Journal of Polymers and the Environment (2022) 30:3523–3533

https://doi.org/10.1007/s10924-022-02444-y

ORIGINAL PAPER

Optimization, Production and Characterization

of Polyhydroxyalkanoate (PHA) from Indigenously Isolated Novel
Bacteria
Faizan Muneer1 · Ijaz Rasul1 · Muhammad Qasim1 · Arfaa Sajid2 · Habibullah Nadeem1

Accepted: 26 March 2022 / Published online: 28 April 2022

© The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2022

Abstract
Synthetic plastics have multiple applications in modern world. However, being non-degradable in nature, these have turned
into environmental pollutants. Natural ecosystem and global biodiversity are facing serious challenges due to the plastic
pollution. Effective degradation and replacement of synthetic plastics with natural and ecofriendly biomaterial is crucial.
Polyhydroxyalkanoates (PHAs) are the microbial polyesters and have great potential as biopolymers for the development
of bioplastics. PHA produced and accumulated as granules in the cytoplasm of various microbes under limited supply of
nutrients can serve the purpose of bioplastic production. Biosynthesis of PHA using bacteria needs well-optimized nutrient
and growth conditions for which, studies optimizing the parameters of bacterial growth using various carbon and nitrogen
sources are required. Current study optimized the production of PHA by two indigenously isolated strains of Pseudomonas
sp. AK-3 and AK-4. The strain AK-3 produced 1.08 g/L of PHA with a yield of 54.82% in the presence of 2% sucrose as
carbon source and 1% of ­(NH4)2SO4 as nitrogen source (C/N ratio of 4:1). The yield, however, reduced to 7.73% when 2%
­(NH4)2SO4 was added as a nitrogen source in the production medium (C/N ratio of 4:2). Pseudomonas sp. AK-4 produced
0.92 g/L of PHA with a yield of 43.80% for 2% sucrose as carbon source. Addition of 1% ­(NH4)2SO4 had negligible effect
on the yield. Considerable increase in cell dry mass was observed when high concentrations of various carbon sources were
used. Biosynthesis of PHA was declined when 1% concentrations of nitrogen sources such as ammonium chloride ­(NH4Cl),
ammonium nitrate ­(NH4NO3) and ammonium sulphate ­(NH4)2SO4 were used. The optimum temperature and pH for pro-
duction of PHA were found to be 38 °C and 8.0 respectively, with an optimum incubation period of 72 h. FTIR results of
the extracted polyesters showed transmittance peaks at wavenumbers of 1725 ­cm−1, 1375 ­cm−1, 1278 ­cm−1, 1132 ­cm−1,
1054 ­cm−1 and 977 ­cm−1. Based on FTIR analysis it was concluded that the polyester produced by both Pseudomonas sp.
AK-3 and AK-4 was poly-3-hydroxybutyrate P(3HB).

Keywords Biopolymers · Bioplastics · Polyesters · Biodegradation · P(3HB) · Plastics

Introduction synthetic polymers that are derived from the petrochemical

Industrialization has resulted in a fast track developing world
where uncontrolled and rapid use of fossil fuels has acceler-
ated the rate of climate change and pollution [1]. Plastics are
* Habibullah Nadeem
habibullah@gcuf.edu.pk
1 ruptions and human health need immediate response from
Department of Bioinformatics and Biotechnology,
Government College University Faisalabad, Faisalabad,
Pakistan
2
Department of Chemistry, The University of Lahore, Lahore,
Pakistan
products and have serious impacts on the environment due to
their non-degradability [2]. Used plastic products are recy-
cled occasionally on a smaller scale while the large amount
of plastic waste discarded primarily in open landfills and
dumpsites [3]. Being a billion dollar global industry it is a
hard task to achieve its counterpart for a sustainable purpose
[4]. The challenge to counter the impacts of plastics on the
environment and particularly climate change, ecological dis-
the global community [5, 6]. The growing concerns of envi-
ronmental pollution and loss of economy due to the plastics
have to be addressed immediately by introducing natural,
biomass derived sustainable and ecofriendly products [7].

