Bioresource Technology Reports 15 (2021) 100783

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Bioresource Technology Reports

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Enhanced recovery of polyhydroxyalkanoates from secondary wastewater

sludge of sewage treatment plant: Analysis and process
parameters optimization
Vineet Kumar a, b, Shaili Srivastava c, Indu Shekhar Thakur a, c, *
a
Environmental Microbiology and Biotechnology Laboratory, School of Environmental Sciences, Jawaharlal Nehru University, New Delhi 110067, India
b
Department of Botany, School of Life Science, Guru Ghasidas Vishwavidyalaya, Koni, Bilaspur, Chattisgarh 495009, India
c
Amity School of Earth & Environment Science, Amity University Haryana, Manesar, Gurugram, Haryana 122413, India

A R T I C L E I N F O A B S T R A C T

Keywords: The present study was aimed to recover and characterize the polyhydroxyalkanoates (PHA) from secondary
13
C NMR spectroscopy sludge biomass of sewage treatment plant (STP) extracted by organic solvent under optimized conditions. The
3-Hydroxyvalerate maximum recovery yield and purity of PHA were 8.3% and 96%, respectively under optimized conditions of
3-Hydroxybutyrate
incubation time 72 h, reaction temperature incubation at 75 ◦ C, and sludge solid to chloroform ratio 1:20 (w/v).
Municipal wastewater sludge
Wastewater Treatment
The maximum PHA concentration was 9.58 g/L, under optimized conditions. Spectroscopic analyses of recovered
polymer were revealed the presence of 3-hydroxybutyrate (90%) and 3-hydroxyvalerate (10%) monomers
content. PHA was thermally stable at below 260 ◦ C, as confirmed by TGA and DSC analyses. This study
concluded that sewage sludge can be a sustainable substitute to recover PHA due to enormously maximum PHA
yield, inexpensive feedstock, and subsequently decrease the excess sludge burden generated from STP.

1. Introduction
Conventional petrochemical-based synthetic plastic has a wide array
of applications in our daily life, from automobiles, packaging, machin­
ery housings, and furniture to accessories. The annual global production
of petrochemicals-based synthetic plastic has been increasing extremely,
that was about 4.0 × 102 million tons in 2018 and it is expected that
worldwide production of plastic will reach approximately 18 × 102
million tons annually by 2050 (Jem and Tan, 2020). Synthetic polymers,
made from petrochemicals are highly resistant to microbial destruction,
posing a major threat to the environment (Kedzierski et al., 2018; Eu­
ropean bioplastics, 2020). Previous research has demonstrated that
microplastics can absorb metals and organic chemicals in the environ­
ment which may be enriched in the food chain to hazard not only
aquatic organisms but also human beings (Smith et al., 2018; Yu et al.,
2019). All these issues are providing a strong initiative to search for a
substitute of petrochemical-derived plastics.
Due to their great biodegradability, strong resilience to temperature,
pressure, and chemical solvents, PHA have gained a lot of attention in
recent years as naturally occurring biomaterials that might be used to
replace traditional petroleum-based plastics (Ruiz et al., 2019; Alsafadi
et al., 2020). PHA has been marketed as eco-friendly completely
biodegradable bioplastic with less greenhouse gas emission and rapidly
replaces the usage of petroleum-based synthetic plastic (Arumugam
et al., 2018; Pernicova et al., 2019). PHB and PHV and their copolymers
e.g., P(3-HB-co-HV) are the most widely spread PHA in the environment.
The PHA did not need specific environmental conditions as compared to
bio-degradable plastic. In addition, PHA can be quickly degraded in both

