1.

INTRODUCTION
Due to the advancement in the economy and living standards of our society, plastics
have turned out to be an indispensable material in people's daily life and production supplies
(1). Synthetic polymers are being widely used in many fields including agriculture, industry,
national defense, transportation, etc (2). They exhibit excellent properties like low density, high
strength, good wear, and corrosion resistance, hence they cannot be fully degraded (3). So they
must be recycled to make the environment clean (4). If not recycled properly, these plastics can
be broken down into smaller particles (< 5 mm) on exposure to sunlight (5). These
microplastics can cause many adverse effects on plants and animals and cause harm to the
natural environment (6). The "white pollution" caused by discarded plastics has become a
public hazard in the world, posing a potential hazard to the ecological environment (3, 5, 7).
The discarded plastic film remains in the soil to reduce soil permeability, hinder the absorption
of water and nutrients by the crop roots, and lead to crop yield reduction (8). Animals that have
eaten discarded plastic films are prone to intestinal obstruction (9). Synthetic fiber fishing nets
and fishing lines that are lost or discarded into the ocean have caused considerable harm to
marine life (10). Managing white pollution and seeking new environmentally friendly polymers
has become a global problem to be solved (11). Improper disposal of plastics can cause serious
environmental pollution (12). This problem can be solved by treating conventional plastic waste
and using biodegradable plastics (13). Landfilling, incineration, blending with new materials
after granulation, chemical degradation, recycling is among the few methods used for treating
polymer waste (14). Among them, some methods are very costly, while others can cause
secondary pollution problems. To overcome this problem biodegradable polymers can be the
best solution (15). Using plastics that can degrade naturally in the environment by the action of
different microorganisms are termed biodegradable (16). Therefore, scientists started to design a
plastic that possesses all the beneficial properties like consistency and resilience as well as more
vulnerability to microbial degradation in nature without harming the environment (17). Using
these plastics instead of conventional polymers can be an effective way of solving plastic
problems as compared to treating polymer waste. The waste of biodegradable plastics is utilized
by microbes as a carbon source (18). Considering this, biodegradable plastics have now been
widely researched, manufactured, and used in almost every country (19). Since the 1980s,
biodegradable polymers have been widely manufactured and used in every field of life. In 2020,
biodegradable and non-biodegradable polymers account for 941,000 metric tons and 1.17
1
million metric tons, respectively (20). European plastic statistics showed that global bioplastics
production capacity will probably increase from around 2.1 million tons in 2019 to 2.4 million
tons in 2024 (21). Many fields of life including agriculture, medicine, tissue engineering, and
industry widely used biodegradable polymers (22). Biodegradable polymers have the advantage
of conserving fossil resources as well as reducing environmental pollution (23).
Poly (butylene adipate-co-terephthalate (PBAT) is among the most important aliphatic-
aromatic copolymer with a wide range of importance (24). It contains terephthalic acid, adipic
acid, and, 1, 4-butanediol monomers in its structure. The terephthalate group in its structure is
responsible for its stability and mechanical properties (25). High elongation at break, good
hydrophilic and processing properties make it a desirable material for industry. Its flexibility
can be enhanced by blending it with other polymers (26). It can replace polyethylene which is
non-biodegradable but has similar properties to PBAT. PBAT is biodegradable due to the
presence of the butylene adipate group (27). Eco flex (trade name) is widely used for making
bags, mulching films, laminated sheets, plastics for wrapping food, toothbrushes, and many
more items like this (28). It is environmentally friendly. Naturally occurring enzymes consume
it within weeks (12). Biodegradation of polymers requires microorganisms to metabolize all
organic components of the polymer. Biodegradation in soil occurs in several steps like
microbial colonization on the surface of polymer and secretion of extracellular enzymes to
depolymerize the polymer into low–molecular weight compounds, the microbial uptake, and
utilization of these compounds, and then incorporation of this polymer carbon into biomass or
releasing it as CO2 (29). Microorganisms can degrade biodegradable polymers either aerobically
or anaerobically and convert them to bio decomposed products including CH4, H2O, and some
inorganic compounds (17). It may also involve hydrolysis, photodegradation, oxidation, etc.
Degrading bacteria secrete lipase which cleaves the ester bonds present in PBAT polymer and
converts them to smaller particles (11). Degradation products like terephthalic acid and adipic
acid can change the pH of the environment and can cause physiological toxicity in plants and
animals (30). Many PBAT-degrading microorganisms have been characterized in previous
studies for their biodegradation behavior (28). The pure aromatic polyesters are quite insensitive
to any hydrolytic degradation (31). It was also observed that direct microbial or enzymatic
attack of pure aromatic polyester is not significant but, other scientists claimed that aromatic
polyester could be disintegrated by microbial strains of Trichosporum and Arthrobacter in a
time scale of weeks (32). On the contrary, aliphatic polyester is considered to be more

2
susceptible to microbial attack (33). Aliphatic polyester degradation is seen as a two-step
process: the first is polymerization or surface erosion. The second is enzymatic hydrolysis,
which produces water-soluble intermediates that can be assimilated by microbial cells (34).
PBAT is used as mulch film in a natural environment, so understanding PBAT-degrading
mesophiles is important in the context of onsite degradation (35). Studies showed that two
PBAT hydrolases from the anaerobic mesophilic bacteria Pelosinus fermentans and
Clostridium botulinum are α/β hydrolases with a lid domain (30). In compost, a thermophilic
actinomycete, Thermobifida fusca K13g, and Thermomonospora fusca DSM43793 play an
important role in the degradation of PBAT (36). Characterization of PBAT hydrolase from T.
fusca showed that the enzyme was cutinase (37), which degrades the plant polyester cutin.
Likewise, Thermobifida alba, Saccharomonospora viridis, Thermobifida cellulosilytica, and
Humicola insolens are all cutinases (38). Another study showed that PBAT can be degraded and
mineralized in the natural environment by fungus Isaria fumosorosea strain NKCM1712 and
Paraphoma-related fungus cutinase-like enzyme (PCLE) and Cryptococcus flavus cutinase-like
enzyme (CfCLE) can degrade PBAT under mild conditions (39). It has been reported that
mesophilic bacteria also contribute to PBAT degradation under mild conditions. Few types of
PBAT-degrading microorganisms have been reported i.e Sphingopyxis ginsengisoli, Bacillus
pumilus, Pseudomonas pseudoalcaligenes, Cryptococcus, and Trichoderma asperellum (40). A
low degradation rate was among the main drawbacks. Therefore, the identification of new
lipase-producing and PBAT-degrading bacteria has become a top priority to provide a
theoretical basis and application prospect for the bioremediation of PBAT in the environment.
r, Liu et al. (9) study the degradation of PBAT films and reported that microbes could degrade
PBAT by secreting enzymes that hydrolyzed the ester bonds. In 2010, Sun Qi and others
isolated Alternaria (fungi) that degraded the aliphatic-aromatic polyesters and carried out
preliminary research on its available carbon source species, degradation temperature, and pH
(41). In another study, PBAT/PLA) blend (Ecovio®) was evaluated for degradation ability
under laboratory conditions (oxic and anoxic conditions) (42). Surface erosion was more
obvious under anaerobic degradation conditions. Hence it was proved that the addition of
biochar filler improved the properties of the blend without affecting its biodegradability (43).
Biodegradable blends can be an alternative solution to conventional plastic. This approach was
used to minimize environmental pollution. In this study (22), they reported the blends were
insoluble in water, but strong interaction was found between their components. In another

3
study, compatibilization of PLA/PBAT was studied. Results of the study showed that vegetable
oil derivatives can be used as a stabilizer for PLA/PBAT blends (44).

2. Enzymatic degradation of poly (butylene adipate

terephthalate) (pbat) copolymer using lipase b from Candida
Antarctica (CALB)
In this study, we investigated the enzymatic degradation of poly (butylene adipate
terephthalate) (PBAT) copolymer using lipase B from Candida Antarctica (CALB). Results
of the study displayed approximately 5.16 % loss in PBAT mass after 2 days which
significantly increased to approximately 15.7 % at the end of the experiment (12 days) as
compared to blank. The pH of the degradation solution also displayed significant reduction
and reached the minimum value of 6.85 at the end of the experiment. The structure and
morphology of PBAT after degradation were characterized by FTIR, XRD, SEM, and TGA.
FTIR analysis showed that after degradation many peaks become weaker and the peak at
2950 cm-1 almost disappeared after 12 days. The XRD results indicated that as the
degradation time increases the intensity of diffraction peaks slightly increases as compared
to the blank PBAT. TGA analysis also confirmed the successful degradation of PBAT with
time. SEM micrographs further confirmed that degradation has occurred. Hence,
biodegradable polymers can widely be used.
2.1 Materials and Methods
2.1.1 Materials
Polybutylene Adipate terephthalate (PBAT) was purchased from Anqing Hexing
Chemical. Ltd. Acetone (AR) was purchased from Sino Pharm Chemical Reagent Co., Ltd. P.
cepacia lipase (900 U/g) was purchased from SIGMA. Chloroform (CHCl3) HPLC was supplied
by Tianjin Oumi Chemical Reagent Co., Ltd. P230 GPC was procured from Dalian Yilite
Analytical Instruments. The OS-1002 constant temperature oscillator was obtained from
Shanghai Bilang Instruments.
2.1.2 Preparation of PBAT film.

4
 The PBAT films were prepared by melt pressing (fig. 2.1) at 120 ºC and then cut into the
rectangle-shaped pieces of about 20 mm length, 10 mm width, and 0.7 mm thickness.
2.1.3 Enzymatic degradation of PBAT polymer.
Degradation analysis of the samples was carried out in phosphate-buffered saline (pH
7.20±0.01, 0.1 mg mL-1) at 45 ºC. The polymeric films were prepared by melt pressing at 120
ºC and then cut into the rectangle-shaped pieces of about 20 mm length, 10 mm width, and 0.7
mm thickness. These films were immersed in a separate tube in triplicate containing 12 mL of
phosphate-buffered saline with CALB (6 mg mL-1) and incubated at a constant temperature
oscillator at 45 ºC. The samples were removed at regular intervals (0, 2, 4, 6, 8, 10, 12 days)
washed with deionized water to remove lipase, gently wiped with paper, and then dried under
the vacuum at 50 ºC to attain a constant weight for analysis. The degradation process was
followed by determining the mass loss of the materials, Molecular weight, and change in pH.
Changes in the surface structure of degraded samples were evaluated from SEM images, FTIR
and XRD. For this purpose, surface sections of degraded PBAT were gold-sputtered before
scanning. Finally, thermal parameters by TGA were also determined by using experimental
conditions described in the next section.

Fig 2.1. Represents steps for preparation of pure PBAT film for degradation experiment
2.1.4 Characterization
2.1.4.1 Mass Loss rate (%)
The determination of mass loss or gravimetric method gives a quantitative measurement
of biodegradation (45). Mass of PBAT films before and after degradation was recorded as.
Percent mass loss rate was calculated as;
Mass loss (%) = M1-M2/M1 x100

5
Where M1 is the mass of the sample before degradation, M 2 is the constant weight of the sample
after degradation at different time intervals.
2.1.4.2 Change in pH
Samples were removed at a different time interval (2, 4, 6, 8, 10, and 12) and the change
in pH was recorded by inserting electrodes of the pH meter [12].
2.1.4.3 Fourier transforms infrared (FT-IR) spectroscopy
Spectra were obtained in a VECTOR-22, in attenuated total reflection (ATR) mode. A
spectral width of 400–4000 cm-1, 16 accumulations, and 2 cm-1 resolution were used in the
analyses.
2.1.4.4 X-ray Diffraction Analyses (XRD).
XRD analyses were performed with Cu Kα (λ=1.54 A°) radiation in a D/Max-3c
diffractometer (46). Every scan was recorded in the range of 2θ=5-50°at a scan speed of 2°min -1
with an X-ray tube operated at 40 kV and 40 mA.
2.1.4.5 Thermal gravimetric analysis (TGA)
Thermal gravimetric analysis (TGA) was done with a Q500 system (TA, USA) to
evaluate the thermal stability of the copolymers (47). The analysis was carried out in a nitrogen
atmosphere. About 5 mg of the sample was heated from 25 ºC to 500 ℃ at a heating rate of 10
ºC min-1.
2.1.4.6 Scanning electron microscopy (SEM)
After spraying the sample surface with gold, the sample surface topography of PBAT
samples collected at different intervals were examined using a scanning electron microscope
(SEM, S-4800, Rigaku Co. Ltd, Japan) operated at 10kV with a spot size of 10 nm (48).

2.2 RESULTS AND DISCUSSION

2.2.1 Mass loss rate (%)
The progress of enzymatic degradation was monitored at regular intervals. The mass-
loss rate (%) of PBAT during different times is presented in fig. 2.2. Mass loss rate (%)
significantly increased with time and the maximum value of 15.7 % at the end of 12 days (fig.
2.2). It was found in enzymatic degradation that the % weight loss of enzyme-treated PBAT
was significantly higher as compared to blank PBAT. Because of the existence of ester bonds in
the polymer chain, biodegradable polyester-based materials undergo biodegradation.
2.2.2 Change in pH

6
 Change in pH was PBAT degradation during different time intervals is presented in
fig.2.3. It was found that as the degradation time proceeds pH of the solution decreased as
compared to blank. Minimum pH was recorded after 12 days in enzyme-treated PBAT i.e. 6.85
which was significantly lower than blank PBAT treatment.
18

16 15.7

M ass loss rate (% )

14

12

10

4
2 4 6 8 10 12
Time (d)

Fig.2.2 Change in the mass loss rate of PBAT during biodegradation at the different time interval

7.12
7.08
7.04
7.00
pH

6.96
6.92
6.88
6.84
2 4 6 8 10 12
Time (Days)

Fig.2.3 Change in pH of the solution during PBAT at the different time interval
2.2.3 FTIR Analysis of Composites
FTIR is a useful tool to detect the interaction of polymer matrix and filler. The width,
intensity, and peak position of the vibrational band are sensitive to molecular conception and
molecular environment changes. As shown in fig. 2.4. Before degradation PBAT FTIR
information showed the following: 2950 cm-1 represents the asymmetric stretching vibration of
methylene (-CH2-), 1710 cm-1 the stretching vibration of ester carbonyl (-C=O) groups belongs
to a carbonyl group on ester bond in PBAT, 717 cm -1 represents the external bending vibration
absorption peak of the (-C-H) group which is attributed to the (-C-H) group on the benzene ring
in PBAT likewise peaks at 1507 cm-1, corresponds to the stretching vibration absorption peak
corresponding to the group (-C=C-) on the benzene ring while peaks at 1268 cm-1, 1124 cm-1,
1099 cm-1, and 929 cm-1 corresponds to (-C-O-) groups, which belong to the (-C-O-) groups

7
connected to the benzene ring in PBAT. The peak at 802cm -1 is caused by the para-substitution
of the benzene ring. A peak at 1455 cm -1 is the in-plane bending vibration absorption peak of (-
CH2-CH2-) in PBAT. The 2950 cm-1, 1388 cm-1, and 1413 cm-1 were caused by the stretching
vibration and in-plane bending vibration of methylene (-CH2-) on the PBAT molecular chain,
respectively.
After degradation, the PBAT peaks become weaker and the peak at 2950 cm -1 almost
disappeared after 12 days, indicating that some have been degraded. After 12 days, positions of
the PBAT peaks was 2948 cm -1, 1709 cm-1, 1505 cm-1, 800 cm-1, and 716 cm-1, respectively.
Further, after PBAT degradation, ester carbonyl (-C=O) groups stretching vibration position
shifted from 1710 cm-1 to 1709 cm-1, which supported our results that degradation had occurred.
As the degradation time goes by, many peaks become weaker and the peak at 2950 cm -1 almost
disappeared after 12 days, indicating that some have been degraded.