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3524
One solution to plastic pollution lies in biopolymers such
as starch, cellulose, chitin, chitosan, lignin and microbial
polyesters that can be used to produce bioplastics and vari-
ous biomaterials [3]. Polyhydroxyalkanoates (PHAs) are the
microbial polyesters and can be used as excellent biomateri-
als for the production of sustainable and ecofriendly bioma-
terials such as bioplastics [8]. A wide range of prokaryotes
such as bacteria can accumulate PHA as water insoluble
granules inside the cytoplasm, with a size of approximately
0.2–0.5 μm [9]. Maurice Lemoigne, a French researcher
first isolated and characterized poly-3-hydroxybutyrate from
Bacillus megaterium in 1920’s [10]. Lemoigne showed that
the insoluble granules can be used to make a transparent
film of plastic [11]. Latter research suggested that a wide
range of microorganisms can produce these polyesters under
nutrient stress [12]. Since the discovery of PHA, a wide
range of microbes have been reported for their production
and characterization [12–14].
Nutrient stress and the availability of rich carbon source
leads to the production of PHA inclusions in various
microbes [15]. Apart for being energy rich, one of the major
function of PHA is their ability to save the bacterial cell
from the imbalances and osmotic stress conditions in the
environment [16]. Hundreds of monomers, varying in num-
ber of carbon and hydrogen atoms yield a diverse polymeric
class of PHA [17]. Due to the presence of large number of
carbon atoms in the main chain, these polymers are referred
as “carbonosomes” [18].
PHAs are biosynthesized by the polymerization of
hydroxyalkanoate (HAs) where the hydroxyl (–OH) group
of HAs in these polymers is usually present at the β-carbon
of the polymer [18]. In nature, microorganisms such as bac-
teria, fungi and algae can produce PHA and more than 300
such microbes are studied for the production of these polyes-
ters [19]. Due to their excellent polymeric properties such as
toughness, strength, flexibility and thermo-mechanical char-
acteristics, PHA are natural alternatives of synthetic plastics
[20]. PHA such as poly-3-hydroxybutyrate (P3HB), polyhy-
droxyvalerate (PHV), poly-4-hydroxybutyrate (P4HB) and
their copolymers like poly (hydroxybutyrate-co-hydroxy-
valerate) (PHB-co-HV) and poly-3-hydroxybutyrate-co-
3-hydroxyvalerate (P3 (HB-co-HV)) are ecofriendly, bio-
degradable and non-toxic materials for the production of
bioplastics [21–23].
Due to the diverse polymeric properties and character-
istics, PHA can be employed in various industries such as
pharmaceuticals, health and agriculture [24]. Due to their
excellent properties that are quite similar to that of synthetic
plastics, PHA are of great importance, as they can save the
environment from the disastrous impacts of the plastic pol-
lution [25]. Environmental sustainability, development of
ecofriendly biomaterial and bioplastics, PHA can promise
a sustainable future in industry and the environment [26].
13
Journal of Polymers and the Environment (2022) 30:3523–3533
United Nations sustainable development goals (SDGs) can
be achieved if we replace the petrochemical-based plastics
with naturally derived bioplastic materials such as polyhy-
droxyalkanoates [21].
Although microbes capable of producing PHA are natu-
rally present in various environments however, only few
are efficient enough to produce these polyesters with high
production rates and efficiency [22, 27]. Some of the most
common and industrially important microbes studied for
PHA production and characterization include bacteria such
as B. megaterium, Cupriavidus nector, Pseudomonas aerugi-
nosa, P. fluorescens, P. oleovorans, P. putida and Ralstonia
eutropha [28, 29]. Microalgal sources of PHA production
include Botryococcus braunii, Chlorella minustissima and
Nostoc muscorum. Fungal sources such as Aspergillus fumig-
atus, Saccharomyces cerevisiae and Yarrowia lipolytica have
been reported in various studies for PHA production [9]. In
the current study two indigenously isolated bacterial strains,
namely Pseudomonas sp. AK-3 and Pseudomonas sp. AK-4
were used to produce PHA after optimization of nutrients
(carbon and nitrogen) and growth conditions.
Materials and Methods
Microorganisms
Two bacterial isolates namely Pseudomonas sp. AK-3and