Abbreviations: PHA, polyhydroxyalkanoates; PHB, poly-β-hydroxybutyric acid; PHV, polyhydroxyvalerate; WWTPs, wastewater treatment plants; 13C NMR,
13
carbon nuclear magnetic resonance; 1H NMR, proton nuclear magnetic resonance; FTIR, fourier transform infrared spectroscopy; GC–MS, gas chromatography-
mass spectrometry; DSC, differential scanning calorimetry; STP, sewage treatment plant; NIST, National Institute of Standard and Technology; AIRF, Advanced
Instrumentation Research Facility; SAIF, Sophisticated Analytical Instrument Facility; TGA, thermogravimetric analysis; Tm, melting temperature; Tg, glass transition
temperature; Td, decomposition temperature; COD, chemical oxygen demand; BOD, biochemical oxygen demand; TSS, total suspended solids; VSS, volatile sus­
pended solids; VFAs, volatile fatty acids; P(3HB-co-HV), poly 3-hydroxybutyrate-co-hydroxyvalerate; CHCl3, chloroform; HPI, hydraulic pressure instrument; CDCl3,
deuterated chloroform; HB, 3-hydroxybutyrate; HV, 3-hydroxyvalerate.
* Corresponding author at: Amity School of Earth & Environment Science, Amity University Haryana, Manesar, Gurugram, Haryana 122413, India.
E-mail address: isthakur@hotmail.com (I.S. Thakur).

