35.5%,
4.5 g/L CDM, and 1.6 g/L PHA content with 0.5% rice bran after 24 h of incubation at
37 ◦ C. The maximum CDM and PHA content was obtained from P. aeruginosa after 72 h
of incubation with 2 and 1.5% rice bran, respectively, 21.7 g/L and 3.8 g/L.
Under optimized conditions of sugarcane molasses, P. aeruginosa produced the
maximum PHA yield of 95%, 1.9 g/L PHA content, and 2.0 g/L CDM in the presence
of 2% sugarcane molasses after 96 h, at 37 ◦ C. Ali & Jamil (2017) reported a 45.74%
production yield of PHA, with 1.40 g/L CDM in the presence of glucose as the carbon
source, which is less than the presented results in PHA production yield obtained from
Alcaligenes sp. was 90.9%, 1.0 g/L PHA content and 1.1 g/L CDM with 8% cane molasses,
after 72 h at 37 ◦ C. PHA production yield obtained from B. subtilis was 91.6%, 1.1 g/L PHA
content, and 1.2 g/L CDM with 2% cane molasses after 24 h at 37 ◦ C. PHA production
yield obtained from B. cereus was 80%, 1.2 g/L PHA content, and 1.5 g/L CDM with 2%
cane molasses after 24 h at 37 ◦ C. The maximum CDM and PHA content was obtained

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 Figure 1 Bacterial strains cultured, stained and viewed under microscope. From left to right, (A)
streaking on nutrient agar, (B) Sudan Black-B staining and (C) microscopic images (left to right) of B.
subtilis, B. cereus, Alcaligenes sp., P. aeruginosa. Photographed by Ansa Naseem.
Full-size DOI: 10.7717/peerj.17936/fig-1

from P. aeruginosa after 96 h with 10% cane molasses, respectively, 5.9 g/L and 4.3 g/L.
To find the optimum pH, the PHA production was studied from pH 4–9 (Figs. 3 and 4).
PHA production was maximum at pH 8 with sugarcane molasses; in the case of rice bran,
maximum PHA was produced at pH 9. Hence, Alcaligenes sp. effectively utilized the cane
molasses for PHA production.

FTIR spectroscopy
The functional groups of the extracted PHA from B. subtilis, B. cereus, Alcaligenes sp.,
and P. aeruginosa were analyzed by the FTIR spectrophotometer. Figure 5 indicated the
presence of functional groups, which represented the more intense absorption band
at 1,038.65 cm−1 , 1,056.51 cm−1 , 1,063.49 cm−1 and 1,064.46 cm−1 respectively, that
described the -C-O- stretch of the ester group present in the structure of PHA. This

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 Figure 2 Extracted PHA polymer. PHA polymer extracted from the (A) B. subtilis (B) B. cereus (C) Al-
caligenes sp. (D) P. aeruginosa. Photographed by Ansa Naseem.
Full-size DOI: 10.7717/peerj.17936/fig-2

absorption spectrum is similar to the previous studies in which the absorption band at
1,046 cm−1 , 1,053 cm−1 , and 1,043 cm−1 indicates the presence of C-O carbonyl groups
(Mohapatra et al., 2017; Liu et al., 2021; Tyagi & Sharma, 2021). Other absorbance peaks
of specific characteristics are shown between the 1,500–2,000 (C =O stretch), 2,000–2,500
(-C ≡C- stretch), 2,500–3,000 (C-H stretch), 3,000-3,500 (O-H group), 600–1,000 (C-C
stretch) which are similar to previous reports (Maaloul et al., 2013; Abid, Raza & Hussain,
2016). From the prior review of the literature and in the comparison to standard PHA,
it can be concluded that the presence of these functional groups indicated the presence
of poly-3-hydroxybutyrate (Ojha & Das, 2018; Evangeline & Sridharan, 2019). Hence the
polymer extracted from B. subtilis, B. cereus, Alcaligenes sp., and P. aeruginosa was P(3HB),
i.e., poly-3-hydroxybutyrate.