Fig. 2.4. Infrared (FTIR) spectra of PBAT (Polybutylene Adipate Terephthalate) biodegradation at different
time intervals using lipase B from Candida Antarctica (CALB).
2.2.4. XRD X-rays diffraction
The XRD diffract grams of PBAT polymers are shown in fig.2.5. Blank PBAT has
characteristic peaks at 011, 010, 101 100, and 111 corresponding to diffraction planes of the
PBAT crystals, and five diffraction peaks (16.2, 17.9, 20.9, 23.2, and 25.1, respectively). The
intensities of PBAT diffraction peaks are weak due to their low crystallinity. As the degradation
time increases, the locations of diffraction peaks are almost the same, and it suggested that their
crystal structures are unchanged. The intensities of PBAT diffraction peaks increase slightly

8
compared with that of blank PBAT. This indicates that the amorphous phase of PBAT decreases
due to degradation by enzymes (fig 2.5).

Fig.2.5. XRD diffract grams of the PBAT biodegradation at different time intervals using lipase B from
Candida Antarctica (CALB).
2.2.5 Thermal Gravimetric Analysis of PBAT (TGA)
The thermal stability of the copolymer before and after degradation is shown in fig.2.6.
It showed the quickest decomposition temperature of PBAT was at 300 ºC. It was observed that
PBAT after 4 days has similar TGA traces with blank PBAT. With the increase of degradation
time, the initial decomposition temperature of PBAT decreased. The TG temperatures of the
PBAT sample slightly decreased after degradation. That is, the molecular weight of the PBAT
sample was basically without major change. This indicated that the thermal stability of PBAT
was affected after degradation. However, the decomposition temperature is still higher than 300
° C.
3.2.6 Morphology Analysis of Composites
Scanning electron microscopy (SEM) analysis was conducted to investigate the
degradation of PBAT during different times. SEM micrographs of the different degradation
times at 1000 x are shown in Fig.2.7 (a–d) while fig. 2.7 (a'–d') represents SEM micrographs of
PBAT degradation at 2000 x. From fig.2.7 (a, a'), is the micrographs of PBAT without
degradation. The surface of the PBAT is clean and no fractures are present. But as the
degradation time increased fractures start appearing and became more obvious at the end of the
12-day experiment (fig.2.7). After 4 days of degradation, the PBAT surface showed some
scratches but after 8-day degradation, small holes were observed which become more obvious
as the degradation time further increased. The PBAT sample surface had become very rough

9
after 12 days of degradation, and numerous cracks were observed (fig. 2.7 (d, d')) which
showed that PBAT has been successfully degraded by the action of an enzyme.

100
100

M ass loss (% )
80
80

M ass loss (% )
60

40

60 20

300 350 400 450 500

40 o
Temperature ( C)

Before degradation
20 4 days
8 days
12 days
0
100 200 300 400 500
Temperature (oC)

Fig. 2.6. TGA curves of the PBAT biodegradation at different time intervals using lipase B from Candida
Antarctica

Fig 2.7. SEM image of PBAT biodegradation at different time intervals using lipase B from Candida
Antarctica (CALB).
a': PBAT (before degradation): b': PBAT (4 days degradation) c': PBAT (8 days degradation) d': PBAT (12
days degradation) at 2000x while a: PBAT (before degradation) b: PBAT (4 days degradation) c: PBAT (8
days degradation) d: PBAT (12 days degradation) at 1000x.

2.3 Summary
Our work describes the enzymatic degradation of poly (butylene adipate co-
terephthalate) (PBAT) copolymer. The enzymatic degradation of PBAT in phosphate buffer
solution showed degradation at regular intervals. Mass loss (%) and change in pH displayed

10
significant changes throughout the experiment. The results of FTIR demonstrated that there was
possible degradation of PBAT with time. As the degradation time proceeds, many peaks
become weaker and the peak at 2950 cm-1 almost disappeared after 12 days, indicating that
PBAT peaks have been degraded by the enzyme. SEM observations showed clear symptoms of
PBAT degradation with time as compared to blank PBAT. XRD indicated that the intensities of
PBAT diffraction peaks slightly increased with time as compared to blank PBAT. It showed that
the amorphous phase of PBAT decreases due to degradation by enzymes. Thermal stability up
to 300 ºC was confirmed by TGA. As degradation time increased, the initial decomposition
temperature of PBAT decreased and the thermal stability of PBAT was affected after
degradation. However, the decomposition temperature is still higher than 300 ° C. PBAT is
copolymers composed of butylene adipate and terephthalate groups. It has both aliphatic and
aromatic groups. It is degradable due to the presence of an aliphatic group and exhibits good
mechanical properties due to the presence of an aromatic ring.

3 Screening and characterization of novel lipase producing Bacillus species

from agricultural soil with high hydrolytic activity against PBAT poly
(butylene adipate co terephthalate) co-polyesters
During this study, we isolated three novel PBAT-degrading bacteria (Bacillus strains)
from farm soil (Shaanxi (yuan Jia cun) by the serial dilution method. Microbial colonies were
spread and streaked many times to get pure colonies. The colonies grew well at 25–45 °C and
did not require high nutrition. The colonies were yellow, round, raised, opaque, neat, and sticky.
SUST B1 and SUST B3 gram-positive bacillus while SUST B2 was gram-negative bacillus. The
strains were sequenced using the NCBI online BLAST tool, and the results showed that the 16S
rRNA gene sequence of the strain SUST B1, SUST B2, and SUST B3 was identical to Bacillus
spp up to 98.3, 99.5, and 97%, respectively. The results showed that SUST B1 resembled
Bacillus thuringiensis and SUST B2 resembled Bacillus cereus while SUST B3 resembled
Bacillus paramycoides, the most. In this study, PBAT-degrading bacteria (SUST B 1, B2, and B3)
were isolated and identified from a cultivated field from Shaanxi. These results provide
technical support for the highly efficient degradation of PBAT in the environment.
11
3.1 Material and Methods
3.1.1. Soil collection and Sample preparation
The soil sample was collected randomly from different locations using a sterile soil
sampler from the cultivated farm in yuan Jia cun, Shaanxi, China. The upper soil was removed
and the lower part was collected in a sterile container. We thoroughly mixed the soil and sieved
using a 60-mesh sieve and vacuum dried. The sample was preserved at 4 °C until bacteria
isolation. The tape-casting method (17) was used to prepare a (200 mm x100 mm x 0.7mm)
PBAT film for the degradation test, and the PBAT (number-average molar mass of 1.0 × 105)
was purchased from Zhuhai Wantong Chemical Co., Ltd. (Guangdong, China) (3, 7).
3.1.2 Media used for bacterial isolation
Salt medium (SM) (pH 7-7.4) containing PBAT as the sole carbon source was used. The
ingredients (g/L) used for preparing SM media were as follow: K 2HPO4, 1.00; MnSO4.7H2O,
1.5; NH4Cl, 3.0; CaCl2. H2O; 0.5, KCl; 0.2, MgSO4.7H2O, 0.005; FeSO4.7H2O, 3.0;
ZnSO4.7H2O, 2.0; MnSO4.7H2O, 0.001, and agar, 20.00 [24] and LB medium (g/L): tryptone,
10.00; yeast extract, 5.00; NaCl, 10.00; pH 7.2–7.4. For preparing the screening medium (g/L)
SM medium containing PBAT as a sole carbon source was used. For this purpose 0.10g PBAT
was dissolved in 10 mL of chloroform. After attaining complete dissolution 100ml of SM
medium and 0.5ml of tween80 (as an emulsifier for phacoemulsification) were added (fig 3.1).
Media was sterilized at 121ºC for 15 min before further process.

Fig.3.1 Depicts mixing of PBAT in MSM

3.1.3. Isolation and identification of PBAT-degrading microorganisms

12
 The soil was configured as bacterial suspension and supernatant was inoculated in
screening medium using serial dilution-based plating method at 30°C and 130 r/min. When the
screening medium become turbid, the solution containing bacteria was added to SM agar plates
and incubated at 30°C. A single colony was picked from these plates and inoculated in a
screening medium following incubation at 30°C.
3.1.3.1 Inoculum preparation
Strains were cultured for 12 hr. in 100 mL of LB medium on a shaker at 37°C and 130 r/min.
3.1.3.2 Morphological observation
Selected strains were observed on the screening medium via scanning electronic
microscopy (SEM, FEI Q45, FEI) (49). A morphological gram staining test was done at 0.8-1
O.D (600nm). A clean grease-free slide was taken and a smear of the bacterial culture was made
on it with a sterile loop. The smear was air-dried and then heat fixed. It was then flooded with
crystal violet for 1min and washed with distilled water then flooded with gram’s iodine for 1
min and washed with alcohol. At last, the slide was counterstained with safranin for 30 seconds
and washed with distilled water. It was then observed under the microscope to determine the
morphology of the selected strain based on shape, size, and color (50).
3.1.4 Physiological and biochemical experiments
Strains were subjected to Gram’s staining, Methyl Red, Voges–Proskauer (V-P
reaction), starch hydrolysis tests, catalase reaction, and lipid hydrolysis (51).
3.1.4.1 Gram’s staining
The Gram stain, the most widely used staining procedure in bacteriology, is a complex
and differential staining procedure. Through a series of staining and decolorization steps,
organisms in the domain Bacteria are differentiated according to cell wall composition. Gram-
positive bacteria have cell walls that contain thick layers of peptidoglycan (90% of the cell
wall). These stain purple. Gram-negative bacteria have walls with thin layers of peptidoglycan
(10% of a wall), and high lipid content. These stain pink. Bacterial cultures were grown. Three
clean greased slides were taken, and bacterial cultures were heat fixed. Slides were flooded with
primary stain (crystal violet) to a heat-fixed smear, followed by the addition of a mordant
(Gram’s Iodine), rapid decolorization with alcohol, and lastly, counterstained with safranin (52).
3.1.4.2 Methyl Red
Methyl Red (MR) is a simple broth that contains peptone, buffers, and dextrose or
glucose. Different bacteria convert dextrose and glucose to pyruvate using different metabolic

13
pathways. Some of these pathways produce unstable acidic products which quickly convert to
neutral compounds. Some organisms use the butylene glycol pathway, which produces neutral
end products, including acetoin and 2,3-butanediol. Other organisms use the mixed acid
pathway, which produces acidic end products such as lactic, acetic, and formic acid. These
acidic end products are stable and will remain acidic. The Methyl Red test involves adding the
pH indicator methyl red to an inoculated tube of broth. If the organism uses the mixed acid
fermentation pathway and produces stable acidic end-products, the acids will overcome the
buffers in the medium and produce an acidic environment in the medium. When methyl red is
added, if acidic end products are present, the methyl red will stay red (53).
To prepare MR-VP broth (pH 6.9) buffered peptone (7.0 g), glucose (5 g), and
dipotassium phosphate (5 g) were added in 1000ml of deionized water. For preparing methyl
red solution (0.02%), 0.1 g of methyl red was dissolved in 300 ml of ethyl alcohol, (95%) in a
500 ml volumetric flask. It was then put in a brown bottle and stored at 4-8 C. It can be stable
for one year. Three sterile test tubes were taken and the nutrient broth was added, each was then
inoculated with respective strain and labeled. These tubes were incubated for 48hrs at 37 °C.
After 48hrs, 1ml of broth was taken in a clean test tube, then 2 to 3 drops of methyl red
indicator were added then a color change was observed immediately (28).
3.1.4.3 Voges–Proskauer (V-P reaction)
The VP test detects organisms that utilize the butylene glycol pathway and produce
acetoin. When the VP reagents are added to MR-VP broth that has been inoculated with an
organism that uses the butylene glycol pathway, the acetoin end product is oxidized in the
presence of potassium hydroxide (KOH) to diacetyl. Creatine is also present in the reagent as a
catalyst. Diacetyl then reacts to produce a red color. Therefore, red is a positive result. If, after
the reagents have been added, a copper color is present, the result is negative. Three sterile test
tubes were taken and the nutrient broth was added, each was then inoculated with respective
strain and labeled. These tubes were incubated for 24hrs at 37 °C. After 24hrs, 2ml of broth was
taken in a clean test tube, then 6 drops of 5% alpha-alpha-naphthol were added, and mixed.
After that, 2 drops of 40% potassium hydroxide were added, and mixed. Then color change at
the surface within 30 min was observed. The tube was robustly shaken during this time (30 min)
(54).
3.1.4.4 Starch hydrolysis tests