Pseudomonas sp. AK-4 having accession numbers of
MW898432 and MW898430 at NCBI respectively, were
collected from the Industrial Biotechnology Laboratory,
Government College University Faisalabad, Pakistan. These
microbial strains along with others were identified after iso-
lation from the waste and plastic dumpsites of Faisalabad
in the previous study [30]. The bacterial strains from the
maintained culture were reactivated and fresh cultures were
prepared on LB-agar plates for the optimization of PHA
production.
Screening of Bacterial Strains for PHA Production
In order to confirm the production of PHA by the Pseu-
domonas sp. AK-3 and AK-4, both the strains were first
grown in nutrient broth medium having; peptone (10 g/L),
beef extract (10 g/L) and NaCl (5 g/L) in two different flasks
and kept in a shaking incubator at 37 °C for 24–48 h. Inocu-
lum from these flasks were used to grow the strains in a
mineral salt medium (MSM) with; ­(NH4)2SO4 (1.0 g/L),
­KH2PO4 (1.0 g/L), ­Na2HPO4·12H2O (12 g/L), ­MgSO4·7H2O
(0.2 g/L) with 1.0 mL/L of micronutrient solution (MES);
­FeCl3·6H2O (9.7 g/L), ­CaCl2·2H2O (7.8 g/L), ­CuSO4·5H2O
(0.15 g/L), ­CoCl2·6H2O (0.2 g/L), N ­ iCl2·6H2O (0.2 g/L)
and ­ZnSO4·7H2O (0.1 g/L) as described by Pernicova et al.,
Journal of Polymers and the Environment (2022) 30:3523–3533
(2018) with selected carbon (glucose, sucrose and maltose)
and nitrogen (ammonium chloride, ammonium sulphate and
ammonium nitrate) sources [31]. The flasks were maintained
in a shaking incubator at 37 °C for 24–48 h. Sudan black-B
staining and fluorescent Nile red staining methods were used
to screen the microbial strains for PHA production.
Sudan Black‑B Staining
Sudan black-B staining solution (0.3%) was prepared by dis-
solving 0.3 g of Sudan black-B in 100 mL of 70% ethanol.
The prepared solution was vortexed vigorously for 5 min in
order to dissolve the dye completely and make it free from
any granular particle. The solution was then filtered using
a filter paper to remove the un-dissolved solid and parti-
cles [32, 33]. Bacterial samples were screened using viable
colony method and smear slide method.
Viable Colony Method
The agar plates with the PHA accumulating and control
strains were flooded with the prepared Sudan black-B solu-
tion for 15–20 min. The plates were washed with sterilized
water. Left upside down the plates for the removal of excess
water and the dye. The black or darkish brown streaked patch
of bacterial spread was confirmed the production of PHA
[28].
Smear Slide Method
Heat fixed bacterial smears on glass slides were prepared for
both bacterial strains. Glass slides were degreased for pos-
sible fatty substances and dirt. Two to three drops of Sudan
black-B solution were poured on to the smears and left for
15–20 min. Washed the slide gently with water and air-dried.
Few drops of xylene were added and left for a minute. Coun-
ter stained with 0.5% (W/V) aqueous solution of safranin for
10–15 s and washed. Examined under a microscope when
dried [34].
Nile Red Staining
Nile red staining method was also used to confirm the PHA
production by Pseudomonas sp. AK-3 and AK-4. 0.25 mg
of Nile red (Sigma, USA) per mL of dimethyl sulfoxide
(DMSO) was added to the sterilized medium to prepare a
final concentration of 0.5 µg/mL. The culture plates were
then prepared by streaking with bacterial strains from the
prepared inoculum and incubated at 34 °C for 24–48 h. The
plates were observed under ultra violet (UV) light at 312 nm
to detect PHA accumulation [29].
3525
Production Media and Culture Conditions
Mineral salt medium (MSM) as described earlier was used
with some modifications for PHA production [31]. The pro-
duction medium was varied with respect to the concentra-
tion and type of carbon and nitrogen sources. The cultiva-
tion was carried out at 37 °C in a rotary shaker at 180 rpm
for 24–72 h. Microelements solution (1.0 mL/L) was also
added to the MSM. Concentrations ranging from 1 to 2% of
glucose, sucrose and maltose were supplemented with the
MSM to optimize the best carbon source for the production
of PHA. Similarly, 0.5 to 1% concentrations of ammonium
chloride ­(NH4Cl), ammonium nitrate (­ NH4NO3) and ammo-
nium sulphate ­(NH4)2SO4) were used to optimize the best
nitrogen source for the production of PHA. All the experi-
ments were performed in triplicates.
Extraction and Purification of PHA