Naseem et al. (2024), PeerJ, DOI 10.7717/peerj.17936 10/21

 Figure 3 Effect on the yield of PHA by using different carbon sources and pH values by various bacte-
rial strains. pH versus CDM (g/L) produced by B. subtilis, B. cereus, Alcaligenes sp., P. aeruginosa with (A)
sugarcane molasses and (B) rice bran as sole carbon sources. The standard bars indicate the standard devi-
ations of triplicate experiments. ∗ p < 0.05, ∗∗ p < 0.03, ∗∗∗ p < 0.01.
Full-size DOI: 10.7717/peerj.17936/fig-3

Figure 4 Effect of pH on PHA yield produced by various bacterial strains. pH versus PHA yield (g/L)
produced by B. subtilis, B. cereus, Alcaligenes sp., P. aeruginosa with (A) sugarcane molasses and (B) rice
bran as sole carbon sources. The standard bars indicate the standard deviations of triplicate experiments.
∗
p < 0.05, ∗∗ p < 0.03, ∗∗∗ p < 0.01.
Full-size DOI: 10.7717/peerj.17936/fig-4

Naseem et al. (2024), PeerJ, DOI 10.7717/peerj.17936 11/21

 Figure 5 FTIR characterization of PHA polymer. FTIR analysis of the PHA polymer extracted from (A)
B. subtilis, (B) B. cereus, (C) Alcaligenes sp., (D) P. aeruginosa.
Full-size DOI: 10.7717/peerj.17936/fig-5

Naseem et al. (2024), PeerJ, DOI 10.7717/peerj.17936 12/21

 Differential scanning calorimetry (DSC) analysis
The PHA extracted from B. subtilis, B. cereus, Alcaligenes sp., and P. aeruginosa was
thermally characterized by differential scanning calorimetry (DSC) to determine its melting
temperature (Tm ). DSC profiles of all four samples are shown in Fig. 6. PHA produced from
Bacillus subtilis showed a sharp endothermic peak in the range of 160−170 ◦ C, without
significant change of overall heat absorption that represented its melting temperature
(Tm ). This data supported the previous reports (Jung et al., 2020; Lorini et al., 2021). In
the case of B. cereus and Alcaligenes sp., no sharp peaks were observed, and the mild
melting temperature curves were observed in the range of 130−150 ◦ C and 230−240 ◦ C.
Recrystallization, crystal modification, and re-melting of thick crystals produce double
endothermic peaks. This is similar to previous studies in which B. megaterium ATCC
14945 showed endothermic peaks at 133.15 and 145.42 ◦ C (Vu et al., 2021; Tian et al.,
2021). PHA produced from P. aeruginosa showed a sharp endothermic peak between the
230−240 ◦ C range. Hence, the PHA produced during this study is thermally more stable at
relatively high temperatures because melting temperature is the most important factor to
describe the product quality and applications (Xu et al., 2021; Karagöz, 2021). The plastic
for daily household applications, engineering, and high-temperature applications has a
melting temperature of 100−300 ◦ C, consistent with the PHA produced during this study
and can be replaced with synthetic plastic (Feldmann, 2016).