14
 This test is used to identify bacteria that can hydrolyze starch (amylose and
amylopectin) using the enzymes a-amylase and oligo-1,6-glucosidase. Often used to
differentiate species from the genera Clostridium and Bacillus. Because of the large size of
amylose and amylopectin molecules, these organisms can not pass through the bacterial cell
wall. To use these starches as a carbon source, bacteria must secrete a-amylase and oligo-1,6-
glucosidase into the extracellular space. These enzymes break the starch molecules into smaller
glucose subunits which can then enter directly into the glycolytic pathway. To interpret the
results of the starch hydrolysis test, iodine must be added to the agar. The iodine reacts with the
starch to form a dark brown color (55).
3.1.4.5 Catalase test
This test demonstrates the presence of catalase, an enzyme that catalyzes the release of
oxygen from hydrogen peroxide (H2O2). It is used to differentiate those bacteria that produce an
enzyme catalase, from non-catalase-producing bacteria. Normally 3% H 2O2 is used for the
routine culture while 15% H2O2 is used for detection of catalase in anaerobes. Hydrogen
peroxide solution (2 ml) was added to a test tube. Several colonies (18-24 hours) were picked
using sterile wooden sticks and immersed in a test tube containing hydrogen peroxide solution.
Bubbles were observed (56).
Positive: Many bubbles produced, active bubbling
Negative: No or very few bubbles were produced.
3.1.4.6 Lipid hydrolysis
Lipids generally are nonpolar molecules that do not dissolve well in water.  Fats are one
type of lipids that are large polymers of fatty acids and glycerol that are too large to enter the
cell membrane.  To utilize fats, bacterial cells secrete exoenzymes known as lipases outside of
the cell that hydrolyzes the lipid to fatty acids and glycerol. These bacteria capable of producing
exoenzyme lipase are called lipolytic bacteria. The lipids form an emulsion when dispensed in
agar, producing opacity while their hydrolyzed end products, glycerol, and fatty acids do not
form such emulsion with agar, for which they do not produce such opacity; rather produce
transparency. In the lipid hydrolysis test, the test bacteria were grown on agar plates containing
tributyrin as the lipid substrate. Tributyrin oil formed an opaque suspension in the agar
medium. If the bacteria could hydrolyze lipids, the bacterial colonies hydrolyzed the tributyrin
in the medium in the areas surrounding them while the rest of the areas of the plates contained
the hydrolyzed tributyrin. The result of hydrolysis was demonstrated as transparent clear zones

15
around the colonies and the rest of the areas of the plates remained opaque indicative of
hydrolyzed tributyrin region [176]. Tributyrin agar was prepared using peptic digest of animal
tissue (5.0 g/L),  yeast extract (3.0 g/L),  agar (15.0 g/L),  final pH (at 25°C) 7.5±0.2. The
tributyrin agar medium with single line streaking technique was inoculated and incubated at
37o C for 24-48 hours (59).
 Positive test: clear zone around the bacterial growth.
 Negative test: absence of clear zone around the bacterial growth.
3.1.5 Identification
3.1.5.1 Sample preparation and analysis of 16S rRNA gene sequence
A loop full of bacterial culture was transferred to 100 µl of filter-sterilized distilled
water, vortex, and boiled for 5 minutes (fig. 3.2). The sample was centrifuged at 10,000 rpm for
2-5 minutes. The supernatant was discarded and the pellet was stored. 100uL of distilling water
was added to the pellet and boiled for 10-20 minutes (60). For the 16S rRNA gene, universal
primers 27F and 1492R were used, and for PCR amplification, a total DNA template was used.
Primer sequences used for bacteria were 6S-27F (5'AGAGTTTGATCCTGGCTCAG-3') and
16S-1492R (5'TACGGTTACCTTGTTACGACTT-3'). The reaction system consisted of 20.0
μL of 2 × Taq PCR Master Mix, 2.0 μL of 27F/1492R (10.0 μmol/L) primers, 3.0 μL of total
DNA, and 23.0 μL of ddH2O. Samples were then placed in a thermocycler for PCR
amplification reaction under the following conditions, pre-denaturing at 95℃ for 3 min,
denaturation at 95℃ for 30 s, annealing at 55℃ for 30 s, extension at 72℃ for 30 s, 27 cycles
and finally extension at 72℃ for 10 min (PCR Amplifier: ABI Gene Amp® 9700 type) (61).
After the PCR reaction, gel electrophoresis was performed. Gel Tank (Bio-Rad Dcode TM
Universal Mutation Detection System), Power supply (Bio-Rad Power PAC 300). 30% (w/v)
Acrylamide: 0.8% (w/v) Bis-Acrylamide (Protoflow Gel, Flowgen Bioscience) was used for gel
electrophoresis (fig 3.3). After that, 10 uL of PCR products were run on the GE. Denaturing gel
was made by adding 0.5% of agarose, 50-55ml of 1xTAE in the gel solution. For staining, 3-5
ul of gel red was added to it until it gets slightly pinkish. The gel was cast immediately after
mixing the solutions. The comb was carefully placed into the gel chamber avoiding any air trap.
After gel solidification, the gel comb was removed carefully to make wells, washed several
times with deionized water (fig. 3.3 a).

16
 Fig. 4.2: a displays pre-PCR treatment steps, b represents sample preparation for PCR amplification
reaction, c and d represents initial and final images of a thermal cycler, respectively
3.1.5.2 Gel electrophoresis
The core was placed into the tank (containing 6 L of 1 x TAE buffer) (62). The
circulation was turned on to warm up the buffer up to 60 °C. As the buffer attained the required
temperature, the core was taken out of the tank. The gel plates were attached to the core and
placed with the gel into the tank. The samples were loaded into the wells (fig. 3.3 b). The
experimental conditions used were 100-120 V, 400mA, and 15 min for bacteria (fig. 3.3 c).
DNA was run on agarose gel with the Hyperladder1 (Bioline Ltd., London, United Kingdom) to
determine the size of the amplified DNA segment. The gel (fig. 3.2 d) was then visualized on a
gel documentation system (UVI Tec Gel Doc) (fig. 3.3 e).
3.1.5.3 Homology
The amplified products were sent to Biotechnology Engineering Co., Ltd (Shanghai,
China) for sequencing. The sequencing results were submitted to the GenBank database, and
BLAST was performed using the NCBI (http://www.ncbi.nlm.nih.gov) sequence database
(28). MEGA X was used for constructing the phylogenetic tree by the neighbor-joining
method (63).

17
 Fig. 3.3: a; represents a solidified agarose gel after removal of the comb, b; represents loading the DNA
sample into a well in the gel. c; experimental setup for electrophoresis. d; represents gel ready for
documentation while e represents a gel documentation system for analyzing DNA displacement bands
present in agarose gel.
3.1.6 Lipase activity of selected strains and factors that affect lipase production.
During this study, we accessed the effect of growth factors on the lipase activity of
PBAT-degrading bacteria. The single-factor method was selected to study PBAT degradation
and lipase production (64). The p-NP was used as the standard and the pre-inactivated crude
enzyme solution was used as a blank. The absorbance was measured using a spectrophotometer
at 410 nm. All the treatments were replicated thrice. The effect of the different culture
conditions was studied to test the lipase activity and degradation rate of the PBAT film. Under
certain conditions, the lipase activity of strain SUST B2 was 10.42 U/mL. The secreted lipase
catalyzes the degradation of ester bonds present in the PBAT structure. The effect of the pH
(6.2, 6.4, 6.8, 7.0, 7.2, 7.4, and 7.6), temperature (25°C, 32°C, 37°C, 42°C, and 47°C), the

18
concentration of inoculum (0.5 ml, 1.0 ml, 1.5 ml, 2.0 ml, 2.5 ml, and 3.0 ml) and PBAT
degradation products like terephthalate, adipic acid, glucose, and starch on lipase activity were
studied during the experiment. Strains utilize these products as carbon sources hence completely
degrading PBAT.
To determine the utilization of the PBAT degradation products, the lipase activity of
selected strains was measured by the para-nitro phenol (p-NP) method (28) (40). One unit of
lipase activity (U) is the amount of enzyme that releases 1 μg of p-NP per min. For this purpose
substrate solution (4 ml) (A: B:: 1:9), [A= p-NP (30mg) dissolved in isopropanol (10ml)]; B=
[Tris–HCl buffer with a volume fraction of 0.1% gum Arabic and a volume fraction of 0.4%
Triton X-100 at pH 8.0], and crude enzyme (200 μL) were added into an Erlenmeyer flask and
shaken at 40°C and 150 r/min for 10 min. The p-NP was used as the standard and the pre-
inactivated crude enzyme solution was used as a blank. The absorbance was measured using a
spectrophotometer at 410 nm. All the treatments were replicated thrice.
For measuring bacterial growth (O.D at 600nm), isolated were picked up from SM Petri
plates and transferred to nutrient broth. It was then incubated at 30 ºC, 120 rpm for 24 hours.
Then, 1 ml of culture broth was collected from each culture variant to record O.D values.
3.1.7 Study to check the effect of different conditions on lipase activity
The effect of the different culture conditions was studied to test the lipase activity and
degradation rate of the PBAT film. Following culture conditions were examined (change in pH,
change in temperature, change in inoculum concentration, and degradation products)).
3.1.7.1 Effect of different degradation products on degradation rate, and lipase activity of
SUSTB1, B2, and B3
The effect of PBAT degradation products like terephthalate, adipic acid, glucose, and
starch on lipase activity as well as degradation of PBAT was studied during the experiment. To
study the utilization of degradation products by SUST B 1, SUST B2, and SUST B3, SM media
(100ml), PBAT film, starch, glucose terephthalic acid, 1,4- butanediol, and adipic acid (2 wt.%
each) and inoculum (1.5ml) of isolated strains were added separately in 250 mL Erlenmeyer
flask and shaken at 37°C.
3.1.7.2 Effect of different temperatures on degradation rate, and lipase activity of
SUSTB1, B2, and B3
To check the effect of temperature at pH 7.4, the cultures (1ml inoculum) shook at different
temperatures (25°C, 32°C, 37°C, 42°C, and 47°C) at 130 r/min for 5 days.

19
3.1.7.3 Effect of different pH on degradation rate, and lipase activity of SUSTB1, B2, and B3
The effect of pH on lipase activity was tested for each strain. For this purpose, SM
medium (100 ml), 1,4-butanediol (2 wt.%), and a PBAT film were added to each Erlenmeyer
flask (250 ml) and the pH was adjusted to 6.2, 6.4, 6.8, 7.0, 7.2, 7.4, and 7.6, respectively. After
that, selected strains were inoculated (1ml inoculum) in the respective flask separately and
shaken at 30°C at 130 r/min for 5 days. In summary, when 2 wt. % 1, 4-butanediol was added
to 100 mL of SM medium and the pH, temperature, and inoculum amount were adjusted to 7.5,
37°C, and 1.5 mL, respectively
3.1.7.4 Effect of different inoculum concentration on degradation rate, and lipase activity
of SUST B1, B2, and B3
We changed the concentration of inoculum (0.5 ml, 1.0 ml, 1.5 ml, 2.0 ml, 2.5 ml, and
3.0 ml) by keeping the temperature (37°C) and pH constant (7.4). Then, these Erlenmeyer flasks
were placed on a shaker at 130 r/min for 5 days.
3.2 RESULTS AND DISCUSSION
3.2.1 Isolation and identification of PBAT degrading microorganisms
We have isolated three novel PBAT-degrading bacteria from cultivated soil (Shaanxi,
yuan jia cun) by the serial dilution method. The isolates were designated as strains SUST B 1,
SUST B2, and SUST B3. Fig. 3.4 shows phylogenetic trees of these isolates and related species
based on the 16S rDNA sequence. Strains SUST B 1, SUST B2, and SUST B3 are closely related
to bacillus species. Microbial colonies were spread and streaked many times to get pure
colonies. SUST B1 and SUST B3 gram-positive bacillus while SUST B2 was gram-negative
bacillus.
The strains were sequenced using the NCBI online BLAST tool, and the results showed that the
16S rRNA gene sequence of the strain SUST B 1, SUST B2, and SUST B3 was identical to
Bacillus spp up to 98.3, 99.5, and 97%, respectively. It was found that strain SUST B1 was
closer to Bacillus thuringiensis, B. cereus, Bacillus sp. enrichment culture clone, B. tropics, B.
anthracis strain bacterium, and Bacillus sp. while SUST B2 resembled, Bacillus cereus,
Bacillus sp. GZT, Bacterium CWISO11, Bacillus thuringiensis, Bacillus paramycoides,
Bacillus tropicus, Bacillus paranthracis, Bacillus paramycoides, Bacillus sp. (in Bacteria) while
B3 resembles Bacillus paramycoides, Bacillus sp. (in Bacteria) strain, Bacillus sp. CMJ2-8 16S,
Bacillus subtilis strain, Bacillus cereus strain, Bacillus thuringiensis, Bacillus sp. GZT, Bacillus
anthracis, Bacillus sp. bai7, Bacillus sp. hb38, Bacillus anthracis, respectively. These results

20
showed that SUST B1 resembles Bacillus thuringiensis and SUST B2 resembles Bacillus cereus
while SUST B3 resembles Bacillus paramycoides the most. (Fig. 3.4 (a, b, c)). The evolutionary
history was inferred by using the maximum likelihood method and the Poisson correction model
(65). The bootstrap consensus tree inferred from 1000 replicates was taken to represent the
evolutionary history of the taxa analyzed (66). Branches corresponding to partitions reproduced
in less than 50% bootstrap replicates were collapsed. The percentage of replicate trees in which
the associated taxa clustered together in the bootstrap test (1000 replicates) were shown next to
the branches. Initial tree(s) for the heuristic search were obtained automatically by applying
neighbor-Join and bioNJ algorithms to a matrix of pairwise distances estimated using a JTT
model and then selecting the topology with a superior log-likelihood value. This analysis
involved 26 amino acid sequences.