https://doi.org/10.1016/j.biteb.2021.100783
Received 5 June 2021; Received in revised form 17 July 2021; Accepted 19 July 2021
Available online 24 July 2021
2589-014X/© 2021 Elsevier Ltd. All rights reserved.
V. Kumar et al.
anaerobic and aerobic conditions.
PHA is a non-toxic promising polyesters of hydroxyl fatty acids that
are synthesized mainly by numerous bacteria or archaea species under
the unbalance nutritional condition as inclusion bodies and serve as
energy and/or carbon intracellular storage reserve inside the vegetative
cells (Mitra et al., 2020). Although PHA production at an industrial scale
grew rapidly, their share in the global market is still marginal. The main
obstacle of PHA is its high manufacturing cost compared to their
petrochemical analogs and largely associated with feed-stock prepara­
tion, downstream processing, polymer extraction, and recovery rate that
prevents the full global market of PHA (Abdo and Ali, 2019).
To overcome this issue, cheap and renewable substrates such as solid
waste, and/or wastewater are being widely used as cheaper feedstock
for microorganisms to produce PHA. Several researchers reported that
various types of waste products such as food waste (Venkateswar Reddy
and Venkata Mohan, 2012), molasses from sugarcane industries (Albu­
querque et al., 2007), tomato cannery effluent (Liu et al., 2008), kraft
mill effluent (Pozo et al., 2011), olive mill effluent (Ntaikou et al., 2014),
pulp-paper mill activated sludge (Bengtsson et al., 2008), palm oil
effluent (Setiadi et al., 2015), and cassava starch effluent (Chaleomrum
et al., 2014) have been used as a promising alternative for the produc­
tion of PHA in a laboratory and/or pilot-scale experiments.
Sewage sludge is a common and hazardous waste by-product pro­
duced in enormous quantities by WWTPs around the world, and it is a
serious environmental concern (Demirbas et al., 2017). However, it
contains high levels of complex polymeric organic materials. It has been
reported that the treatment and disposal of plenty of waste spend about
60% of the overall operational expenditure of treatment plants (Pinheiro
et al., 2018). In the next few years, the generation of sludge increased
due to the implementation of new WWTPs that is a matter of global
concern. Thus, there is requirement to search for cost-effective and
sustainable solutions to utilize wastewater sludge and also decrease the
burden of waste disposal into the environment. The possible strategy for
the management of waste sludge towards its reuse that contains mainly
mixed microbial cells as the renewable carbon sources for PHA synthesis
and/or recovery (Venkateswar Reddy and Venkata Mohan, 2012). In
recent years, there has been growing attention in production of PHA by
using the excess municipal sludge from WWTPs as carbon source (Yuan
et al., 2015; Valentino et al., 2019). Although a few studies revealed the
feasibility of PHA production recovery from municipal solid waste and
sewage sludge (Morgan-Sagastume et al., 2014; Valentino et al., 2019).
Till now, few research works have been reported on the possibility of
utilizing municipal secondary sludge for maximum recovery of PHA and
optimization of process parameters. Kumar et al. (2018) reported a high
recovery of PHA (0.605 g) extracted from municipal sludge under
optimized conditions. Proper optimization of bioprocess parameters
such as pH and substrate concentration is vital for increasing PHA
productivity. Thus, the aim of present study to investigate the feasibility
of PHA synthesis using secondary municipal wastewater sludge to sub­
stitute as a cheap feedstock for PHA's production. Further, present
research work focus on process parameters optimization and assessing
the impact of each process variable on improving PHA recovery from
secondary sludge. Furthermore, various spectroscopic techniques were
used to determine the purity and stability of the polymer at high tem­
peratures, followed by TGA and DSC analyses.
2. Materials and methods
2.1. Chemical and materials
All the HPLC-grade organic solvents of hexane (purity 99.9%),
chloroform (purity 99.9%), and normal (n) hexane (purity 99.9%) used
to extract and purify PHA was collected from Sigma–Aldrich Chemical
Co. Ltd. (St. Louis, USA). All the chemicals were used in this study of
HPLC grade reagents supplied by HiMedia Laboratories, Mumbai, India.
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Bioresource Technology Reports 15 (2021) 100783
2.2. Collection of samples
Secondary sewage sludge samples were sampled aseptically in clean
plastic jerrycan (capacity 10 L; Tarson Production Pvt. Ltd., USA) from
the outlet of secondary sedimentation tank of the STP-300 KLD, situated
near Shipra Hostel, a Girls' hostel located on premises of Jawaharlal
Nehru University, India, used for extraction of PHA. The collected