DISCUSSION
These experiments showed that B. subtilis, B. cereus, Alcaligenes sp., and P. aeruginosa
strains produced PHA granules stained black due to Sudan Black-B under the compound
microscope. Bacterial PHA was isolated from cellular biomass using sodium hypochlorite
and chloroform digestion method. Under optimized conditions of rice bran, P. aeruginosa
effectively utilized 0.5% rice bran and produced the maximum PHA yield, which was
93.7%, 3.2 g/L CDM, 3.0 g/L PHA content, after 72 h at 37 ◦ C. According to previously
reported studies, Pseudomonas sp. P (16) produced a 90.9% PHA yield (Aljuraifani, Berekaa
& Ghazwani, 2019b), which has little difference from the research results presented in PHA
production yield obtained from Alcaligenes sp. was 87.1%, 3.1 g/L CDM, and 2.7 g/L PHA
content with 0.5% rice bran, after 72 h at 37 ◦ C. PHA production yield obtained from B.
subtilis was 69%, 2.9 g/L CDM, and 2.0 g/L PHA content with 0.5% rice bran after 24 h at
37 ◦ C. PHA production yield obtained from B. cereus was 35.5%, 4.5 g/L CDM, and 1.6 g/L
PHA content with 0.5% rice bran after 24 h of incubation at 37 ◦ C. The maximum CDM
and PHA content was obtained from P. aeruginosa after 72 h of incubation with 2 and
1.5% rice bran, respectively, 21.7 g/L and 3.8 g/L. Under optimized conditions of sugarcane
molasses, P. aeruginosa produced the maximum PHA yield of 95%, 1.9 g/L PHA content,
and 2.0 g/L CDM in the presence of 2% sugarcane molasses after 96 h, at 37 ◦ C. Ali & Jamil
(2017) reported a 45.74% production yield of PHA, with 1.40 g/L CDM in the presence of
glucose as the carbon source, which is less than the presented results in PHA production
yield obtained from Alcaligenes sp. was 90.9%, 1.0 g/L PHA content and 1.1 g/L CDM with
8% cane molasses, after 72 h at 37 ◦ C. PHA production yield obtained from B. subtilis was

Naseem et al. (2024), PeerJ, DOI 10.7717/peerj.17936 13/21

 Figure 6 Characterizing PHA polymer using DSC thermogram. DSC thermogram of the PHA extracted
from (A) B. subtilis, (B) B. cereus, (C) Alcaligenes sp. (D) P. aeruginosa.
Full-size DOI: 10.7717/peerj.17936/fig-6

Naseem et al. (2024), PeerJ, DOI 10.7717/peerj.17936 14/21

 91.6%, 1.1 g/L PHA content, and 1.2 g/L CDM with 2% cane molasses after 24 h at 37 ◦ C.
PHA production yield obtained from B. cereus was 80%, 1.2 g/L PHA content, and 1.5 g/L
CDM with 2% cane molasses after 24 h at 37 ◦ C. The maximum CDM and PHA content
was obtained from P. aeruginosa after 96 h with 10% cane molasses, respectively, 5.9 g/L
and 4.3 g/L. To find the optimum pH, the PHA production was studied from pH 4-9.
PHA production was maximum at pH 8 with sugarcane molasses; in the case of rice bran,
maximum PHA was produced at pH 9. Hence, Alcaligenes sp. effectively utilized the cane
molasses for PHA production.
The functional groups of the extracted PHA from all the selected bacterial strains
were analyzed by the FTIR spectrophotometer. The presence of functional groups, which
represented the more intense absorption band at 1,038.65 cm−1 , 1,056.51 cm−1 , 1,063.49
cm−1 and 1,064.46 cm−1 respectively, described the -C-O- stretch of the ester group
present in the structure of PHA. This absorption spectrum is similar to the previous
studies in which the absorption band at 1,046 cm−1 , 1,053 cm−1 , and 1,043 cm−1 indicates
the presence of C-O carbonyl groups (Mohapatra et al., 2017; Liu et al., 2021; Tyagi &
Sharma, 2021). Other absorbance peaks of specific characteristics are shown between the
1,500–2,000 (C =O stretch), 2,000–2,500 (-C ≡C- stretch), 2,500–3,000 (C-H stretch),
3,000–3,500 (O-H group), 600–1,000 (C-C stretch) which are similar to previous reports
(Maaloul et al., 2013; Abid, Raza & Hussain, 2016). From the prior review of the literature
and in the comparison to standard PHA, it can be concluded that the presence of these
functional groups indicated the presence of poly-3-hydroxybutyrate (Ojha & Das, 2018;
Evangeline & Sridharan, 2019). Hence the polymer extracted from B. subtilis, B. cereus,
Alcaligenes sp., and P. aeruginosa was P(3HB), i.e., poly-3-hydroxybutyrate. The PHA
extracted from various these bacterial strains were thermally characterized by differential
scanning calorimetry (DSC) to determine its melting temperature (Tm ). PHA produced
from B. subtilis showed a sharp endothermic peak in the range of 160−170 ◦ C, without
significant change of overall heat absorption that represented its melting temperature
(Tm ). This data supported the previous reports (Jung et al., 2020; Lorini et al., 2021). In
the case of B. cereus and Alcaligenes sp., no sharp peaks were observed, and the mild
melting temperature curves were observed in the range of 130−150 ◦ C and 230−240 ◦ C.
Recrystallization, crystal modification, and re-melting of thick crystals produce double
endothermic peaks. This is similar to previous studies in which B. megaterium ATCC
14945 showed endothermic peaks at 133.15 and 145.42 ◦ C (Vu et al., 2021; Tian et al.,
2021). PHA produced from P. aeruginosa showed a sharp endothermic peak between the
230−240 ◦ C range. Hence, the PHA produced during this study is thermally more stable at
relatively high temperatures because melting temperature is the most important factor to
describe the product quality and applications (Xu et al., 2021; Karagöz, 2021). The plastic
for daily household applications, engineering, and high-temperature applications has a
melting temperature of 100−300 ◦ C, consistent with the PHA produced during this study
and can be replaced with synthetic plastic (Feldmann, 2016).