(a)

21
 (b)

(c)
Fig. 3.4: a, b, and c represent the phylogenetic tree of isolate SUST B1, B2, and B3, respectively.
All positions containing gaps and missing data were eliminated (complete deletion
option). There were a total of 181 positions in the final dataset. Evolutionary analyses were
conducted in MEGA X (65). Phylogenetic relationships could be inferred through the alignment
and the approximate phylogenetic position of the strains as shown in fig. 3.4 (a, b, c).
3.2.2 Physicochemical tests

22
 Based on grams staining and physicochemical tests (Table. 3.1), all strains secrete
lipase, which can degrade aliphatic-aromatic polyesters. Results showed that SUST B1 and B3
were positive while both SUST B 2 were negative for the gram staining test, All the three strains
SUST B1, SUST B2, and SUST B3 were negative for the MR test. Results showed that SUST B 1
was positive while both SUST B2 and B3 were negative for the V-P test. Catalase and lipid
hydrolysis tests were positive for all three strains while the starch hydrolysis test was positive
for SUST B1 and negative for both SUST B2 and B3.
Table 3.1. Physiological and biochemical experiments
Tests SUST B1 SUST B2 SUST B3
Gram staining + - +
Methyl red - - -
V-P reaction + - -

Starch hydrolysis test + - -

Catalase reaction + + +
Lipid hydrolysis test + + +
Where “+”= positive, and “-”= negative

3.2.3 Gel electrophoresis

For Gel electrophoresis, PCR samples were used to observe DNA bands of microbes.
Several DNA bands were observed along the increasing gradient of denaturing agent that
separates DNA fragments according to their melting point and GC content. The gel picture was
observed under UV light (fig. 3.5 (a)). Compared with universal ladder bp was found between
900-1200. The strains were preserved in NA slants at 4 °C and in liquid glycerol at -20 °C.
Nucleotide sequences were subjected to BLAST study at the national center for biotechnology
information NCBI database web.www.blast.ncbi.com which depicted the high similarity
percentage of B1, B2, and B3 with already isolated microbes from the Genus: Bacillus; Domain:
Bacteria; Family: Bacillaceae; Class: Bacilli. Bacterial phylogenetic trees of SUST B1, B2, and
B3 were made by using DNAMAN and MEGA X software, respectively (fig. 3.4 (b, c, d)).

23
 (a)
Fig 3.5. (a) Image of a gel post electrophoresis. 2, 4, 9 and10 bands represent SUST B1, B2, and B3,
respectively. Compared with universal ladder bp was found between 900-1200.
3.2.4 Lipase assay of selected strains and PBAT degradation ability of selected strains
Lipases are extensively been used in different applications such as detergents, cosmetics,
food flavorings, diesel, papers, and pulps. Thus, measurement of lipase activity is an important
process for the quantification of lipases (67).
3.2.4.1 Effect of degradation products on degradation rate, and lipase activity of SUSTB 1,
B2, and B3
Effect of degradation products showed a significant increase in degradation rate, lipase
activity, and bacterial growth as compared to control treatment (without carbon source) (fig. 3.6
(a, b, c)). The PBAT degradation rate of SUST B 1, B2, and B3 was 1.01, 1.5, and 0.96 % within
5 days, and each strain produces 0.430 U/mL lipases in the control treatment. However, the
addition of degradation products like terephthalate, adipic acid, butanediol, and glucose
enhanced the degradation rate and lipase activity of respective strains. Maximum degradation
rate and lipase activity were recorded in adipic acid and butanediol treatment. This is due to the
efficiency of selected Bacillus strains in utilizing PBAT products as a carbon source (growth
substrate) which ultimately enhanced its biomass and lipase activity.

24
 (a) (b) (c)
Fig. 3.6 (a, b, c) represents the effect of degradation products (CK= control, terephthalate, adipic acid,
butanediol, and glucose) on degradation rate, and lipase activity of SUSTB 1, B2, and B3, respectively at 37 ºC
and pH 7.5, respectively.
3.2.4.2 Effect of temperature on degradation rate, and lipase activity of SUSTB 1, B2, and
B3
The temperature had a considerable influence on the PBAT degradation rate and lipase
activity of the strain SUST B 1, B2, and B3 fig. 3.7 (a, b, c). At 27 °C, the PBAT degradation rate
of SUST B1, B2, and B3 was 4.1, 4.6, and 4.2 % while the lipase activity was 3.60, 4.3, and 4.2
U/ml, respectively. When the temperature was 32 °C, the PBAT degradation rate of SUST B 1,
B2, and B3 was 4.5, 6.6, and 5.3 % and the lipase activity (U/ml) was 5.8, 5.8, and 4.8 U/ml,
respectively. Maximum degradation rate and lipase activity were recorded at 37°C. Degradation
rate (%) was 10.6, 11.5, and 8.5, and lipase activity (U/ml) was 9.40, 10.1, and 8.7, respectively
for PBAT degrading strains SUST B1, B2, and B3, respectively. At 42°C and 45°C, the lipase
activity of selected strains was found to be low as compared to 37°C. Low metabolic growth
and lipase production ultimately resulted in a reduced degradation rate. Temperature enhanced
the growth of enzymes up to a certain limit. As enzymes are protein in nature, at higher
temperatures, denaturation of protein occurs which ultimately reduces the enzyme activity.
These results showed that 37°C is the best temperature for the activity of these selected strains.

25
 (a) (b) (c)
Fig. 3.7 (a, b, c) represents the effect of temperature i.e. 27, 32, 37, 42, 47 ºC on degradation rate and lipase
activity of isolated strains SUST B1, B2, and B3,
3.2.3.3 Effect of pH on degradation rate, and lipase activity of SUSTB1, B2, and B3
The effect of pH (6, 6.5, 7, 7.5, and 8) is shown in fig. 3.8 (a, b, c). At pH 6, the PBAT
degradation rate of SUST B1, B2, and B3 were 4.1, 4.3, and 4.1 % while the lipase activity was

4.3, 4.3, 3.9, and 3.5 U/ml, respectively (Fig. 3.8 (a, b, c)). SUST B2 showed maximum
degradation rate and lipase activities at pH 7.5 i.e. 10.5 and 9.4, respectively. Both degradation
rate and lipase activity rates were lower at low pH as compared to neutral and high pH. Gram-
positive and Gram-negative bacteria of the Bacillus genera showed lipolytic activity (68). Many
previous studies had confirmed the effect of the initial pH of the growth medium on lipase
activity and reported that optimum pH was very crucial for lipase production. Gupta et al. [23]
reported that though the production of lipase depends on the pH and temperature of the growth
medium, however, for their host they perform well in a wide range of temperature and pH.
Another group of scientists reported that lipase production was reduced when the pH was higher
than 7.5. Largely, bacteria prefer pH around 7.0 for best growth and lipase production, such as
in the case of Bacillus sp. but, many studies also showed that the maximum activity was found
at pH (>7.0) (30)

26
 (a) (b) (c)
Fig. 3.8 (a, b, c) represents the effect of different pH (6, 6.5, 7, 7.5, and 8) on lipase activity of isolated strains
SUST B1, B2, and B3, respectively
3.2.3.4 Effect of inoculum concentration on degradation rate, and lipase activity of
SUSTB1, B2, and B3
The effect of inoculum (1, 1.5, 2, and 2.5 ml) is presented in fig. 3.9 (a, b, c). inoculum
concentration of 1.5 ml was found to be more effective for all strains. In an experiment lipolytic
activity of 4.58 U /ml for Bacillus sp., 3.51 U/ml for Ralstonia paucula, and 1.80 to 2.62 U/ ml
for other unidentified bacteria (69). The variation in the production of lipase by the isolates
showed their ability to tune the growth and metabolic activities. Many microorganisms are
capable of producing lipase but Bacillus sp. is the most widely studied group. A group of
scientists had reported many bacterial genera like Acinetobacter sp., Yersinia sp., Arthrobacter
sp., Brevibacterium sp., Staphylococcus sp., Aeromonas sp., Acidomonas sp., Lactobacillus sp.,
Bacillus sp., Streptococcus sp., Bifidobacterium sp., Acetobacterium sp., and Citrobacter sp.
can produce lipase. Ankit et al. (21) also reported that Pseudomonas sp. and Bacillus sp. from
highly contaminated water samples can produce lipase. Both Gram-positive and Gram-negative
bacteria of the Bacillus genera showed lipolytic activity (68).

(a) (b) (c)

Fig. 3.9 (a, b, c) represents the effect of different inoculum concentrations (0.5, 1, 1.5, 2, and 2.5) on lipase
activity of isolated strains SUST B1, B2, and B3, respectively.
3.3 Summary
We have reported for the first time three Bacillus sp. that could secrete lipase and
cleaved the ester bonds of PBAT. We worked on the isolation and characterization of poly
(butylene adipate co-terephthalate) (PBAT) copolymer degrading bacteria from Shaanxi (yuan
jia cun) agriculture soil, the effect of degradation products, temperature, pH, and inoculum
concentration on degradation rate, and lipase activity . The results showed that each strain had a
significant degrading effect on PBAT. Under certain conditions, the lipase activity of strain
27
SUST B2 was 10.42 U/mL and degraded 10.5 % of PBAT films. Results of the study displayed
a significant change in PBAT properties throughout the experiment. In summary, when 2 wt. %
1, 4-butanediol was added to 100 mL of SM medium and the pH, temperature, and inoculum
amount were adjusted to 7.5, 37°C, and 1.5 mL, respectively, This is related to optimum
conditions required for the growth of microorganisms.
4 The study of PBAT degradation using isolated strains SUST B1, B2, and B3,
respectively, and its mechanism.
During this study, the most active strains (SUST B 1, SUST B2, and SUST B3) were
selected for degradation study. The degradation mechanism was investigated using attenuated
total reflection Fourier transform infrared spectroscopy, scanning electron microscopy, X-ray
diffraction, and thermogravimetric analysis. The results showed that each strain had a
significant degrading effect on PBAT. Under certain conditions, the lipase activity of strain
SUST B2 was 10.42 U/mL and degraded 10.5 % of PBAT films. Results of the study displayed
a significant change in PBAT properties throughout the experiment. The pH of the degradation
solution also displayed significant reduction throughout the experiment and reached a minimum
value at the end of the experiment. The secreted lipase enzyme catalyzed the degradation of
ester bonds present in the PBAT structure. Terephthalic acid, 1, 4-butanediol, and adipic acid
were the by-products of this reaction. Strains utilize these products as carbon sources hence
completely degrading PBAT. The proposed PBAT degradation mechanism was also studied.
The bioremediation of PBAT in the environment can be achieved using these strains.
4.1 Material and Methods
4.1.1 Preparation of PBAT film for degradation study
PBAT film used for degradation experiment was prepared in the laboratory of the school
of chemistry and chemical engineering. Raw PBAT granules and chloroform were mixed in a
three-necked flask and shaken at 600 rpm for 24 hours until completely dissolved followed by
subsequent purification using industrial alcohol. Then weighed amount of purified PBAT and
chloroform was added in a 3-necked flask and shaken for 4-6 hours to let it dissolve completely.
After that, it was poured on smooth Petri plates to make the films of desired thickness. A screw
gauge was then used to measure the thickness of all films (24).
4.1.2 PBAT-degradation using isolated strains microbes.
Degradation analysis of the samples was carried out in phosphate-buffered saline (pH
7.20±0.01, 0.1 mg mL-1) at 37 ºC. The polymeric films were prepared by melt pressing at 120
28
ºC and then cut into the rectangle-shaped pieces of about 20 mm length, 10 mm width, and 0.7
mm thickness. To study the degradation rate of SUST B1, SUST B2, and SUST B3, SM media
(100ml), inoculum (1.5ml) of respective strain were added in 250 mL Erlenmeyer flask
separately. Flasks were placed on a shaker at 37°C. The samples were removed at regular
intervals (0, 2, 4, 6, 8,12 days), washed with deionized water to remove the enzyme, wiped
gently with filter paper, and then dried under the vacuum at 50 ºC to attain a constant weight for
further analysis. The experiment was replicated thrice to minimize the error. The degradation
process was followed by determining the mass loss of the film, and the change in pH of the
degradation solution. Changes in the surface structure of degraded samples were evaluated from
SEM images, XRD and FTIR. Finally, thermal parameters by TGA were also determined by
using experimental conditions described in the next section.
The degradation rate was calculated by the weight loss method given below
Degradation rate (r) (%) = (w0 −w1)/w0 × 100
Where, r, w0, and w1 are the degradation rates of PBAT (%), the weight before
degradation (g), and the weight after degradation (g), respectively (70).
4.1.3. Characterization of PBAT Degradation.
PBAT degradation was characterized by recording different parameters.
4.1.3.1 Degradation rate of PBAT by mass loss method (%)
The degradation rate of PBAT was determined using mass loss or gravimetric
method. It gives a quantitative measurement of the biodegradation rate. Mass of
PBAT films before and after the degradation experiment was recorded. Values were
computed using the following formula;
wo−wt
Degradation rate (%)= x 100
wo
Where wO was the weight of the sample before degradation, wt was the constant
weight of the sample after degradation at different time intervals.
4.1.3.2 Change in pH
PBAT samples were removed at a different time interval (2, 4, 6, 8, 10, and 12) and a
change in pH was recorded by inserting electrodes of pH meter (Japan grade) (71).
4.1.3.3 X-ray diffractometer (XRD) analysis
The PBAT films were tested using XRD (D/max 2200 PC, Rigaku, Japan), analyses
were performed with Cu Kα (λ=1.54 A°) radiation in a D/Max-3c diffractometer. Every scan

29
was recorded in the range of 2θ=5-50° at a scan speed of 2° min -1 with an X-ray tube operated at
40 kV and 40 mA (72).
4.1.3.4 Fourier transforms infrared (FT-IR) spectroscopy
Fourier transforms infrared (FT-IR) spectroscopy analysis was also performed for PBAT
films before and after different time intervals. Spectra were obtained in a VECTOR-22, in
attenuated total reflection (ATR) mode. A spectral width of 400–4000 cm -1, 16 accumulations,
and 2 cm-1 resolution were used in these analyses (73).
4.1.3.5 Thermogravimetric Analysis (TGA)
Thermal measurements were carried out using a TA instruments Auto-MTGA Q500 Hi-
Res thermogravimetric analyzer (TGA). The analysis was carried out in a nitrogen atmosphere
(60 mL min-1). About 5 mg of the sample was heated from 25 ºC to 450 ℃ at a heating rate of
10 ºC min-1 (3).
4.1.3.6 Scanning Electron microscope (SEM)
The Surface topography of PBAT samples collected at different intervals was examined
using a scanning electron microscope (SEM, S-4800, Rigaku Co. Ltd, Japan) operated at 10kV
with a spot size of 10nm (74).
4.2 Results and Discussions
4.2.1 Characterization of degradation study using isolated microbes.
4.2.1.1. Mass loss (%)
The progress of enzymatic degradation was monitored at regular intervals. The mass-
loss rate (%) of PBAT during different times is presented in fig. 4.1 (a). Mass loss rate (%)
significantly increased with time and the maximum value of 8, 10.5, and 9 % was recorded
using isolated strains SUST B1, B2, and B3, respectively at the end of 12 days. It was found that
% weight loss in enzyme-treated PBAT was significantly higher as compared to blank PBAT.
Enzymes secreted by microorganisms can cleave polymeric molecules and reduce their
molecular weight. Due to the presence of ester bonds in the polymer chain, biodegradable
polyester-based materials undergo biodegradation. Several enzymes have already been reported
to effectively degrade copolymers in organic solvents, including Mucor miehei lipase, Rhizopus
delemar lipase N435, Candida Rugosa lipase, and Lipolase (75). PBAT can be degraded in the
environment by the intervention of microbial lipases. The biodegradation behavior of PBAT
was studied previously by many scientists. Kijchavengkul et al [18] group found that the PBAT
biodegradation was mainly caused by microbial degradation and reported that the

30
biodegradation of the non-crystalline portion was faster than that of the crystalline portion.
Likewise, Weng et al (69) also worked on biodegradation of polybutylene adipate-terephthalate
(PBAT). The results of the study showed visible signs of PBAT biodegradation.
4.2.1.2. Change in pH
Change in pH during PBAT degradation at different time intervals is presented in fig.
4.1 (b). It was found that as the degradation time proceeded the pH of the solution decreased as
compared to blank. However, no obvious change was observed in blank throughout the
experiment. Minimum pH was recorded after 12 days in SUST B 2 strain treated PBAT i.e. 6.91
was significantly lower than blank PBAT treatment. The change in pH might be due to the
production of acidic intermediates formed during the degradation process. PBAT degradation
involved several steps and the process could stop at each step (76). The process of degradation
generated oligomers, dimers, and monomers. After that, microbial receptors cells recognized
these molecules and ingested them to get energy and certain secondary metabolites. The other
molecules stayed in the extracellular surroundings and underwent different modifications. Some
simple and complex metabolites were excreted and reached the extracellular surroundings (e.g.
organic acids, aldehydes, terpenes, antibiotics, CO2, N2, CH4, H2O, and different salts, etc.).
This might be the reason for the change in pH that was observed throughout our study (41).