For the extraction of PHA from the bacterial cells, solvent
extraction method as described earlier was used with some
modifications [35]. Bacterial cells were collected from the
sample by centrifugation for 10–15 min at 12,298×g. After
washing with acetone, the obtained pellets were washed
with Triton X-100 to make cells permeable. The cell mass
was then suspended in 50 mL chloroform and shifted to a
250 mL flask with incubation at 60 °C for 3 h to extract
PHA. The extract was cooled down to room temperature and
filtered to remove the cell debris [36, 37]. Chilled metha-
nol was added in a ratio of 1:10 to the filtrate drop wise to
precipitate the PHA [36]. Chloroform and methanol in the
solution was evaporated leaving behind the white powdered
of PHA. The extracted PHA was dried at 40–50 °C to a
constant mass.
Optimization of Different Parameters for PHA
Production
In order to optimize the parameters for maximum production
of PHA, different culture conditions such as carbon, nitrogen
sources, incubation time, pH and temperature were used to
determine the optimum conditions for PHA production by
Pseudomonas sp. AK-3 and AK-4.
Optimization of Carbon and Nitrogen Sources
Carbon and nitrogen sources are crucial for the produc-
tion of PHAs [38]. In order to check the effect of vari-
ous carbon sources on the production of PHA, glucose,
sucrose and maltose were used. 1 and 2% concentrations
of the selected carbon sources were added in the produc-
tion (MSM) medium. For optimizing the nitrogen sources
ammonium chloride (­ NH4Cl), ammonium nitrate (­ NH4NO3),
13
3526
and ammonium sulphate [(NH4)2SO4] were used. 0.5% and
1.0% of each salt was added in the production medium as a
source of nitrogen, keeping 1% glucose as the carbon source.
Optimization of the carbon and nitrogen sources were done
by following the procedure as reported previously by Wei
et al. [39].
Cell dry mass (CDM) was calculated by centrifugation of
the production medium in a pre-weighed tube. The superna-
tant was discarded and obtained cell pellet was dried over-
night at 37 °C. The difference between the weights of the
tube having cell pellets and pre-weighed tube was taken as
CDM [35]. Amount of cell dry mass was noted for both
strains after regular time intervals i.e., 24, 48, and 72 h along
with the percentage yield of PHA. Percentage yield of PHA
was calculated using the formula;
Extracted PHA (g∕L)
% Yield ofPHA = × 100
Cell Dry Mass (g∕L)
Optimization of Temperature and pH
Cultural conditions such as temperature, pH and incubation
time were optimized according to the method reported by
Wei et al. with some modifications [39]. Flask cultures were
maintained at a range of parameters. A temperature range of
25–40 °C, pH 5–9 and incubation time 24–72 h were pro-
vided to find the optimum conditions for PHA production.
Cell dry mass as well as percentage yield of PHA were noted
for the provided parameters.
Characterization of PHA
The extracted PHA was characterized by using Fourier trans-
form infrared spectrophotometry (FTIR) and crotonic acid
test.
FTIR Analysis
FTIR analysis is an important technique for the identifi-
cation of extracted PHA [40]. The extracted PHA in the
form of granular powder was analyzed at national textile
research center, National Textile University (NTU) Faisal-
abad, Pakistan.
Crotonic Acid Confirmation of PHA
Confirmation of PHA was done by crotonic acid test as
reported earlier by [41, 42]. Approximately 1 g of the
extracted polyester was dissolved in chloroform. Evapo-
rated the chloroform and acid hydrolyzed by adding 10 mL
concentrated sulphuric acid ­(H2SO4) in a capped glass tube
and heated in a water bath for 10 min at 100 °C. The color
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Journal of Polymers and the Environment (2022) 30:3523–3533
of the solution changed from yellow to brown because of
acid hydrolysis of the polyester to crotonic acid. Diluted
solution was screened using UV/Vis spectrophotometer
from 200–400 nm. Absorption peak obtained between
230–240 nm confirmed the presence of PHA.
Results and Discussions
Microorganisms and Screening of microbes for PHA
Production
Two bacterial isolates namely Pseudomonas sp. AK-3 and
Pseudomonas sp. AK-4 grown on freshly prepared LB agar
plates from the cultural stock (Supplementary Fig. 1a, b).
These strains were further used for the production of poly-
hydroxyalkanoate (PHA).