Naseem et al. (2024), PeerJ, DOI 10.7717/peerj.17936 15/21

 CONCLUSION
This research was conducted for the cost-effective production of PHA from B. subtilis, B.
cereus, Alcaligenes sp. and P. aeruginosa by using rice bran and sugarcane molasses as the
carbon source. Increased concentrations of rice bran and sugarcane molasses decreased
the PHA yield. All the selected bacterial strains utilized the cane molasses more effectively
than rice bran. Hence, sugarcane molasses proved to be the best carbon source for the
maximum production of PHA under optimized conditions of incubation period at pH 7
and 37 ◦ C. The 24–48 h incubation period was best suited for full PHA production from
B. subtilis and B. cereus, while the 72–96 h incubation period was best suited for maximum
PHA production from Alcaligenes sp. and P. aeruginosa. FTIR and DSC analysis confirmed
the production of P(3HB) and thermal stability of the produced P(3HB).

ADDITIONAL INFORMATION AND DECLARATIONS

Funding
The authors received no funding for this work.

Competing Interests
The authors declare there are no competing interests.

Author Contributions
• Aansa Naseem conceived and designed the experiments, performed the experiments,
analyzed the data, prepared figures and/or tables, and approved the final draft.
• Ijaz Rasul performed the experiments, authored or reviewed drafts of the article, and
approved the final draft.
• Zulfiqar Ali Raza performed the experiments, authored or reviewed drafts of the article,
and approved the final draft.
• Faizan Muneer conceived and designed the experiments, analyzed the data, prepared
figures and/or tables, and approved the final draft.
• Asad ur Rehman analyzed the data, authored or reviewed drafts of the article, and
approved the final draft.
• Habibullah Nadeem conceived and designed the experiments, analyzed the data,
authored or reviewed drafts of the article, and approved the final draft.

Data Availability
The following information was supplied regarding data availability:
Raw data is available as a Supplementary File.

Supplemental Information
Supplemental information for this article can be found online at http://dx.doi.org/10.7717/
peerj.17936#supplemental-information.