watery sewage sludge samples were immediately transported to the
laboratory under ice-cold conditions for further analyses.
2.3. Waste sludge pre-processing step
The sludge biomass was recovered from collected watery secondary
sewage sludge according to the methodology described earlier (Kumar
et al., 2016; Liao et al., 2018). Briefly, the raw sludge samples were
concentrated overnight at room temperature by gravity settling to settle
sludge solids. Thereafter, the upper unconcentrated supernatant was
removed with the help of a glass pipette and the resulting wet settled
sludge-solids were then centrifuged (5000 ×g, 4 ◦ C, and 25 min) for
further dewatering as well as ruptured the microbial cells present in
sludge- solids for maximum extraction of PHA. After discarding the su­
pernatant, the dewatered wet sludge solids were spread in an iron tray
and oven-dried at 105 ◦ C to eliminate water content. Subsequently, to
reduce the particle size and make a fine powder of oven-dried sludge-
solids, the sludge-solids were compressed and homogenized by mortar
and pestle, Thereafter, the dried biomass of homogenized sludge-solids
was preserved at 4 ◦ C until the analyses were conducted. pH, TSS,
VSS, BOD, COD, VFAs, protein, and carbohydrate of collected watery
secondary sewage sludge were measured as per the protocol described in
APHA-AWWA-WPCF (2017). The pH value was measured using a bench-
scale pH meter (Orion Star™ A212, ThermoFisher Scientific, FL, USA).
BOD test method (5210B) was carried out to monitor the dissolved ox­
ygen concentration in wastewater. A UV–Vis spectrophotometer based
colorimetric closed reflux COD method (No. 5220D) was used to mea­
sure the absorbance of COD of samples at 600 nm. The TSS and VSS were
determined following method No. 2540D and 2540G, respectively.
Table 1 summarizes the average physicochemical characteristics of the
dewatered sludge solid.
2.4. Extraction of PHA from sludge
To extract PHA, a fixed amount (2 g) of sludge-solid was taken in
tight neck screw-cap flasks (500 mL) and subjected to extraction in
presence of 200 mL chloroform at 70 ◦ C for 72 h and 180 rpm on a rotary
shaker water bath. Subsequently, the reaction mixture was filtered using
a Whatman GF/C glass microfiber filter (1.2 μm; Millipore Ltd., Bedford,
MA, United States) to separate the organic phase enriched with PHA.
Afterward, the collected filtrate was condensed on a rotary evaporator at
60 ◦ C until 1/4 of the total volume. To determine the composition of
monomers in the recovered sample, PHA was purified by the following
Table 1
A general characteristics of secondary sewage sludge and discharge standards in
India.
Parameter Mean value Waste discharge permissible limit
CPCB (2016)
pH 6.5 ± 0.1 5.5-9.0
TSS g/L 5.8 ± 0.3 100.00
VSS g/L 3.7 ± 0.5 –
BOD mg/L 240 ± 0.8 30.00
COD mg/L 575.1 ± 15.2 250.00
VFAs mg/L 29.1 ± 2.2 –
Protein mg/L 85.2 ± 2.6 –
Carbohydrate mg/L 19.5 ± 0.9 –
TSS: total suspended solids; BOD: biochemical oxygen demand; COD: chemical
oxygen demand; VSS: volatile suspended solids; VFAs: volatile fatty acids.
V. Kumar et al.
method described earlier (Berger et al., 1989). Furthermore, the filtrate
was precipitated by adding n-hexane. The tube was vortex mixed until
the pellets completely disintegrate. Thereafter, the upper n-hexane layer
was aseptically removed from the biopolymer, and the remaining pre­
cipitate was subjected to drying at 60 ◦ C to measure the weight of the
recovered polymer.
2.5. Optimization of process parameters for PHA recovery
To enhance the yield and recovery of PHA, the numerous process
parameters such as temperature (60–80 ◦ C), incubation time duration
(24–120 h), and sludge solid to chloroform ratio (2:50–2:250 w/v) were
optimized. The preliminary parameters of optimization experiments
opted from the earlier study conducted by Kumar et al. (2018).
2.6. Calculations of different parameters
Recovery of PHA was determined using the following equation.
PHA Yield × PHA Purity
Recovery of PHA (%) = × 100 (1)
PHA Amount in sludge
The yield of PHA polymer recovered after extraction was calculated
as per the following equation (Neves and Muller, 2012).
MPHA
PHA Yield (%) = × 100 (2)
MSludge
where, MPHA and MSludge are the mass of recovered PHA and sludge solid
used in experiments, respectively.
The purity of PHA in the extracted polymer was calculated using the
below equation.
MPHA
PHA Purity (%) = × 100 (3)
MMEP
where, MPHA is the mass of recovered PHA, and MMEP is the Mass of
extracted polymer.
The PHA content in sludge was calculated as per the following
equation.
MPHA
PHA Content (%) = × 100 (4)
MSludge
where, MPHA is the mass of recovered PHA and MSludge is the mass of
sludge.
2.7. Characterization of polymer
2.7.1. Infrared spectroscopy
Infrared spectroscopy was performed for the functional groups in