7.20 B1
12 B1 B2
7.15
B2 10.5
10 B3
B3 9 7.10
8.1
Mass loss (%)

8
7.05
pH

6
7.00
4
6.95 6.95
2 6.93
6.90 6.91
0
0 2 4 6 8 10 12 14
Time (d) 2 4 6 8 10 12 14
Time (d)

(a) (b)
Fig. 4.1: (a) represents the change in percent mass loss and b represents the change in pH of the solution
during PBAT biodegradation at different time intervals, respectively using isolated strains SUST B 1, B2, and
B3, respectively at different time intervals.
4.2.1.3. X-rays diffraction (XRD)
The XRD diffractograms of PBAT polymers are shown in Fig. 4.2 (a, b, c). Blank
PBAT has characteristic peaks at 011, 010, 101 100, and 111 correspondings to diffraction
31
planes of the PBAT crystals, and five diffraction peaks (16.2, 17.9, 20.9, 23.2, and 25.1,
respectively). The intensities of PBAT diffraction peaks were weak due to their low
crystallinity. As the degradation time increased, the locations of diffraction peaks were almost
the same, and it suggested that their crystal structures were unchanged. The intensities of PBAT
diffraction peaks increased slightly compared with that of blank PBAT. This indicated that the
amorphous phase of PBAT decreased due to degradation by enzymes (fig. 4.2 (a, b, and c)).

(a) (b) (c)

Fig. 4.2 (a,b,c) represents XRD diffractograms of the PBAT biodegradation at different time intervals using
isolated strains SUST B1, B2, and B3, respectively

5.2.1.4 FTIR analysis of PBAT polymer.

FTIR is a useful tool to detect the changes after degradation (fig. 4.3 (a, b, c). The width,
intensity, and peak position of the vibrational band are sensitive to molecular conception and
molecular environment changes. Before degradation PBAT FTIR information showed the
following: 2975 cm-1 represented the asymmetric stretching vibration of methylene (-CH 2-),
1750 cm-1, the stretching vibration of ester carbonyl (-C=O) groups belonged to a carbonyl
group on ester bond in PBAT, 717 cm -1 represented the external bending vibration absorption
peak of the (-C-H) group which was attributed to the (-C-H) group on the benzene ring in
PBAT, likewise peaks at 1510 cm-1, corresponding to the stretching vibration absorption peak
equivalent to the group (-C=C-) on the benzene ring while peaks at 1290 cm -1, 1124 cm-1, 1099
cm-1 and 929 cm-1 corresponded to (-C-O-) groups, which belonged to the (-C-O-) groups
connected to the benzene ring in PBAT. The peak at 802 cm -1 was caused by the para-
substitution of the benzene ring. The peak at 1510 cm-1 was an in-plane bending vibration
absorption peak of (-CH2-CH2-) in PBAT. The 2975 cm-1, 1750 cm-1, 1510 cm-1, and 1290 cm-1
were caused by the stretching vibration and in-plane bending vibration of methylene (-CH2-) on
the PBAT molecular chain, respectively.

32
 After degradation using SUST B1, the PBAT peaks became weaker and the peak at 2975
cm-1 almost disappeared after 12 days, indicating that some have been degraded. After 12 days,
positions of the PBAT peaks were 2910 cm -1, 1695 cm-1, 1490 cm-1, and 1275 cm-1, respectively.
Further, after PBAT degradation, ester carbonyl (-C=O) groups stretching vibration position
shifted from 1750 cm-1 to 1695 cm-1, which supported our results that degradation had occurred
(fig. 4.3 c). Likewise, results of degradation showed that after degradation using isolated strain
SUST B2 showed that the PBAT peaks became weaker and the peak at 2975 cm -1 almost
disappeared after 12 days, indicating that some have been degraded. After 12 days, positions of
the PBAT peaks were 2900 cm-1, 1710 cm-1, 1475cm-1, respectively. Further, after PBAT
degradation, ester carbonyl (-C=O) groups stretching vibration position shifted from 1750 cm -1
to 1710 cm-1 (fig 4.3 b). Using SUST B 3, the PBAT peaks became weaker and the peak at 2975
cm-1 almost disappeared after 12 days, indicating that some have been degraded. After 12 days,
the position of the PBAT peaks were 2910 cm -1 and 1690 cm-1, respectively. Further, after
PBAT degradation, ester carbonyl (-C=O) groups stretching vibration position shifted from
1750 cm-1 to 1690 cm-1 (fig 4.3 c), these all supported our results that SUST B 1, B2, and B3
successfully degraded PBAT polymer (fig. 4.3 (d, e, f). Weng et al [19] also reported similar
results of PBAT degradation.

(a)

(b)
33
 (c)
Fig. 4.3 (a, b, c) represents Infrared (FTIR) spectra of PBAT (Polybutylene adipate terephthalate)
biodegradation at different time intervals using isolated strains SUST B 1, B2, and B3, respectively
5.2.1.5. Thermal Gravimetric Analysis of PBAT (TGA)
The thermogravimetric (TG) curves of the PBAT degradation using isolated strains
SUST B1, B2, and B3, respectively are shown in Fig. 4.4 (a, b, c, d) and the 50% and 95%
thermogravimetric temperatures are given in Table 4.1. It showed the quickest decomposition
temperature of PBAT was at 300 ºC. The 50%, and 95% thermal gravimetric (TG) temperatures
of the PBAT sample before degradation were 409 ºC, and 372 ºC, respectively. After
degradation using isolated strain B1 the temperatures were recorded as 400 ºC, 368 ºC,
respectively. Meanwhile, the 50% and 95% TG temperatures of the isolated strain B2 after
degradation were 394ºC and 361ºC, respectively. Likewise, the 50% and 95% TG temperatures
of the PBAT sample degraded by isolated strain B 3 were 395 ºC, and 361 ºC, respectively. It
means that with the increase in degradation time, the initial decomposition temperature of
PBAT also decreased. This indicated that the thermal stability of PBAT was affected after
degradation. The TGA temperature of this PBAT sample slightly decreased after degradation,
but the changes were slight. That is, the molecular weight of the PBAT sample was basically
without major change.
As can be seen from the TG curve, all degraded PBAT samples shifts to lower
temperatures as compared to PBAT sample without degradation. Many previous studies showed
that PBAT curves shift to lower decomposition temperature after degradation which confirmed
our results that degradation has occurred (44, 47, 48).

34
 100 100
Blank Blank
80 B1 80 B1

Mass loss (%)

Mass loss (%)

B2
60 B3 60
100 100
95 Blank 95
40 B1
Blank

Mass loss (%)

40 90 90 B1

Mass loss (%)

85
85
80
80

20
75
75
20 70
300 320 340 360 380 400 420
Temperature (°C )
70
360 380 400 420
Temperature (°C )

0
50 100 150 200 250 300 350 400 450 500 0
100 200 300 400 500
Temperature (°C ) Temperature (°C )

(a) (b)

100 100
Blank Blank
80 B2 100
B3
80 90

Mass loss (%)

Mass loss (%)

Mass loss (%)

80

60 100 100
60 70 100
90 90 100
60

Mass loss (%)

Mass loss (%)

80
Mass loss (%)

80 50

Mass loss (%)

360 80380 400 420 440 460
40
80
70 70
40 Temperature (°C )

60 60
60 60
50 50
300 320 340 360 380 400 420 440 460 360 380 400 420
20 Temperature (°C ) Temperature (°C )
20
300 320 340 360 380 400 420 440 460
Temperature (°C )
360 380 400 420
Temperature (°C )

0 0
100 200 300 400 500 100 200 300 400 500
Temperature (°C ) Temperature (°C )
(b) (d)
Fig. 4.4 (a, b, c, d) represents the TGA of the PBAT biodegradation at different time intervals using isolated
strains SUST B1, B2, and B3, respectively.
Table 4.1: 50%, and 95% thermo gravimetric temperatures.
Samples 50% 95%
PBAT0 409 372
PBAT1 400 368
PBAT2 394 361
PBAT3 395 361
PBAT0: Before degradation; PBAT1: After degradation with SUST B1; PBAT2: After degradation with
SUST B2; PBAT3: After degradation with SUST B3
4.1.3.6 Morphology Analysis of Composites
Scanning electron microscopic (SEM) analysis was conducted to investigate the
degradation of PBAT during different times using isolated strains SUST B 1, B2, and B3,
respectively (fig. 4.5 (A-C)). Here we isolated strains from three different microbes. Each lipase
has its optimum concentration where it can produce degradation spots on PBAT with the
highest density on the film [31]. These hydrolysis spots formed on the film by microbial strains
35
were observed using SEM to verify the changes of film surface after incubation with enzyme
solutions. PBAT that acted as negative control did not produce any change on the film surface
as the film surface remained smooth. The film became rougher with increasing time and more
holes were formed on the film due to a higher rate of polymer degradation.
SEM micrographs of the different degradation times at 1000 x are shown in fig. 4.5 (a–
c). The fig. 4.5 (A (a-e)) represents the micrographs of PBAT using isolated strain SUST B 1.
The surface of the PBAT was clean and no fractures were present initially (fig. 4.5 A-C (a)).
But as the degradation time increased fractures appeared and became more obvious at the end of
the 12-day experiment (fig 4.5. A-C (e)). After 4 days of degradation, the PBAT surface showed
some scratches but after 8-day of degradation small holes were observed which become more
obvious as the degradation time further increased. The PBAT sample surface had become very
rough after 12 days of degradation, and numerous cracks were observed (fig. 4.5. A-C (e))
which showed that PBAT has been successfully degraded by the action of an enzyme.
In 2007, it was reported that obvious symptoms of PBAT biodegradation were observed
during the macroscopic and microscopic biodegradation behavior of films in composting
conditions. Many degrading microorganisms were isolated and screened from the compost [8].
Due to polymer size and water insolubility, microorganisms were not able to pick up the
polymers directly into the cells where most of the biochemical processes took place. Firstly,
they secreted the extracellular enzymes to depolymerize the polymers outside the cells. .
Extracellular enzymes were too large to penetrate deeper into the polymer material so they only
acted on the polymer surface (classical surface erosion process) and reduced the molar mass of
the polymer. When the molar mass of the polymers was sufficiently reduced to generate water-
soluble intermediates, these could be transported into the microorganisms. Due to this process,
microbial metabolic products such as water, carbon dioxide, methane (anaerobic degradation),
etc. are produced (77).

(A)
36
 (B)

(C)
Fig. 4.5 (A, B, C). SEM image of PBAT (Polybutylene Adipate Terephthalate) biodegradation at different
time intervals using isolated strains SUST B1, B2, and B3, respectively. a: PBAT (without degradation) b:
PBAT (blank) c:PBAT (4 days degradation) d: PBAT (8 days degradation) e: PBAT (12 days degradation)

4.2 Proposed PBAT degradation mechanism

4.2.1 Biodegradation mechanism and factors affecting.
Biodegradation is the mechanism by which polymers break down into gases, salts, and
biomass (78). Generally, there are two types of biodegradation. During biodegradation,
polymers change into monomers and dimers (76). Complete biodegradation is one in which no
monomers or oligomers are left at the end of the reaction (30). Many factors affect the rate of
degradation of the polymer (79). Physical and biological degradation are the main types of
biodegradation (21). Physical degradation involves hydrolysis or photodegradation which leads
to partial or complete degradation of the polymer (78). While biological degradation involves
microorganisms that degrade the polymer under aerobic or anaerobic conditions.
Microorganisms secrete enzymes either endoenzyme or exoenzyme, these enzymes get
attached to the surface of the polymer and break down the polymers into fragments (oligomers
and dimers). This is called mineralization (80). After this microbes assimilate these products to
37
make biomass. There are several processes involved in the biodegradation mechanism.
Oxidation (addition of oxygen, removal of hydrogen or loss of electrons) (81), Hydrolysis
(breakdown in the presence of water)(75), photo-oxidation (oxidation in the presence of light)
(82), thermal hydrolysis (hydrolysis in the presence of heat)(83), and enzymatic hydrolysis
(breakdown by enzymes) (fig. 4.6) (84). Vast research on polymer degradation showed that
besides the structure and morphology, environmental conditions had a great influence on the
degradation of polymers (85)(86). The chemical structure of the polymer showed a direct
relation with the ability of microbes to degrade a polymer (77). Biodegradation of functional
groups showed the following trend:
Aliphatic ester > Peptide bond > Carbamate > Aliphatic ether > Methylene.
Hydrophilic groups (amino, hydroxyl, carboxyl) are readily attacked by microbes and
more degraded as compared to hydrophobic groups (87). Similarly, branched and cross-linked
polymers were difficult to degrade as compared to linear polymers (87). The structural
configuration of polymer is also very important. Polymers with flexible chains degrade fast, as
compared to amorphous while amorphous areas degrade faster than crystalline. Likewise,
saturated compounds biodegrade more easily than unsaturated compounds, while aliphatic
polyesters biodegrade more readily (12). Previous studies showed that aliphatic polymers
degraded faster than aromatic polymers as aromatic polymers are bio-inert (88). Blending these
compounds with other polymers results in their degradation (89). Many scientists had added
special additives and plasticizers to enhance their biodegradation (44). This is becoming an
attractive topic in the polymer research field. The result of previous studies proved that the
polymerization degree of polymer also played an important role (90). Biodegradation also
depends on the molecular weight of the polymer (27). The higher the molecular weight lower
will be the rate of biodegradation and vice versa (73, 87). Natural polymers do not have high
molecular weight hence can be biodegraded easily as compared to synthetic polymers (which
have higher molecular weight). In most cases, polymers having a higher molecular weight
(exceeding 400–500 daltons) were first disintegrated by physical and chemical means into a
smaller fraction and then microbial degradation occurred (91). Environmental factors are very
essential for the biodegradation process. Previously reported that all the factors that affected the
enzyme activity also affected the rate of biodegradation (92). As water is an essential
component of life. Under certain humidity, microbes can efficiently degrade polymers (26).
Temperature is another important factor that can affect the rate of biodegradation (93). It was

38
reported that every enzyme could work on a narrow range of temperatures e.g. PLA requires
approximately 55 °C for effective biodegradation (94). Most enzymes performed best at
optimum temperature (47). An increase in temperature up to a certain limit increased the growth
and metabolic processes and hence the rate of enzyme activity and biodegradation (40). A
further increase in temperature can result in the denaturation of enzymes and reduce the
biodegradation process (95). The pH was also reported as an influential factor in biodegradation
(96). Like temperature, enzymes can work only on a specific range of pH, below and above
which enzyme denatured and reaction slows down or completely stops (97). Previous studies
showed that the microbes which degraded poly (lactic-co-glycolic acid) (PLGA) and
polyglycolic acid (PGA) work best at pH 5–9 (53).