Pseudomonas sp. AK-3 and AK-4 were stained using
Sudan black-B and fluorescent Nile red dyes [43, 44]. The
results clearly indicated the production of PHA by both
strains. Viable colony method and smear slide method were
used to stain the bacterial samples using Sudan black-B for
both strains. In viable colony method, the whole culture
plate was flooded with Sudan black-B dye (Supplemen-
tary Fig. 2a, b). Viable colony method was performed as
described by Zargoun et al. [45] while smears (Supplemen-
tary Fig. 3a, b) were prepared and observed under a micro-
scope in smear slide method [32]. The PHA accumulating
bacteria were seen in dark color after fluorescent Nile red
staining (Supplementary Fig. 4a, b). The Pseudomonas sp.
storing inclusions of PHA when streaked on a medium con-
taining fluorescent Nile red dye glows due to the refractile
inclusions as a result of UV light [41].
Extraction and Purification of PHA
PHA was extracted using solvent extraction method [46].
PHA obtained after extraction (Supplementary Fig. 5) from
both strains of Pseudomonas sp. AK-3 and AK-4 were kept
in separate Eppendorf tubes for further analysis.
Optimization of Carbon and Nitrogen Sources
Carbon and nitrogen sources were optimized by varying
the nature and concentration of every selected source for
the production of PHA by Pseudomonas sp. AK-3 and
AK-4. Effect of 1% and 2% carbon sources showed that
higher cell dry mass (CDM) was obtained for 2% concen-
trations of all used carbon sources where as 2% sucrose
gives the maximum CDM after 48 h of incubation while
maltose yields the minimum CDM for both Pseudomonas
sp. AK-3 and AK-4 (Fig. 1a–d). Pseudomonas sp. AK-3
produced 50% yield of PHA while Pseudomonas sp. AK-3
Journal of Polymers and the Environment (2022) 30:3523–3533
Fig. 1  Production of cell dry
mass at various time intervals
(24–72 h.) by Pseudomonas sp.
AK-3 a at 1% concentration of
carbon sources and b at 2% con-
centration of carbon sources; by
Pseudomonas sp. AK-4 c at 1%
concentration of carbon sources
and d at 2% concentration of
carbon sources. The standard
bars indicate the standard
deviation of triplicate experi-
ments. *p < 0.05, **p < 0.03,
***p < 0.01
produced 47% yield of PHA when 2% sucrose was used
as a carbon source. The maximum percentage yields of
PHA (50% and 47%) were produced by Pseudomonas sp.
AK-3 and AK-4 respectively, when 2% sucrose was used as
carbon source (Fig. 2a, b). Cell dry mass (CDM) produced
by Pseudomonas sp. AK-3 and AK-4 at various time inter-
vals (24–72 h) with 0.5 and 1% nitrogen sources showed
that increase in nitrogen concentration increases the CDM
however, the corresponding PHA yield was decreased
(Fig. 3a–d). It was concluded that ammonium sulphate
­(NH4)2SO4 yields highest CDM while ammonium chloride
­(NH4Cl) yields lowest CDM for both species (Fig. 4a, b).
The higher concentration of nitrogen sources increased
the CDM but the percentage yield of PHA was found to
be decreased, which suggested that nitrogen limits the bio-
synthesis of PHA. Optimized parameters for Pseudomonas
AK-3 showed that the highest CDM (1.46 g/L) was
obtained at 2% sucrose concentration and 1% ammonium
sulphate with 72 h of incubation. Pseudomonas sp. AK-3
yielded 1.08 g/L (54.82% of CDM) of PHA when C/N
ratio of 4:1 was used. Pseudomonas sp. AK-4 produced
2.4 g/L CDM with 2% sucrose as carbon source yielding
0.92 g/L (43.80% of CDM) of PHA. 50.27% yield of PHA
was produced by P. aeruginosa [37] which is lower than
3527
the yield obtained for Pseudomonas sp. AK-3 and higher
than that produced by Pseudomonas sp. AK-4 in this study.
These results confirmed previous studies that the increase
in carbon concentration increases PHA production by pro-
moting the formation of CDM [9]. Optimized conditions
for nitrogen sources showed that increase in nitrogen con-
centration resulted in high CDM production however, it
hindered the PHA production. Pseudomonas AK-3 at 0.5%
­NH4NO3 produced 1.14 g/L CDM with 0.19 g/L of PHA
(16.66% yield). By increasing the nitrogen concentration
to 1% the yield of PHA was decreased. 1% (­ NH 4) 2SO 4
yielded the lowest amount of extracted PHA with 7.73%
yield, while it was considered the best source in a previ-
ous study [28]. Studies conducted by Costa et al., 2018 on
microalgae for checking the effects of nitrogen sources on
PHA production reported that increase in nitrogen concen-