PHA sample (Arumugam et al., 2018; Nwinyi and Owolabi, 2019).
Approximately 2.0 mg of oven-dried PHA was dissolved in CHCl3 and
allowed to evaporate. Subsequently, PHA was gently mixed with
spectral-grade pure anhydrous potassium bromide (KBr) crystals
(Merck, Darmstadt, Germany) in a ratio of 5:100 (w/w), and then the
resulting mixture was compressed by applying pressure (8–10 tons
cm− 2) using an HPI so that a thin disc of KBr was obtained. The IR
spectrum of PHA was collected for ambient equilibrated samples using a
spectrophotometer (Thermo Nicolet Nexus-870ESP; SpectralLab, Can­
ada) at 25 ◦ C in 4000 to 400 cm− 1 scanning range at 0.15 cm− 1 reso­
lution to investigate the elemental nature of recovered biopolymer
(Akdogan and Çelik, 2018).
2.7.2. NMR techniques
1
H NMR and 13C NMR spectroscopy was employed to identify the
individual monomer units in the recovered and purified biopolymer.
Briefly, about 20 mg of PHA polymer was mixed in CDCl3 (concentra­
tion; 10 mg mL− 1) and filtered using a glass syringe filter (0.22 μm).
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Bioresource Technology Reports 15 (2021) 100783
Afterward, the filtrates were analyzed on an NMR spectrometer (700
MHz DD2, Agilent Technologies, US). 1H spectrum was recorded at
25 ◦ C with a pulse duration of 450, pulse repetition of 1.36 s, 16 scans,
and 300 MHz frequency, whereas 13C (75.46 MHz) spectrum was
recorded at 45 pulse duration, 0.28 s pulse repetition, and 300 scans.
The signals of resonance (δ) were recorded in ppm.
2.7.3. GC–MS analysis
To carry out the characterization of PHA by GC–MS, lyophilized and
dried sludge-solids were processed following to known methodology as
described previously (Kumar et al., 2018). Briefly, the sludge-solids (10
mg) were taken into glass vials (10 mL) and subjected to methanolysis in
presence of chloroform (2.0 mL), 98% sulfuric acid (0.3 mL), and
methanol (1.7 mL) for 140 min at 100 ◦ C. The reaction mixture was
continually heated at a temperature-controlled water bath shaker at
65 ◦ C for 72 h. After cooling the mixture, distilled water (1.0 mL) was
added to the mixture consequently the reaction mixture was separated
into two organic phases. Thereafter, the lower organic phase of the PHA
was collected for GC–MS analysis.
GC–MS analyses were performed using a QP2010 Plus gas chro­
matograph (Shimadzu) coupled to a Quattro Micro triple quadrupole
mass spectrometer and equipped with a capillary column (25 m) con­
tained stationary phase of crosslinked (5%) phenylmethyl silicone. An
aliquot of methanolized sample (1.0 μL) was automatically inserted into
the GC column. The oven and column temperature programs were set
according to literature (Tan et al., 2014). The detected PHA was iden­
tified by combined Electron Ionization (EI) mass spectra matching of
compounds eluted from the GC with the EI mass spectra from the
compounds available in the inbuilt NIST mass spectra library. The mass
spectra were operated in EI mode (70 eV) and mass range was scanned
from 40 to 250 m/z.
2.7.4. Differential scanning calorimetry analysis
To determine the Tm and Tg values of purified PHA, the polymer was
subjected to DSC analysis by a DSC instrument (DSC 4000; PerkinElmer,
USA). In this analysis, about 8.0 mg PHA was heated up to 185 ◦ C to
achieve an absolute melting of polymer and kept isothermal for 0.1 min
at the first heating ramp where heating rate was 10 ◦ C min− 1. Subse­
quently, the sample was cooled (185 ◦ C to -70 ◦ C) at a rate of 10 ◦ C
min− 1 and isothermal was kept for 5 min (Akdogan and Çelik, 2018).
After this, the sample was reheated up to 185 ◦ C at 10 ◦ C min− 1 at the
second heating ramp, followed by fast cooling to -70 ◦ C at 10 ◦ C min− 1.
Further, the sample was heated at 10 ◦ C min− 1 up to 200 ◦ C in the final
ramp to determine the Tg and Tm, the heating ramps were analyzed.
2.7.5. Thermogravimetric analysis
To evaluate the Td of purified PHA polymer, TGA was carried out
with a TGA instrument (TGA 4000, Perkin-Elmer, USA). About 0.1 g of
purified PHA was heated from 25 ◦ C to 700 ◦ C with an isothermal halt of
10 min at 100 ◦ C and 10 ◦ C min− 1 a heating rate (Tappel et al., 2014).
The flow rate of nitrogen was 20 mL min− 1. Td was recorded by
observing the peak in the weight loss slope (dw dT− 1). The decreasing
value of the weight and the first derivative data of TGA were noted to
calculate the thermal degradation behavior of polymers.
2.8. Statistical analysis
Each experiment was conducted twice in triplicate to avoid any
experimental error and confirm the validity of the results. Further, the
obtained data are articulated as the computation mean values and
standard-deviation. All the data were analyzed using Microsoft Excel
Program (v. 2019; Microsoft (MS) Office).
V. Kumar et al. Bioresource Technology Reports 15 (2021) 100783