Fig. 4.6. Mechanism of biodegradation of Polymer

4.4 Summary
The use of biodegradable films in agriculture can promote sustainability and reduce soil
contamination. During this project, we worked on the degradation of PBAT using SUST B1, B2,
and B3 under laboratory conditions. All the experiments were replicated thrice to minimize the
error. The degradation of PBAT in phosphate buffer solution has shown the degradation at
regular intervals. Mass loss (%) and change in pH had displayed a significant change
throughout the experiment. XRD indicated that the intensities of PBAT diffraction peaks
slightly increased with time as compared to blank PBAT. It showed that the amorphous phase of
PBAT decreased due to degradation by enzymes. The results of FTIR demonstrated that there
was possible degradation of PBAT with time. As the degradation time proceeded, many peaks
became weaker and almost disappeared after 12 days, indicating that PBAT peaks have been
39
degraded by the enzyme. The SEM observations had shown clear symptoms of PBAT
degradation with time as compared to blank PBAT. These isolated strains had successfully
degraded the PBAT polymer. The TG curves showed that degraded PBAT curves bend towards
the lower decomposition temperature. Microorganisms utilize polymer as their nutrient source,
by using this approach microorganisms can be isolated. PE and PBAT closely resemble each
other due to similar mechanical properties compared to other biodegradable polyesters. Due to
the good mechanical properties and biodegradability, aliphatic-aromatic polymers are being
widely commercialized. PBAT has excellent mechanical properties as well as good
biodegradability. Mechanical property is attributed to the presence of the benzene ring while
biodegradation is due to the presence of the aliphatic group. PBAT is copolymers composed of
butylene adipate and terephthalate groups. It has both aliphatic and aromatic groups. It is
degradable due to the presence of an aliphatic group and exhibits good mechanical properties
due to the presence of an aromatic ring. Microorganisms utilize polymer as their nutrient
source, by using this approach microorganisms can be isolated.

5 Effects of Polybutylene terephthalate-

adipate - (PBAT) degradation on soil microorganisms
Polybutylene terephthalate-adipate - (PBAT) as a thermally stable, biodegradable,
mechanically excellent material, has important use value in combating the problem of "white
pollution", whether the use of the process will have an impact on the soil environment, it is not
clear. In this project, PBAT is proposed to use Raman spectroscopy (46) to analyze the
PBAT degradation process, and then investigate the PBAT degradation process of different
particle sizes and addition amounts by high-throughput sequencing (54) method. The effect on
the structure of soil microbial communities, the analysis, and evaluation of the microplastic
effect of PBAT provide a way for the development of microplastic resources for effective
degradation of microplastics, the assessment of the potential impact of microplastics on soil
ecosystems, the understanding of their degradation process, and the maintenance of soil
environmental health. The results showed that the increasing particle size and the amount of
PBAT affected its degradation rate; PBAT micro plastics affected the structure of soil microbial
communities, which significantly inhibited the proteobacteria phylum and actinomycete phylum
in the soil at the gate level, and the abundance of the acid bacillus phylum was increased.
Nocardia spp. abundance has declined.
40
5.1 Materials and methods
5.1.1 Experimental samples
The microplastic sample required for the experiment PBAT was made up of plastic
purchased by Guangdong Kingfa Co., Ltd., which removed possible impurities from the surface
of the plastic before use, and then crushed it using a pulverizer, sifted to obtain the microplastic
of the required particle size, and sealed at 4 °C for preservation (98).
5.1.2 Experimental reagents
The reagents used in this experiment are shown in Table: 5.1.
Table 2.1: Experimental reagent
Reagent name specification Origin
Sodium bromide Analyze purely Tianjin Tianli Chemical Reagent Co., Ltd
Dichloromethane Analyze purely Tianjin Fuyu Fine Chemical Co., Ltd
Acetone Analyze purely Tianjin Tianli Chemical Reagent Co., Ltd
5.1.3 Experimental instruments and equipment
The instruments and equipment used in this experiment are shown in Table 5.2.
Table 5.2: Experimental instruments and equipment
Device name Model factory
Electronic analysis balances AR223CN OHAUS Instruments (Changzhou)
Co., Ltd
High-speed multi-function DLH-250 Wuyi QiTeng Electric Appliance
shredder Co., Ltd
Circulating water vacuum SHZ-Ⅲ Shanghai Yarong Biochemical
pump Instrument Factory
Magnetic stirrer CJJ78-1 Jintan Dadi Automation Instrument
Factory
Laser confocal micro-Raman-
LabRAM HR
transient - fluorescence HORIBA FRANCE S.A.S
Evolution
spectrometer
5.2 Experimental methods
5.2.1 Preparation of PBAT microplastics
The experiment used a multi-function pulverizer to process the plastic samples after
washing and drying. Firstly, the raw plastic materials were poured into the pulverizer shell and
41
then covered with a lid. After that, the switch was opened so that the motor drives the hinge and
start working. when the material was crushed to a certain extent, the switch was turned off to
scrutinize the pulverized plastic particles, and then the plastic particles with larger particle size
were poured back into the pulverizer for the second crushing, and finally sieved and collected.
The brush was used to clean up the inside of the pulverizer.
The following should be noted when using a shredder (21)
(1) The pulverizer and the power unit should be firmly installed: if it is necessary to fix the
pulverizer for a long time, it should be fixed on the cement foundation; if the pulverizer is
movable, the device should be installed on a base made of angle iron, and the power engine
(diesel engine or motor) and the pulley groove of the pulverizer should be in the same rotation
plane.
(2) After the installation of the shredder, the tightening of each clasp should be
checked. Tighten it if it is loose, and at the same time check the belt tightness;
(3) Before starting the shredder, first rotate the rotor by hand, check whether the tooth claw,
hammer blade, and rotor are flexible and reliable, whether the shell has collisions, whether the
rotor rotation direction is consistent with the direction indicated by the machine arrow, and
whether the lubrication of the power machine and the shredder is good.
(4) Do not replace the pulley, to avoid the explosion of the crushing chamber.
(5) After starting the crusher, it should be left for 2-3 minutes to monitor its working.
(6) Pay attention to the operation of the crusher at any time during the working process, evenly
feed, and prevent blockage and long-term overload. If there is vibration, noise, high
temperature, construction, and other phenomena between the bearing and the body, it should be
stopped immediately for inspection, and the work should continue after troubleshooting.
5.2.2 Extraction of PBAT microplastics from soil
Based on density separation, the density floatation method uses the density difference
between microplastics and impurities in the sample to achieve the separation of light component
microplastics and recombinant impurities, and finally flotation of microplastics to the upper
layer. Usually, a saturated salt solution is added to the sample to be extracted, stirred, and
shaken to make it mixed thoroughly, and then left to be delaminated, and the microplastics are
separated by filtration and other means. This experiment used the patent of this laboratory: a
separation and extraction device and method for microplastics in soil, and the flotation solution

42
was selected for saturated sodium bromide (NaBr) solution fig (5.2). The separation steps used
were as follows:
(1) Flotation: flotation solution was added to separation cell 1, the magnetic stirring device 17
was started, and the sample to be processed was added to separation cell 1, stirred evenly, and
then left until it reached separation cell 1. The mixed solution in the upper layer was divided
into an upper non-precipitated layer and a lower precipitated layer. The non-precipitated layer
contained the microplastic particles.
(2) Filtration: The flotation solution was added to reservoir 13, which transferred the flotation
solution into the non-precipitated layer in separation tank 1 through the inlet pipe 6. Air was
allowed to pass through it 3 times. The upper solution containing microplastics in the separation
pool 1 was driven by the bubbles generated at the aeration head 3 into the suction filter unit
through the infusion tube 12 for filtration, the device was kept running for 20-30 minutes.
(3) Cleaning: The instrument was turned off, the sediment was poured out at the bottom of the
separation cell 1. After that, distilled water was added to separation pool 1 and the extraction
device was started. The magnetic stirring device 17 was started at the same time as the
separation cell 1 /13. the filter residue on reservoir 13 and the extraction device were cleaned,
and the cleaned filter residue was dried to obtain microplastic particles (99).
5.2.3 Degradation performance analysis of PBAT microplastics
The degradation rate of PBAT was determined using weightlessness (89). The
weightlessness method refers to measuring the quality change of a material sample and studying
the decomposition properties of a substance. PBAT microplastics in soils with landfill times
of 20d, 40d, 60d were extracted, picked, weighed, and compared with theoretical values,
and PBAT was calculated separately.

43
 Fig. 5.2: Schematic diagram of the extraction device
Note:1 is the separation cell, 2 is the aeration pump,3 is the aeration head I, and 4 is the aeration
head II, 5 is the peristaltic pump I, 6 is the inlet tube,7 is the snorkel, 8 is the vacuum pump,
9 for suction bottles, 10 for funnels, 11 for the filter paper,12 for infusion tubes, 13 for
reservoirs, 14 is peristaltic pump II, 15 is the outlet pipe, 16 is the hose diameter reducer switch,
and 17 is the magnetic stirring device
5.2.4 Monitoring of PBAT decomposition process
In this study, Raman spectroscopy was used to analyze the degradation process
of PBAT microplastics. Raman spectrometer (46) measured a scattering spectrum. Based on the
Indian scientist C.V's Raman scattering effect discovered by Raman, the Raman spectroscopy
method came into being. It can analyze scattering spectra different from the frequency of
incident light, obtain information on molecular vibration and rotation, and can be used for the
study of molecular structure. The theoretical explanation of Raman spectroscopy is: inelastic
scattering occurs between incident photons and molecules, molecules absorb U-
frequency photons, emit U-U photons (absorb energy is greater than released energy), and
molecules change from low-energy states to high-energy states; when molecules transit from
high-energy states to low-energy states, molecules release U-frequency photons and
emit /U+U photons (release energy is greater than absorbed energy). The transition of a
molecular energy level involves only rotational energy levels, emitting a small Raman
spectrum; when it comes to - energy levels, it emits a large Raman spectrum. Unlike molecular
infrared spectra, both polar and non-polar molecules can produce Raman spectra. Raman
spectroscopy can identify the composition and types of microplastics, and can also achieve the
distribution characterization of polymers in microplastics. At the same time, Raman
44
spectroscopy is a non-destructive testing technique with relatively low instrument cost. The
focused laser spot can reach several microns, enabling the analysis of smaller size microplastics.
Advantages of Raman spectroscopy:
The sample does not need to be pretreated, no sample preparation is required, some
errors are avoided, the analysis process is simple to operate, the measurement time is short, and
the sensitivity is high.
Refer to HJ 834-2017 determination of soils and sediments, Semi-volatile organic
compounds by mass spectrometry, HJ 911-2017 Soils and Sediments, Extraction of organic
matter Ultrasonic extraction method", "HJ 783-2016 Soil and sediment Extraction of organic
matter Pressurized fluid extraction method", using dichloromethane - Acetone 1:1 mix solvent
as extractant. 20 g of the soil sample was taken, sieved through a 0.25 mm screen. Then 100
ml of extractant was added and left for 16-18 hours, After that, centrifuged (10 min, 3000 r/min)
to get the supernatant for assay.
5.2.5 Analysis of soil microbial community structure
In this study, high-throughput sequencing was used to analyze different sample
soils. High-throughput sequencing (100) was achieved by using DNA polymerase
or DNA ligases and primers to extend the template through a microscope to observe and record
light signals during continuous sequencing cycles. High-throughput sequencing has the
characteristics of large throughput, a high degree of automation, and requires a small sample
volume. High sensitivity and accuracy can be achieved through the repeated
determination of DNA fragments in the same region. It can detect point mutations, changes in
gene copy number, gene recombination, and other genetic changes.
Advantages of high-throughput sequencing technology:
(1) Chip sequencing, through ordered or disordered array configuration, can achieve large-scale
parallelism, can read sequencing at millions of points at the same time, the ultimate realization
of parallel processing greatly improved the sequencing throughput.
(2) Quantitative function, the number of times a certain nucleic acid in the sample is sequenced
reflects the abundance of the nucleic acid.
(3) Low cost, sequencing the human genome through high-throughput sequencing technology
costs less than 1% of the traditional sequencing method.
In summary, the specific steps of this study are as follows:

45
Crushing PBAT → sieved the PBAT microplastics, and divided into three groups according to
the particle size: <0.1 mm (D1), 0.1-0.2 mm (D2), 0.2-0.5 mm (D3) → sieved the soil (2 mm)→
plastic was added to the soil and divided into two groups: 0.02% (M 1) and 0.2% (M2) according
to the added amount→ soil without plastic was used as the blank and terephthalic acid (PTA)
was added as the control group (addition 0.2%)→ the degradation process of PBAT
microplastics was analyzed by Raman spectrometer→ soil microbial community structure was
analyzed by high-throughput sequencing method→Comprehensively evaluated and analyzed
the influence of PBAT microplastics degradation process on soil microbial community
structure.
5.3 Results and discussions
5.3.1 PBAT degradation performance
The PBAT microplastics extracted by the sodium bromide density flotation method were
selected and weighed at 20 d, 40 d, and 60 d, compared with the theoretical values, and the
degradation rate was calculated separately, and the results are presented in Table 5.3.
From table 5.3, it can be seen that the degradation rate of PBAT microplastics
with different particle sizes and different additions increases with the increase of landfill time,
and the particle size is the same as the landfill time, and the amount of PBAT is 0.2%. The
degradation rate of microplastics is slightly higher than that of 0.02% PBAT microplastics.
PBAT microplastics of different particle sizes have different degradation rates. It can be seen
that the degradation rate of PBAT microplastics is slightly higher at the size < 0.1 mm.
In summary, it is shown that the increase in the amount and particle size of PBAT
microplastics will affect their degradation rate. The larger the additional amount, the smaller the
particle size, the higher the degradation rate of PBAT microplastics. It is speculated that the
reason for this may be that the smaller the particle size of PBAT microplastics, the larger the
specific surface area, the stronger the adsorption capacity, and the greater the impact on the
microbial community in the soil, which in turn affects the degradation rate of PBAT
microplastics.
Table 5.3 Degradation rates of PBAT microplastics
Sample name 20 d 40 d 60 d
M1D1 27.3% 35.4% 40.5%
M1D2 26.4% 33.9% 41.4%
M1D3 29.5% 31.3% 39.5%

46
M2D1 38.5% 43.0% 48.8%
M2D2 28.1% 39.2% 43.8%
M2D3 33.5% 36.2% 41.2%
5.3.2 PBAT degradation process monitoring
The PBAT microplastics with three particle sizes after crushing were determined by
Raman spectroscopy, and the results are as shown in fig. 5.1

6000 1614cm
-1
D1
D2
5000 -1 D3
1732cm

-1
2938cm
Intensity (a.u.)

4000
-1
3085cm
3000

2000

1000

0
0 1000 2000 3000 4000
-1
Raman shift (cm )

Fig. 5.1: PBAT Solid Raman Spectrogram

The PBAT microplastic samples with different particle sizes were studied at 0 d, 20 d,
40 d, and 60 d, respectively. The soil leaching solution was determined using Raman
spectroscopy it was observed that the peaks of the three particle sizes of PBAT microplastics
were roughly the same, according to the literature review, 1186 cm -1, 1614 cm-1 (fig. 5.1). At
the C-C single bond of the benzene ring, the telescopic vibration, 632 cm-1, 702 cm-1, 796 cm-1,
1094 cm-1 is the contraction vibration of the C-C single key, and 1732 cm-1 is C= O double bond
telescopic vibration, CH2 telescopic vibration at 2938 cm-1, which coincides with the molecular
structure of PBAT.
7000
0d
6000 20d
40d
60d
5000
Intensity (a.u.)

4000

3000

2000

1000

0 1000 2000 3000 4000 5000 6000 7000

-1
Raman shift (cm )

47
Fig. 5.2 Raman spectrometry of 0.02% addition, particle size < 0.1 mm PBAT

5000 0d
20d
40d
4000 60d

In ten sity (a .u .)
3000

2000

1000

0 1000 2000 3000 4000 5000 6000 7000

-1
Raman shift (cm )

Fig. 5.3 Raman spectrogram of 0.02% addition and particle size 0.1-0.2 mm

14000 0d
20d
12000 40d
60d
10000
Intensity (a.u.)

8000

6000

4000

2000

0 1000 2000 3000 4000 5000 6000

-1
Raman shift (cm )

Fig. 5.4 Raman spectrogram of 0.02% addition and particle size 0.2-0.5 mm

7000
0d
20d
6000
40d
60d
5000
Intensity (a.u.)

4000

3000

2000

1000

0 1000 2000 3000 4000 5000 6000 7000

-1
Raman shift (cm )

Fig. 5.5 Raman spectrogram of 0.2% addition, particle size < 0.1 mm PBAT

7000
0d
6000 20d
40d
60d
5000
Intensity (a.u.)

4000

3000

2000

1000

0 1000 2000 3000 4000 5000 6000 7000

-1
Raman shift (cm )

48
 Fig. 5.6 Raman spectrogram of 0.2% addition and particle size 0.1-0.2 mm
7000
0d
6000 20d
40d
60d
5000

Intensity (a.u.)
4000

3000

2000

1000

0 1000 2000 3000 4000 5000 6000 7000

-1
Raman shift (cm )

Fig. 5.7 Raman spectrogram of 0.2% addition and particle size 0.2-0.5 mm

12000

0d
10000 20d
40d
60d
8000
Intensity (a.u.)

6000

4000

2000

0 1000 2000 3000 4000 5000 6000

-1
Raman shift (cm )

Fig. 5.8. PTA Raman spectrogram

The Raman spectrogram of the landfill time of 20 d added to the PBAT microplastic soil
leaching solution is significantly different from the landfill time of 40 d and 60 d (fig.5.2-5.8).
Increasing PBAT microplastic landfill time changed the telescopic vibrations of microplastic
chemical bonds, such as weakening of telescopic vibration of C-C single at 796 cm-1, and
CH2  at 2938 cm-1. Raman spectrogram of soil leaching solution with PBAT with PTA content
is presented in fig. 5.8. Comparing the Raman spectrograms, it was noted that there was a large
difference between the two, which showed that the expansion vibration of the chemical bonds
present in PBAT microplastics changed during the degradation process.
5.3.3 Soil microbial community changes
5.3.3.1 Taxonomic analysis of microbial OTU
The taxonomic results of the microbial OTU of each soil sample are shown in table 5.4.
The number of species in each horizontal community of PBAT microplastic soil samples with
landfill times of 20 d and 40 d increased relative to the blank soil samples. In contrast, the
number of community species varied in the soil samples. For PBAT microplastic soil samples
with a landfill time of 20 d and particle size < 0.1 mm and 0.1-0.2 mm,  the 0.2 % added
PBAT microplastic soil samples species in each horizontal community were higher
49
than the 0.02 %  with a particle size of 0.2-0.5 mm. 0.2% added PBAT microplastics soil
samples showed less than 0.02% added community species at each level. In 40
d PBAT microplastic soil samples, the number of species in each horizontal community was
higher in the sample with 0.2% PBAT as compared to 0.02% PBAT in microplastic soil
samples. In summary, it was noted that PBAT microplastics with different additions and
particle sizes have different effects on soil microbial communities.
Table 5.4: Microbial OTU Taxonomic Analysis Table
Sample world door class eye section genus seed OTU
name
KB-20 1 35 97 229 349 639 1269 2921
M1D1-20 1 36 104 237 368 655 1280 3009
M1D2-20 1 34 100 226 355 642 1304 3186
M1D3-20 1 36 111 247 383 675 1346 3309
M2D1-20 1 39 107 242 373 654 1272 3071
M2D2-20 1 34 101 234 365 651 1294 3077
M2D3-20 1 36 102 231 354 634 1271 3043
PTA-20 1 36 99 220 344 595 1153 2568
KB-40 1 36 100 235 365 639 1308 3189
M1D1-40 1 36 102 236 375 662 1327 3184
M1D2-40 1 37 111 255 379 666 1324 3291
M1D3-40 1 37 107 247 375 666 1333 3317
M2D1-40 1 36 103 244 378 663 1331 3327
M2D2-40 1 38 112 257 401 705 1387 3484
M2D3-40 1 38 111 252 391 687 1340 3235
PTA-40 1 36 88 209 334 605 1171 2549

5.3.3.2 Microbial Community Alpha Diversity Index

The microbial diversity indices of each soil sample are shown in table 5.5.
Alpha diversity analysis reflected the richness and diversity of microbial communities in soil
samples. The diversity indices for soil samples with landfill times of 20 and 40d are presented
in table 5.5. The Ace and Chao indices reflect community richness, and the higher
the Ace and Chao indices, the higher the community richness; the Shannon. The Simpson index
reflects community diversity, the higher the Shannon value, the higher the community diversity,
and the smaller the Simpson value indicates the higher community diversity. The index reflects
the community coverage, i.e. the coverage of each sample library, the greater the value, the
higher the probability of detecting the sequence in the sample, the lower the probability of
50
undetected sequences, and the index reflects whether the sequencing results represent the true
situation of the microorganisms in the sample or not. All of the PBAT microplastic soil
samples in Table 3-3 covered more than 97.7%, indicating that the sequencing results represent
the true condition of the bacteria in the samples. For soil samples with landfill times of 20
d and 40 d, add Ace and Ace of the PBAT microplastic soil sample. The Chao index was mostly
higher than that of the blank soil sample, while the soil sample with PTA was lower than
the white soil sample; the Shannon with the addition of PBAT microplastic soil sample.
The Simpson index is not much different from the blank soil sample, and the soil sample
with PTA is quite different from the blank soil sample, which shows that the addition
of PBAT microplastics can improve the richness and diversity of microbial communities in the
soil.
5.3.3.4 Microbial community composition analysis
A columnar plot of the community at the level of each soil sample phylum is shown in
fig. 5.9. Ten phyla were identified in the communities of 16 soil samples, namely
Proteobacteria, Actinobacteria, Acidobacteriota, Phylum Acidobacteriota, Phylum Aguscus
aurifolia Chloroflexi, Bacteroidota, Gemmatimonadota, Mycoccus phylum cota, the firmicutes,
Verrucomicrobia, and Planctomycetota (fig. 5.9). Among them, the first three phyla were
relatively high in abundance in 16 soil samples and were the main dominant bacterial phyla.
Proteobacteria accounted for a relatively large proportion of the soil samples, between 23.51%
and 43.14%, which was the dominant bacterial phylum in the PBAT microplastic soil sample,
blank soil samples, and additions while the proportion of the proteobacteria phylum in the soil
sample having PTA increased 29.97% to 43.14% with the extension of the landfill time
of 20 d to 40 d, respectively. This showed that the addition of PBAT microplastics reduced the
abundance of the phylum Proteus in the soil, while the addition of PTA increased the abundance
of the phylum Proteomics in the soil. Actinobacteria also accounted for a relatively large
proportion of the soil samples, between 23.80% and 31.52%, with a landfill time of 20 d of
added PBAT.
Table 5.5 Microbial diversity index table
Sample name Shannon Simpson Ace Chao coverage
KB-20 5.776658 0.006896 1386.770795 1421.007937 0.993962
M1D1-20 5.729737 0.008597 1439.911286 1485.426357 0.993489
M1D2-20 5.736159 0.008384 1396.075153 1390.317568 0.994041

51
 M1D3-20 5.773401 0.007737 1446.066506 1469.1875 0.993621
M2D1-20 5.718313 0.009237 1409.931582 1420.77305 0.993962
M2D2-20 5.837391 0.007177 1439.681641 1448.054054 0.993909
M2D3-20 5.847289 0.006218 1390.055863 1374.006173 0.994277
PTA-20 5.476704 0.012066 1323.350917 1333.285714 0.993988
KB-40 5.898282 0.006371 1460.801618 1481.88806 0.993542
M1D1-40 5.809842 0.007849 1469.801718 1474.903226 0.993804
M1D2-40 5.758139 0.008592 1431.392506 1431.377483 0.994014
M1D3-40 5.854079 0.007252 1442.473473 1463.223776 0.994093
M2D1-40 5.890438 0.006031 1426.269989 1412.860759 0.994198
M2D2-40 5.908565 0.006373 1508.6688 1539.108696 0.9932
M2D3-40 5.866476 0.00636 1460.392709 1514.079365 0.993489
PTA-40 5.570676 0.011094 1359.240961 1353.559441 0.993988
The proportion of microplastic soil samples decreased compared with the proportion of
actinomycete phylum in soil samples with PTA (25.51%). as compared to a blank (31.52%).
The decline was even greater, the proportion of actinomycete phylum in the PBAT microplastic
soil sample with a landfill time of 40 d decreased mostly compared with the blank soil sample
(28.25%), and the amount added was the proportion of actinomycete phylum in PBAT soil
samples with a particle size of 0.02% and a particle size of <0.01 mm was increased compared
with that of blank soil samples. The addition of PBAT microplastics reduced the abundance of
actinomycete phylum in the soil. Acidobacteriota occurred between 4.66% and 22.74% of the
soil samples, and the landfill time was 20 d and 40 d. The proportion of acid bacillus phylum in
the PBAT microplastic soil samples was higher than that of the blank soil samples in the same
period; secondly, the abundance of the acid bacillus phylum in the PBAT microplastic soil
samples increased with the extension of the landfill time.

52
 Fig. 5.9: Community columnar diagram at the gate level
The abundance of acid bacillus phylum in soil samples decreased with the increase in
landfill time, and the addition of PBAT microplastic increased the abundance of acid bacillus
phylum in soil. This was due to the decrease in soil pH by intermediate products generated
during the degradation of PBAT microplastics and its inhibitory effect on microorganisms. A
community columnar plot of each genus is shown in fig. 5.10. Forty-four (44) genera
were identified in the16 soil samples, and most of the soil samples with PBAT microplastics
increased with the time of landfill (Arthrobacter). Sphingomonashas an extremely wide
metabolic capacity for aromatic compounds, which can be used for the biodegradation of
aromatic compounds, and it is shown from the figure that the abundance of Sphingomamas in
soil samples decreases with the length of landfill time; blank soil samples and PBAT. The
abundance of Nocardioides in the microplastic soil sample decreased with the increase of
landfill time, while the PTA soil sample was the opposite; in summary, the addition
of PBAT has indicated microplastics have an impact on soil community composition.

5
5.3.3.5 Venn plot analysis of microbial communities
According to the landfill time, the soil samples with PBAT microplastics were divided
into two groups of 20 d and 40 d, and the Venn figure at the genus level is shown in fig. 5.11.
The number of species in the common genus of the microbial community
of PBAT microplastic soil samples with landfill times of 20 d and 40 d is 802 (fig. 5.11). 