tration increased the CDM production however, it does not
support an increase in PHA accumulation or production
[42]. The PHA production by Pseudomonas sp. in this
study was found to be decreased with increase in nitrogen
concentration and 0.21 g/L PHA with 13.2% yield was
produced at 1% ­NH4Cl. Similarly, an increase in nitrogen
concentration in the PHA culture media by P. hydrogeno-
vora actively decreased the yield [35].
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3528
a are not just good enough to obtain purified PHA but also for
b and hydroxyl group stretches (Figs. 6 and 7). The analysis
Fig. 2  Percentage yield of PHA produced by Pseudomonas sp. a
AK-3 and b AK-4 with respect to cell dry mass (CDM) and PHA
obtained from it for various concentrations of carbon sources. The
standard bars indicate the standard deviation of triplicate experi-
ments. *p < 0.05, **p < 0.03, ***p < 0.01
Effect of pH and Temperature on PHA Production
Temperature and pH was optimized for PHA production
by Pseudomonas sp. AK-3 and AK-4. Pseudomonas sp.
AK-3 showed an abrupt increase in CDM between pH 5
to 6 with maximum production at pH 9. Pseudomonas sp.
AK-4 showed a continuous increase in CDM with maxi-
mum production at pH 9 (Fig. 5a). Production of CDM was
found to be maximum at 37–40 °C, when effect of tempera-
ture was studied for both the strains (Fig. 5b). Previously
Bacillus cereus NRRL-B-3711 produced maximum PHA
at 37 °C and pH value of 8.0 [47] whereas study conducted
by Joyline, 2019 reported a decline in PHA production at
temperature above 35 °C [43]. The temperature of 37 °C and
pH 8 was found to be the optimum conditions for PHA pro-
duction by indigenously isolated strains of Pseudomonas sp.
AK-3 and AK-4 in the present study. Elevated temperatures
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Journal of Polymers and the Environment (2022) 30:3523–3533
the higher production rates [44]. An increase in tempera-
ture favors PHA production by increasing the accumulation
in cells up to 30% regardless of the inoculum used [48].
In the present study, we concluded that higher temperature
(35–40 °C) not only increased the CDM production but also
supported the higher yield of PHA within the cell biomass.
The other important aspect is the pH that varies greatly for
various microorganism used to produce PHA. A pH range
varying between slightly acidic to slightly basic is required
to grow microbes in order to produce PHA. In case of Pseu-
domonas sp. AK-3 and AK-4, it was noted that the CDM
production and the PHA yield increased sharply between a
pH of 5–9. Montiel-Jarillo reported the effect of pH (5–9)
on the production of PHA and found maximum yield at ≥ 7.5
pH [49]. Similarly, Muneer et al. suggest higher pH value
(8–9) for maximum PHA production [9].
Characterization of PHA
FTIR Analysis
FTIR analysis of the PHA obtained from both bacterial
strains showed that the polyester produced by both Pseu-
domonas sp. AK-3 and AK-4 was poly-3-hydroxybutyrate
(P3HB). The transmittance and corresponding wavenumbers
represented various carbon-to-carbon; carbon to hydrogen
of these stretches confirmed the production of P3HB. FTIR
results of the extracted polymers showed highest transmit-
tance peaks at wavenumbers of 1725 ­cm−1, 1375 ­cm−1,
1278 ­cm−1, 1132 ­cm−1, 1054 ­cm−1 and 977 ­cm−1. Analysis
of these wavenumbers correspond to the functional groups
that compose poly-3-hydroxybutyrate i.e. P(3HB), polymer.
The band at 1725 ­cm−1 represented the ester carbonyl group
(C=O) while the transmittance band at 1375 ­cm−1 is for –CH
group [47]. The characteristic bands for C–O, –OH and C–C
stretches were found between 1285 and 970 ­cm−1. Similar
results have been reported by various studies for PHA pro-
duction [48].
Crotonic Acid Test
Extracted polyesters on acid hydrolysis with concentrated
­H2SO4 and chloroform changed the color of solution from
yellow to brown confirmed the formation of crotonic acid
from the polyhydroxyalkanoate (Supplementary Fig. 6).
Qualitative confirmation of the extracted PHA was done
using crotonic acid test. The change of sulfuric acid color
on acid hydrolysis of the polyester to dark brown confirmed
the formation of crotonic acid from the PHA [50]. Further-
more, a strong absorption curve for the crotonic acid formed
Journal of Polymers and the Environment (2022) 30:3523–3533 3529