Naseem et al. (2024), PeerJ, DOI 10.7717/peerj.17936 16/21

 REFERENCES
Abid S, Raza ZA, Hussain T. 2016. Production kinetics of polyhydroxyalkanoates by
using Pseudomonas aeruginosa gamma ray mutant strain EBN-8 cultured on soybean
oil. 3 Biotechnology 6(2):1–10 DOI 10.1007/S13205-016-0452-4/FIGURES/5.
Ahn J, Jho EH, Nam K. 2015. Effect of C/N ratio on polyhydroxyalkanoates (PHA)
accumulation by Cupriavidus necator and its implication on the use of rice straw
hydrolysates. Environmental Engineering Research 20(3):246–253.
Ali I, Jamil N. 2017. Biosynthesis and genetics of polyhydroxyalkanoates by newly
isolated Pseudomonas aeruginosa IFS and 30N using inexpensive carbon sources.
International Journal of Environmental Science and Technology 14(9):1879–1888
DOI 10.1007/S13762-017-1268-4.
Aljuraifani AA, Berekaa MM, Ghazwani AA. 2019a. Bacterial biopolymer (polyhy-
droxyalkanoate) production from low-cost sustainable sources. Microbiologyopen
8(6):e00755 DOI 10.1002/MBO3.755.
Aljuraifani A, Berekaa M, Ghazwani A. 2019b. Bacterial biopolymer (polyhydroxyalka-
noate) production from low-cost sustainable sources. Microbiologyopen 8(6):e00755
DOI 10.1002/MBO3.755.
Alshehrei F. 2019. Production of polyhydroxybutyrate (PHB) by bacteria isolated
from soil of Saudi Arabia. Journal of Pure and Applied Microbiology 13:897
DOI 10.22207/JPAM.13.2.26.
Amaro TM, Rosa D, Comi G, Iacumin L. 2019. Prospects for the use of whey for
polyhydroxyalkanoate (PHA) production. Frontiers in Microbiology 10:00992
DOI 10.3389/FMICB.2019.00992.
Amulya K, Reddy MV, Rohit MV, Mohan SV. 2016. Wastewater as renewable feed-
stock for bioplastics production: understanding the role of reactor microen-
vironment and system pH. Journal of Cleaner Production 112(5):4618–4627
DOI 10.1016/J.JCLEPRO.2015.08.009.
Angra V, Sehgal R, Gupta R. 2023. Trends in PHA production by microbially di-
verse and functionally distinct communities. Microbial Ecology 85(2):572–585
DOI 10.1007/s00248-022-01995-w.
Basset N, Katsou E, Frison N, Malamis S, Dosta J, Fatone F. 2016. Integrating
the selection of PHA storing biomass and nitrogen removal via nitrite in the
main wastewater treatment line. Bioresource Technology 200(1):820–829
DOI 10.1016/J.BIORTECH.2015.10.063.
Chen GQ, Jiang XR. 2018. Engineering microorganisms for improving polyhy-
droxyalkanoate biosynthesis. Current Opinion in Biotechnology 53(10):20–25
DOI 10.1016/J.COPBIO.2017.10.008.
Cui Y-W, Shi Y-P, Gong X-Y. 2017. Effects of C/N in the substrate on the simultaneous
production of polyhydroxyalkanoates and extracellular polymeric substances by
Haloferax mediterranei via kinetic model analysis. RSC Advances 7(31):18953–18961
DOI 10.1039/C7RA02131C.