3. Results and discussion

3.1. Processing of sludge and PHA extraction

In the present study, the wastewater secondary sludge contained

about 93% solids as estimated after drying the centrifuged sludge in a
hot air oven at 105 ◦ C. Further, the chloroform was used for extraction of
PHA from sludge biomass at 70 ◦ C for 72 h. Results revealed that
chloroform was efficient for extraction of PHA with polymer recovery of
28 ± 2% that corresponded to extraction yields of PHA of 20 ± 1%.
Chloroform mainly modifies the permeability of the plasma membrane
of microbes and consequently dissolves the PHA present in the cell
(Jacquel et al., 2008). However, water residues present in microbial cells
or sludge inhibit the procedure of PHA extraction, thereby resulting in
low recovery and yield of PHA (Koller et al., 2013). Thus, the dehy­
dration step had been employed to increase the extraction efficiency and
yield of PHA polymer from microbial PHA-rich biomass. In our study,
the expected yield for biomass and PHA yield was found to be 10.35 g/L
and 6.98%, respectively.

3.2. Process parameter optimization for PHA recovery one factor at a

time

The initial experiments were conducted in batch reactors at 65 ◦ C for

a period of 72 h to analyze the effect of different process parameters on
the PHA yield. Before optimization of process parameters, the PHA re­
covery at preliminary extraction conditions was found to be 6.58% of
sludge solids concentration. However, the recovery of PHA was
increased up to 8.3% under optimized process parameters. The effect of
sludge solid to solvent ratio on the PHA yield and purity that recovered
secondary sludge is shown in Fig. 1a. The purity, recovery, and yield of
PHA were enhanced by increasing the solvent volume from 50 to 200
mL. Fig. 1b illustrated the effect of temperature on the PHA purity and
yield. It shows that the PHA yield and purity were increased with the
increase of the reaction temperature ranges from 24 to 72 h. However,
the PHA yield and purity reduced when the temperature was greater
than 75 ◦ C. PHA purity and yield was reached maximum levels 95% and
8.5%, respectively. The maximum content of PHA was found 6.98 g/L at
a temperature 75 ◦ C whereas higher temperatures did not support the
recovery of PHA. Fig. 1c shows the effect of the reaction time on PHA
recovery, purity, and yield from wastewater sludge. The highest PHA
purity, yield, and recovery occurred at 72 h. Although, the PHA purity
was increased by increasing temperature from 24 to 72 h, it was slightly
decreased when the temperature was greater than 72 h. The purity of the
Fig. 1. Optimization of various process parameter for maximum yield, recov­
polymer extracted with sludge were overall quite high, ranging from
ery, and purity of PHA extracted from secondary wastewater sludge of STP (a)
55% to a maximum of 95% within 72 h at 75 ◦ C under optimized effect of sludge solid to solvent ratio (chloroform) (b) effect of temperature (c)
conditions. effect of incubation time.