Fig. 3  Production of cell dry

mass at various time intervals
(24–72 h.) by Pseudomonas
sp. AK-3 a at 1% concentration
of nitrogen sources and b at
2% concentration of nitrogen
sources; by Pseudomonas sp.
AK-4 c at 1% concentration
of nitrogen sources and d at
2% concentration of nitro-
gen sources. The standard
bars indicate the standard
deviation of triplicate experi-
ments. *p < 0.05, **p < 0.03,
***p < 0.01

13
3530 Journal of Polymers and the Environment (2022) 30:3523–3533

a
a 2.5

CDM (g/L)
1.5

0.5

0
4 6 8 10
pH

b
2.5

CDM (g/L)
1.5

0.5

0
20 30 40 50
Temprature (oC)

Fig. 5  a Effect of pH and b effect of temperature on the production of

CDM of Pseudomonas sp. AK-3 and AK-4

Fig. 4  Percentage yield of PHA produced by Pseudomonas sp. a

AK-3 and b AK-4 with respect to cell dry mass (CDM) and the PHA
obtained from it for various concentrations of nitrogen. The stand-
ard bars indicate the standard deviation of triplicate experiments.
*p < 0.05, **p < 0.03, ***p < 0.01

Fig. 6  FTIR analysis of the polyester extracted from Pseudomonas

sp. AK-3

13
Journal of Polymers and the Environment (2022) 30:3523–3533
Fig. 7  FTIR analysis of the polyester extracted from Pseudomonas
sp. AK-4
as a result of acid hydrolysis of PHA can be obtained at
λ = 235 nm [40].
Conclusion
In order to produce PHA on industrial scale optimized
growth parameters such as carbon, nitrogen sources, incu-
bation time, pH and temperature are very important. Novel
and indigenous bacterial strains with hyper-production of
PHA are equally important in this regard. The present study,
keeping aforementioned points as its basic target utilized
indigenously isolated strains of Pseudomonas sp. for the
production and optimization of PHA. The study confirmed
that carbon and nitrogen sources along with various growth
parameters highly affected the PHA production rates. Cur-
rently, PHA is expensive in terms of its industrial scale
production due to expensive carbon and nitrogen sources,
therefore, further studies are required to use and explore
cheap and naturally available nutrient sources. Apart from
using indigenously isolated and novel bacterial strains, bio-
engineering of already existing PHA producing microbes
can also be useful in this regard. In short, PHA can ensure
a new horizon of sustainable future and clean environment
if current hurdles in its industrial scale production are well
addressed.
Supplementary Information The online version contains supplemen-
tary material available at https://​doi.​org/​10.​1007/​s10924-​022-​02444-y.
Acknowledgements The results and data presented in this study are
a part of Mr. Faizan Muneer’s MS thesis .The authors would like to
acknowledge the Department of Bioinformatics and Biotechnology,
Government College University Faisalabad, Pakistan, for providing
lab facilities to carry out this research work.
3531
Declarations
Conflict of interest All the authors declare that there is no conflict of
interest in this work.
Ethical Approval This article does not contain any studies with human
participants or animals performed by any of the authors.
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