Naseem et al. (2024), PeerJ, DOI 10.7717/peerj.17936 17/21

 Evangeline S, Sridharan TB. 2019. Biosynthesis and statistical optimization of poly-
hydroxyalkanoate (PHA) produced by Bacillus cereus VIT-SSR1 and fabrication
of biopolymer films for sustained drug release. International Journal of Biological
Macromolecules 135:945–958 DOI 10.1016/j.ijbiomac.2019.05.163.
Feldmann M. 2016. The effects of the injection moulding temperature on the me-
chanical properties and morphology of polypropylene man-made cellulose fibre
composites. Composites Part A: Applied Science and Manufacturing 87:146–152
DOI 10.1016/j.compositesa.2016.04.022.
Gomaa EZ. 2014. Production of polyhydroxyalkanoates (PHAs) by Bacillus subtilis and
Escherichia coli grown on cane molasses fortified with ethanol. Brazilian Archives of
Biology and Technology 57(1):145–154 DOI 10.1590/S1516-89132014000100020.
Guerra-Blanco P, Cortes O, Poznyak T, Chairez I, García-Peña EI. 2018. Polyhydrox-
yalkanoates (PHA) production by photoheterotrophic microbial consortia: effect of
culture conditions over microbial population and biopolymer yield and composi-
tion. European Polymer Journal 98:94–104 DOI 10.1016/j.eurpolymj.2017.11.007.
Jo SY, Sohn YJ, Jina Son SY, Yoo J, Baritugo KA, David Y, Kang KH, Kim H, Choi J,
Rhie M, Kim HT, Joo JC, Park SJ. 2021. Biosynthesis of polyhydroxyalkanoates from
sugarcane molasses by recombinant Ralstonia eutropha strains. Korean Journal of
Chemical Engineering 38(7):1452–1459 DOI 10.1007/s11814-021-0783-7.
Jung HR, Choi TR, Han YH, Park YL, Park JY, Song HS, Yang SY, Bhatia SK, Gurav
R, Namgung S, Kwon-Young C, Yang YH. 2020. Production of blue-colored
polyhydroxybutyrate (PHB) by one-pot production and coextraction of indigo
and PHB from recombinant Escherichia coli. Dyes and Pigments 173:107889
DOI 10.1016/J.DYEPIG.2019.107889.
Karagöz İ. 2021. An effect of mold surface temperature on final product properties
in the injection molding of high-density polyethylene materials. Polymer Bulletin
78(5):2627–2644 DOI 10.1007/S00289-020-03231-2.
Khatami K, Perez-Zabaleta M, Owusu-Agyeman I, Cetecioglu Z. 2021. Waste to
bioplastics: how close are we to sustainable polyhydroxyalkanoates production?
Waste Management 119(1):374–388 DOI 10.1016/j.wasman.2020.10.008.
Koller M, Maršálek L, De Sousa Dias MM, Braunegg G. 2017. Producing microbial poly-
hydroxyalkanoate (PHA) biopolyesters in a sustainable manner. New Biotechnology
37(7):24–38 DOI 10.1016/J.NBT.2016.05.001.
Kourmentza J, Plácido C, Venetsaneas N, Burniol-Figols A, Varrone C, Gavala
HN, Reis MAM. 2017. Bioengineering recent advances and challenges towards
sustainable polyhydroxyalkanoate (PHA) production. Bioengineering 4(2):55
DOI 10.3390/bioengineering4020055.
Liu C, Wang X, Yang H, Liu C, Zhang Z, Chen G. 2021. Biodegradable polyhy-
droxyalkanoates production from wheat straw by recombinant Halomonas
elongata A1. International Journal of Biological Macromolecules 187:675–682
DOI 10.1016/J.IJBIOMAC.2021.07.137.
Lorini L, Martinelli A, Capuani G, Frison N, Reis M, Sommer B, Villano M, Majone M,
Valentino F. 2021. Characterization of polyhydroxyalkanoates produced at pilot