3.3. Spectroscopic study

stretching frequency of ester linkage present in PHB. These findings are
similar to the report of Arumugam et al. (2018). The abruption band
FT-IR spectroscopy confirms the various functional groups that exist
peaks at 1546 cm-1 attribute to the stretching vibration of the asym­
in the recovered biopolymer. FT-IR spectra of PHA showed numerous
metric –CH3 group. Moreover, stretching vibration near 1463.6 cm− 1 is
bands at different wave frequencies in the interval between 4000 and
assigned to the existence of the amide I (–CO–N–) carbonyl group in the
400 cm− 1. The assignment of the chemical group present in the
biopolymer. The signal of the C–H stretching vibration of − CH3 found in
extracted polymer was done following earlier reports (Arumugam et al.,
the biopolymer can also be traced at 1377.4 cm− 1. Besides, the vibra­
2018; Mohapatra et al., 2014). PHA primary contains CH, CH2, and
tional peak at 1172.3 cm− 1 indicates a –C-O-C- bridge present in the
C–– O as three functional groups. The stretching vibrational frequency at
polymer chain (Nair et al., 2014). The findings of the present study are
3438.0 cm− 1 corresponded to the methyl (–CH3) group found in the
following Kumar et al. (2017). The vibrant peaks in the range of
biopolymer. The prominent band peaks at 2921.0 cm− 1 attributed the
900–1100 cm− 1 assigned to the stretching band of C–O and the peak
asymmetric methylene (–CH2) group form by the interaction with the
band detected at 720 cm− 1 were assigned to vibrations of the C–H group.
union of C–H–O and carbonyl (C=O) group. These groups are involved
Although FT-IR analysis indicated the relative intensity and occurrence
in the side-chain formation of monomeric units (Arumugam et al., 2018;
of various functional groups, it does not provide information about the
Nwinyi and Owolabi, 2019). Moreover, the characteristics band peak at
individual compounds and the complexity of the mixture.
2855.3 cm− 1 resembles extending of the C–H bond's vibration of 1
H and 13C NMR spectroscopy of PHA polymers extracted from
symmetric − CH3 groups present in lateral monomeric units. In addition,
sludge was also performed to elucidate the structural monomeric units
the vibrational frequency at 1725.7 cm− 1 corresponded to C– –O