Naseem et al. (2024), PeerJ, DOI 10.7717/peerj.17936 18/21

 scale from different organic wastes. Frontiers in Bioengineering and Biotechnology
9:52 DOI 10.3389/FBIOE.2021.628719/BIBTEX.
Maaloul EZ, Trabelsi I, Elleuch L, Chouayekh H, Ben Salah R. 2013. Purification and
characterization of two polyhydroxyalcanoates from Bacillus cereus. International
Journal of Biological Macromolecules 61:82–88 DOI 10.1016/j.ijbiomac.2013.06.043.
Mannina G, Presti D, Montiel-Jarillo G, Suárez-Ojeda ME. 2019. Bioplastic recov-
ery from wastewater: a new protocol for polyhydroxyalkanoates (PHA) extrac-
tion from mixed microbial cultures. Bioresource Technology 282(6):361–369
DOI 10.1016/J.BIORTECH.2019.03.037.
Maqbool Z, Hussain S, Ahmad T, Nadeem H, Imran M, Khalid A, Abid M, Martin-
Laurent F. 2016. Use of RSM modeling for optimizing decolorization of simulated
textile wastewater by Pseudomonas aeruginosa strain ZM130 capable of simultaneous
removal of reactive dyes and hexavalent chromium. Environmental Science and
Pollution Research 23(11):11224–11239 DOI 10.1007/S11356-016-6275-3/METRICS.
Marang L, Van Loosdrecht MCM, Kleerebezem R. 2018. Enrichment of PHA-producing
bacteria under continuous substrate supply. New Biotechnology 41(3):55–61
DOI 10.1016/J.NBT.2017.12.001.
Masood F, Yasin T, Hameed A. 2014. Polyhydroxyalkanoates –what are the uses?
Current challenges and perspectives. Critical Reviews in Biotechnology 35(4):514–521
DOI 10.3109/07388551.2014.913548.
Mohandas SP, Balan L, Jayana G, Anoop BS, Philip R, Cubelio SS, Singh ISB. 2018.
Biosynthesis and characterization of polyhydroxyalkanoate from marine Bacillus
cereus MCCB 281 utilizing glycerol as carbon source. International Journal of
Biological Macromolecules 119(11):380–392 DOI 10.1016/J.IJBIOMAC.2018.07.044.
Mohanrasu K, Rao GR, Gujuluva DH, Zhang K, Prakash GS, Song SD. 2020. Optimiza-
tion of media components and culture conditions for polyhydroxyalkanoates pro-
duction by Bacillus megaterium. Fuel 271(5):117522 DOI 10.1016/j.fuel.2020.117522.
Mohapatra S, Mohanta PR, Sarkar B, Daware A, Kumar C, Samantaray DP.
2017. Production of polyhydroxyalkanoates (PHAs) by bacillus strain isolated
from waste water and its biochemical characterization. Proceedings of the Na-
tional Academy of Sciences India Section B - Biological Sciences 87(2):459–466
DOI 10.1007/S40011-015-0626-6/FIGURES/5.
Morya R, Sharma A, Kumar M, Tyagi B, Singh SS, Thakur IS. 2021. Polyhydroxyalka-
noate synthesis and characterization: a proteogenomic and process optimization
study for biovalorization of industrial lignin. Bioresource Technology 320(1):124439
DOI 10.1016/J.BIORTECH.2020.124439.
Muiruri JK, Yeo J, Soo X, Wang S, Liu H, Kong J, Cao J, Hoon Tan B, Suwardi A, Li
Z, Xu J, Loh XJ, Zhu Q. 2023. Recent advances of sustainable short-chain length
polyhydroxyalkanoates (Scl-PHAs)–plant biomass composites. European Polymer
Journal 187:111882 DOI 10.1016/j.eurpolymj.2023.111882.
Muneer F, Hussain S, Sidra tul Muntaha , Riaz M, Nadeem H. 2021a. Plastics versus bio-
plastics. Millersville: Materials Research Forum LLC DOI 10.21741/9781644901335-9.