4
V. Kumar et al.
of polymer. The resonance absorption signals of the 1H NMR spectrum of
PHA polymers correspond to the methyl protons of the CH3 group pre­
sent in 3-hydroxyvalerate units (3-HV) and PHB at 0.80 and 1.20 ppm,
respectively. The peak obtained at 7.3 ppm corresponded to the occur­
rence of CDCl3. The obtained signals are analogous to the results re­
ported by various workers (Kumar et al., 2018). 1H NMR spectrum
analysis confirms that the recovered PHA were composed of 3-HB and 3-
HV units however, various unknown peaks were detected that might be
attributed to the occurrence of impurity in extracts (Arumugam et al.,
2018).
The resonance signals of 13C spectrums for PHA polymer were
assessed. Further, the carbon peaks were identified based on the pub­
lished literature. 13C spectrum of the PHA polymer showed three intense
signals at 76.81, 77.23, and 77.65 ppm, which corresponds to the methyl
carbon, methylene, and methine of 3-HB and 3-HV units (Arumugam
et al., 2018). Similar to the spectrum of 1H, various peaks were detected
in the 13C spectrum of the copolymer. These peaks might be due to the
existence of several impurities in the polymer. These observations
demonstrated that recovered PHA was a copolymer of PHB and PHV as
two monomeric units. These results were in agreement with that of
Martino et al. (2014) and Nair et al. (2014).
3.4. Mass spectrometric study
GC–MS analysis was performed for characterization and chemical
composition of PHA monomers from extracted polymer. The TIC chro­
matogram of GC–MS analysis of the extracted polymer shows three
peaks at RT 20.27 and 27.44 and 32.08 which corresponded to that of 3-
HB and 3-HV, respectively. The peaks' acquired area in TIC was 27.59%
and 12.56%, respectively. In addition, two different peaks at RT of 10.87
and 13.54 identified as 3-HB and 3-HV methyl esters in biopolymer,
respectively; while a major peak of 3-HB methyl ester at RT 37.22 cor­
responded to PHB-C. The major monomer constituent of the polymer
was 3-HB and 3-HV. The chief constituent of the recovered PHA was the
mixture of 3-HB and 3-HV monomers. The recovered 3-HV had a purity
and recovery rate of 89% and 83% respectively compared to the PHB
that recovery of 77% and purity of 92%.
3.5. DSC and TGA analysis
To assess the possible differences in thermal degradation behavior
and thermal stability, freeze-dried PHA was subjected to TGA and DSC
analyses. The Tm of the extracted polymer was mainly determined by
DSC analysis. The calorimetric scan of the extracted PHA polymers pu­
rified by n-hexane exhibited the multiple Tm of extracted PHAs to be
43.0 ◦ C and 65.0 ◦ C and a Tg of 21.0 ◦ C. The relatively low Tm and Tg
were attributed to the random incorporation of monomer units of 3-HV
into the polymer chain. The inclusion of the co-monomer HV fraction in
the copolymer can increase polymer flexibility, strength, and toughness
without affecting degradation temperature, allowing better processing
conditions and also reduce Tm value. In the present study, the double
melting peaks might be due to the melting or re-crystallization of a
single co-homopolymer, or again to the existence of two different co- or
homopolymers. The stability of PHA at high temperatures is a key
property to be studied during analyses as the polymer can withstand
before thermal decomposition at the highest temperature. Further, the
TGA was conducted to measure the Td of the biopolymer. The Td of
recovered biopolymer was found in a two-step process with tempera­
tures of decomposition ranging between 245 and 265 ◦ C and 450 ◦ C and
460 ◦ C. The two Td might be because of the integration of 3-HV
monomers in the PHA. The TGA analysis also shows nearly 85.26%
weight loss occurred between 450 ◦ C and 460 ◦ C. No heat change or
weight loss was detected in the temperature range of 0–245 ◦ C. In the
present study, a weight loss was observed between 450 ◦ C and 460 ◦ C.
This might be attributed to the ester cleavage of PHA as well as loss of
water molecules (Choi et al., 2003). This Td analysis confirms the
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Bioresource Technology Reports 15 (2021) 100783
polymer's stability of up to 245 ◦ C. These findings are in good corre­
spond with Nair et al. (2014).
4. Conclusion
The present research work reports recovery of PHA from secondary
sewage sludge which is rich in PHA diversity and readily available raw
material. The results indicated high-value of PHA was recovered under
optimum conditions. The PHA yield and purity was 8.3% and 96%
respectively. Further, the purified PHA was characterized and recovered
as copolymer of 3-HB and 3-HV monomer. The TGA demonstrated the
thermostability of the recovered biopolymer. The study suggested that
secondary sewage sludge is an ideal feedstock that could also reduce the
PHA cost at an industrial scale and simultaneously reduce the environ­
mental burden of excess sludge.
Funding
The experimental work was financially supported by the DBT, Gov­
erment of India as a Grant-in-Aid Major Research Project (BT/PR11762/
PBD/26/420/2014).
CRediT authorship contribution statement
Vineet Kumar: Conceptualization, Validation, Formal analysis,
Writing - original draft, Writing - review & editing, Data curation,
Software; Shaili Srivastava: Conceptualization, Visualization, Soft­
ware, Validation, Formal analysis; Indu Shekhar Thakur: Conceptu­
alization, Validation, Formal analysis, Resources; Writing - original
draft, Writing - review & editing; Visualization, Investigation; Supervi­
sion; Funding acquisition; Project administration.
Declaration of competing interest
None.
Acknowledgments
The authors are thankful to the Department of Biotechnology, Gov­
ernment of India for providing a senior research fellowship as financial
assistance to Vineet Kumar. Authors are also gratefully acknowledged to
the following institutions for timely analysis of the samples; GC–MS
analysis from AIRF at Jawaharlal Nehru University, New Delhi, India,
FT-IR analysis from University Sophisticated Instrumentation Centre at
BBAU, UP, India, and 1H as well as 13C NMR analysis from SAIF at CSIR-
Central Drug Research Institute (CDRI), UP, India.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.biteb.2021.100783.
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