Naseem et al. (2024), PeerJ, DOI 10.7717/peerj.17936 19/21

 Muneer F, Nadeem H, Arif A, Zaheer W. 2021b. Bioplastics from biopolymers, an
eco-friendly and sustainable solution of plastic pollution. Polymer Science, Series C
63(1):47–63 DOI 10.1134/S1811238221010057.
Muneer F, Rasul I, Azeem F, Siddique MH, Zubair M, Nadeem H. 2020. Microbial poly-
hydroxyalkanoates (PHAs): efficient replacement of synthetic polymers. Journal of
Polymers and the Environment 28(9):2301–2323 DOI 10.1007/S10924-020-01772-1.
Muneer F, Rasul I, Qasim M, Sajid A, Nadeem H. 2022. Optimization, production
and characterization of polyhydroxyalkanoate (PHA) from indigenously iso-
lated novel bacteria. Journal of Polymers and the Environment 30:3523–3533
DOI 10.1007/s10924-022-02444-y.
Obruca S, Sedlacek P, Koller M, Kucera D, Pernicova I. 2017. Involvement of polyhy-
droxyalkanoates in stress resistance of microbial cells: biotechnological consequences
and applications involvement of polyhydroxyalkanoates in stress resistance of
microbial cells: biotechnological consequences and applications. Biotechnology
Advances 36(3):856–870 DOI 10.1016/j.biotechadv.2017.12.006.
Ojha N, Das N. 2018. A statistical approach to optimize the production of polyhydrox-
yalkanoates from Wickerhamomyces anomalus VIT-NN01 using response surface
methodology. International Journal of Biological Macromolecules 107:2157–2170
DOI 10.1016/J.IJBIOMAC.2017.10.089.
Pittmann T, Steinmetz H. 2016. Potential for polyhydroxyalkanoate production on
German or European municipal waste water treatment plants. Bioresource Technology
214(4):9–15 DOI 10.1016/J.BIORTECH.2016.04.074.
Raza ZA, Abid S, Banat IM. 2018. Polyhydroxyalkanoates: characteristics, production,
recent developments and applications. International Biodeterioration & Biodegrada-
tion 126(1):45–56 DOI 10.1016/J.IBIOD.2017.10.001.
Rodriguez S, Serrano A, Pantión AA, Alonso-Fariñas B. 2018. Challenges of scaling-
up PHA production from waste streams. A review. Journal of Environmental
Management 205(1):215–230 DOI 10.1016/J.JENVMAN.2017.09.083.
Sagastume FM, Heimersson S, Laera G, Werker AG, Svanström M. 2016. Techno-
environmental assessment of integrating polyhydroxyalkanoate (PHA) production
with services of municipal wastewater treatment. Journal of Cleaner Production
137(8):1368–1381 DOI 10.1016/J.JCLEPRO.2016.08.008.
Shah KR. 2014. Optimization and production of polyhydroxybutarate (PHB) by Bacillus
subtilis G1S1 from soil. International Journal of Current Microbiology and Applied
Sciences 3(5):377–387.
Slaninova E, Sedlacek P, Mravec F, Mullerova L, Samek O, Koller M, Hesko O, Kucera
D, Marova I, Obruca S. 2018. Light scattering on PHA granules protects bacterial
cells against the harmful effects of UV radiation. Applied Microbiology and Biotech-
nology 102(4):1923–1931 DOI 10.1007/S00253-018-8760-8.
Takagi Y, Yasuda R, Maehara A, Yamane T. 2004. Microbial synthesis and charac-
terization of polyhydroxyalkanoates with fluorinated phenoxy side groups from
Pseudomonas putida. European Polymer Journal 40(7):1551–1557
DOI 10.1016/j.eurpolymj.2004.01.030.

Naseem et al. (2024), PeerJ, DOI 10.7717/peerj.17936 20/21

 Tian J, Zhang R, Wu Y, Xue P. 2021. Additive manufacturing of wood flour/polyhy-
droxyalkanoates (PHA) fully bio-based composites based on micro-screw extrusion
system. Materials & Design 199:109418 DOI 10.1016/J.MATDES.2020.109418.
Tyagi P, Sharma A. 2021. Utilization of crude paper industry effluent for Polyhydrox-
yalkanoate (PHA) production. Environmental Technology & Innovation 23:101692
DOI 10.1016/J.ETI.2021.101692.
Verlinden RAJ, Hill DJ, Kenward MA, Williams CD, Radecka I. 2007. Bacterial
synthesis of biodegradable polyhydroxyalkanoates. Journal of Applied Microbiology
102(6):1437–1449 DOI 10.1111/J.1365-2672.2007.03335.X.
Vicente D, Proença DN, Morais PV. 2023. The role of bacterial polyhydroalka-
noate (PHA) in a sustainable future: a review on the biological diversity. In-
ternational Journal of Environmental Research and Public Health 20(4):2959
DOI 10.3390/ijerph20042959.
Vu DH, Wainaina S, Taherzadeh MJ, Åkesson D, Ferreira JA. 2021. Produc-
tion of polyhydroxyalkanoates (PHAs) by Bacillus megaterium using food
waste acidogenic fermentation-derived volatile fatty acids. 12(1):2480–2498
DOI 10.1080/21655979.2021.1935524.
Xu Z, Xu M, Cai C, Chen S, Jin M. 2021. Microbial polyhydroxyalkanoate production
from lignin by Pseudomonas putida NX-1. Bioresource Technology 319(2020):124210
DOI 10.1016/j.biortech.2020.124210.
Zhang J, Shishatskaya EI, Volova TG, Da Silva LF, Chen GQ. 2018. Polyhydroxyalka-
noates (PHA) for therapeutic applications. Materials Science and Engineering: C
86(5):144–150 DOI 10.1016/J.MSEC.2017.12.035.

Naseem et al. (2024), PeerJ, DOI 10.7717/peerj.17936 21/21