53
 Fig.5.10: Community histogram horizontally
Several species of the genus endemic to PBAT microplastic soil samples
were added at 20d and the landfill time was added at 40d. The addition
of PBAT microplastics increased the diversity of microbial communities in the soil and
increased the number of species of the genus as the landfill time increased. Venn plot at the
genus level of microbial communities in all soil samples is shown in fig. 5.12:

Fig. 5.11: Venn plot on the genus level

54
 Fig. 5.12: Venn plot at the genus level
There were 404 species in the 16 soil samples with common genera of microbial
communities, and the column-shaped part represents the total number of species at the genus
level in each soil sample; each PBAT. The total number of species in the microplastic soil
sample was higher than blank, while the PTA soil sample was the opposite; as available, the
addition of PBAT microplastic increased the diversity of microflora in the soil, and the number
of species of its genus increased with the increase in landfill time.
Venn plot at the microbial community species level of all soil samples is shown
in fig.5.13. There were 717 species with common microbial communities in 16 soil samples,
and the landfill time was 20d. The number of endemic species and the total number of
species added to the PBAT microplastic soil sample with 40d was higher than the blank soil
samples over the same period, while the total number of species of soil samples
with PTA addition was smaller than the blank. The number of endemic species in each soil
sample increased with the increase of landfill time, and the addition of PBAT microplastics
increased the microbial diversity of soil samples.

55
 Fig. 5.13: Venn plot on the species level
5.3.3.6 Analysis of microbial communities PCoA plots
PCoA reflects the similarity of community composition between different samples by
analyzing the distance between samples, and the percentage represents the magnitude of the
interpretation of the difference in sample composition by the main coordinate axis, and the
greater the distance between the samples, the greater the difference in community composition.
Soil samples with PBAT microplastics added to the landfill time of 20d and 40 d PCoA are
shown in (fig. 5.14 A, B): The percentage contribution of difference in bacterial sample composition,
represented by the abscissa of the PCoA plot with a landfill time of 20 d, is 60.27% and 30.16%,
respectively (fig.5.14). Unlike soil samples where PTA was added, soil
samples with PBAT microplastics were separated by PC2 (30.16%). On the lower side of the PCoA plot;
the percentage contribution to the difference in bacterial sample composition, expressed in the abscissa
of the PCoA plot with a landfill time of 40 d, is 77% and 4.9%, respectively. Separate soil samples
of PBAT microplastics added by PC1 (77%), on the right side of the figure,
and added PBAT samples were relatively far away from the soil samples where PTA was added.

56
 (A)

(B)
Fig. 5.14: (A). PCoA plot on the gate level of 20 d (B): 40 d
The composition of the soil microbial community with PBAT microplastics was
significantly different from that of the soil samples where PTA was added.
5.3 Summary
This experiment explored the effects of different particle sizes and additive amounts
of PBAT microplastics on soil microbial community structure during
degradation, how PBAT microplastics affected soil microbial community structure, and whether
the microbial community structure in soil, in turn, would affect PBAT. The amount of
PBAT microplastics added and the particle size has an impact on their degradation rate in the
soil, and it is speculated that the larger the amount of PBAT microplastics, the smaller the
particle size, and the higher will be the degradation rate. With the extension of the time
of PBAT microplastics in the soil, the vibration of chemical bond expansion and contraction
will change, and the vibration of chemical bond expansion and contraction will continue to

57
weaken. PBAT microplastics had an impact on the structure of soil microbial communities,
which improved the richness and diversity of microbial communities in soil, significantly
inhibited the phylum proteobacterium and actinomycete in the soil at the genus level, and
improved the abundance of acid bacillus phylum; the abundance of Bacillus spp., sphingosine
monocytogenes and Nocardia spp. has decreased, and the PBAT with different additions of
different particle sizes has decreased. The effects of microplastics on the structure of soil
microbial communities vary.

6 Effect of PBAT degradation products on plant growth

Abstract
During this study, the effect of PBAT degradation products on plant growth was
investigated. Chinese cabbage was selected as a test crop. Three levels of PBAT (0, 0.1, 0.5
g) were studied during this experiment. Every treatment was replicated thrice to minimize
the error. Samples were checked for changes after 15, 30, 45, and 60 days. Results of the
study suggested that there was no harmful effect of PBAT on the growth of Chinese
cabbage. Hence PBAT can be employed in agriculture as a biodegradable mulch film to
achieve a green environment.
6.1 Material and methods
This study was carried out to check the effect of PBAT degradation products on the
growth of Chinese cabbage (Brassica rapa ssp.) grown in pots under controlled conditions.
6.1.1 Soil sampling
Soil samples were collected randomly from Shaanxi (yuan Jia cun). It was then
carried to the environmental sciences and engineering laboratory of Shaanxi university of
science and technology. It was grounded using mortar and pestle and sieved. It was then
poured in disposable glasses for further study. Physico-chemical properties of soil are
presented in table 6.1.
6.1.2 PBAT
PBAT in the powdered form was used for this purpose. Three levels of PBAT (0,
0.1, 0.5 g) were studied during this experiment. Soil without PBAT was considered as
control soil.
6.1.3 Experimental setup
The effect of PBAT degradation products on the growth of Chinese cabbage
(Brassica rapa ssp.) grown in pots under controlled conditions was checked. Three levels of
58
PBAT (0, 0.1, 0.5 g) were studied during this experiment. Every treatment was replicated
thrice to minimize the error. Samples were checked for changes after 15, 30, 45, and 60
days.
6.2 Results and discussions
6.2.1 Effect of degradation products on plant growth
We investigated the effect of different levels of incorporated copolymers (PBAT) on
plant growth (fig. 6.1). To investigate the effect of PBAT degradation products on plant growth
we used Chinese cabbage as a test crop. Three levels of PBAT (0, 0.1, 0.5g) were studied
during this experiment. Soil without PBAT was used as control soil. No significant change in
plant growth was observed during the first 15 days. After 30 days, a significant increase in no.
of leaves was recorded. This means that degradation products did not interfere with plant
growth. On day 30, the number of leaves recorded in the C (1) treatment was significantly
higher than in A (1), and B (1) treated soil. Treatments amended with PBAT showed no toxic
effect on overall plant growth as compared to soil without PBAT (control soil). Our results were
per previous studies (101)(102). Plants continue to grow without any toxic effects. After 45
days, it was observed that all PBAT treated soil exhibited good plant growth. It was found that
the number of leaves in all treatments was the same after 60 days experiment but leaves of C (3)
were greener as compared to other treatments. Which showed that PBAT might enhance the
growth rate. This can be due to the release of PBAT degradation products which enhance the
growth rate. No toxic effect of PBAT was recorded during this experiment. Hence, PBAT is the
best polymer that can be used in agriculture as a mulching film to enhance plant growth (74).

Day 15 Day 30 Day 45 Day 60

59
Fig. 6. 1 Chinese cabbage growth in soil containing (A) Control, (B) 0.1 g P(BAT), (C) 0.5 g P(BAT), during
different time intervals (15, 30, 45, and 60 days).
6.3 Summary
Our work describes the effect of poly (butylene adipate co-terephthalate) (PBAT)
copolymer on plant growth. It was proved during the study that PBAT degradation products
have non-significant effects on plant growth So, PBAT can be used for a wide range of
applications. PBAT can be considered as one of the sustainable materials of the generation of
modern “green materials”.

60
7 Summary and Prospect
7.1 Summary
During this study, we focused on the use of poly (butylene adipate terephthalate)
(PBAT), which is one of the most prospective and prevalent biodegradable polymers instead of
non-biodegradable polymers. We got the following conclusions
(1) Polybutylene-adipate-terephthalate (PBAT) as a thermally stable, biodegradable,
mechanically excellent material, has important use value in combating the problem of
"white pollution".
(2) We investigated the enzymatic degradation of poly (butylene adipate
terephthalate) (PBAT) copolymer using lipase B from Candida Antarctica (CALB).
Results of the study displayed approximately 5.16 % loss in PBAT mass after 2 days
which significantly increased to approximately 15.7 % at the end of the experiment (12
days) as compared to blank. The pH of the degradation solution also displayed
significant reduction and reached the minimum value of 6.85 at the end of the
experiment.
(3) In another study, we isolated three novel PBAT-degrading bacteria (Bacillus strains)
from farm soil (Shaanxi (yuan Jia cun) by the serial dilution method. Strains SUST B 1,
SUST B2, and SUST B3 were closely related to bacillus species. Microbial colonies were
spread and streaked many times to get pure colonies. SUST B 1 and SUST B3 gram-
positive bacillus while SUST B2 was gram-negative bacillus. The strains were
sequenced using the NCBI online BLAST tool, and the results showed that the 16S
rRNA gene sequence of the strain SUST B1, SUST B2, and SUST B3 was identical to
Bacillus spp up to 98.3, 99.5, and 97%, respectively. The results showed that SUST B 1
resembled Bacillus thuringiensis and SUST B2 resembled Bacillus cereus while SUST
B3 resembled Bacillus paramycoides, the most. Different physiochemical tests were also
performed to verify our results.
(4) We then accessed the effect of growth factors on the lipase activity of PBAT-
degrading bacteria. The lipase activity under different degradation products,
temperature, pH, and inoculum concentration was studied. The p-NP was used as the
standard and the pre-inactivated crude enzyme solution was used as a blank. The
absorbance was measured using a spectrophotometer at 410 nm. All the treatments were
replicated thrice.

61
(5) The effect of the different culture conditions was also studied to test the lipase
activity and degradation rate of the PBAT film. Under certain conditions, the lipase
activity of strain SUST B2 was 10.42 U/mL. The secreted lipase catalyzes the
degradation of ester bonds present in the PBAT structure. The effect of the pH (6.2, 6.4,
6.8, 7.0, 7.2, 7.4, and 7.6), temperature (25°C, 32°C, 37°C, 42°C, and 47°C), the
concentration of inoculum (0.5 ml, 1.0 ml, 1.5 ml, 2.0 ml, 2.5 ml, and 3.0 ml) and PBAT
degradation products like terephthalate, adipic acid, glucose, and starch on lipase
activity were studied during the experiment. Strains utilize these products as carbon
sources hence completely degrading PBAT. In summary, when 2 wt. % 1, 4-butanediol
was added to 100 mL of SM medium and the pH, temperature, and inoculum amount
were adjusted to 7.5, 37°C, and 1.5 mL, respectively
(6) Isolated strains (SUST B1, SUST B2, and SUST B3) were selected for
degradation study. The degradation mechanism was investigated using attenuated total
reflection Fourier transform infrared spectroscopy (FT-IR), scanning electron
microscopy (SEM), X-ray diffraction (XRD), and thermogravimetric analysis (TGA).
The results showed that each strain had a significant degrading effect on PBAT. Under
certain conditions, the lipase activity of strain SUST B2 was 10.42 U/mL and degrade
10.5 % of PBAT films.
(7) The structure and morphology of PBAT after degradation were characterized by FTIR,
XRD, SEM, and TGA. FTIR analysis showed that after degradation many peaks become
weaker and the peak at 2950 cm-1 almost disappeared after 12 days. The XRD results
indicated that as the degradation time increases the intensity of diffraction peaks slightly
increases as compared to the blank PBAT. TGA analysis also confirmed the successful
degradation of PBAT with time. SEM micrographs further confirmed that degradation
has occurred. Hence, biodegradable polymers can widely be used. Mechanism of PBAT
degradation and factors that affect the degradation mechanism was also studied
(8) Results of the study displayed a significant change in PBAT properties throughout the
experiment. The pH of the degradation solution also displayed significant reduction
throughout the experiment and reached a minimum value at the end of the experiment.
(9) All three strains showed the biodegradation of PBAT. The bioremediation of PBAT in
the environment can be achieved using these strains.
(10) PBAT degradation mechanism was also studied theoretically
(11) After this, the effect of PBAT degradation products on soil microbial community
and plant growth was also studied in separate experiments.
62
(12) Raman spectroscopy was used for analyzing the PBAT degradation process, and a high-
throughput sequencing method was used for investigating the degradation process of PBAT
polymer of different amounts and sizes. The effect of PBAT microplastic on the soil microbial
communities was also studied. It provided a way for the development of microplastic
resources for effective degradation of microplastics, the assessment of the potential impact of
microplastics on soil ecosystems, and improved the understanding of the degradation process,
as well as the maintenance of soil environmental health. The results showed that the increasing
particle size and the amount of PBAT significantly affected its degradation rate. PBAT
microplastics affected the structure of soil microbial communities, which significantly
inhibited the proteobacteria phylum and actinomycete phylum in the soil. It was noticed that
the abundance of the acid bacillus phylum increased while Nocardia spp. abundance
significantly decreased.
6.2 Innovation
Plastic waste recycling and disposal are costly therefore the use of biodegradable
polymers is is an economical and environment-friendly approach. Polybutylene-adipate-
terephthalate (PBAT) as a thermally stable, biodegradable, mechanically excellent material,
has important use value in combating the problem of "white pollution". Poly (butylene
adipate terephthalate) (PBAT), is one of the most prospective and prevalent biodegradable
polymers instead of non-biodegradable polymers. All three isolated strains showed the
biodegradation of PBAT. The bioremediation of PBAT in the environment can be achieved
using these strains.
6.3 Prospects
The following fields of biodegradable polymers should be researched in the future. (1)
Controlled biodegradation (2) The blending of non-biodegradable polymers to make the best
performing biodegradable polymers (3) To reduce the production cost of biodegradable
polymers to increase their application.

63
 Acknowledgment
This project would not have been possible without the support of many people. Firstly, I
offer my sincerest gratitude to my supervisor Prof. Min Zhang, whose perpetual support and
guidance, enabled me to finish my Ph.D. studies. I am also very thankful to my co-supervisor
Dr. Chengtao Li for his help during my degree. He help me a lot and enabled me to complete
my research work on time. I spent a very good time of my life in an excellent research group
under the supervision of Prof.Min Zang. I appreciate the flexibility and the extent of
independence she gave me to conduct my Ph.D. work and her reactivity to provide valuable
feedback and perpetual help in conducting and planning experiments and in writing my research
papers. Furthermore, I am very thankful to all my labmates of the College of Chemistry and
Chemical Engineering as well as Environmental Science and Engineering who provide me with
a very friendly environment and helped me in every difficult time. Sincere thanks to a special
group of friends, especially, Faisal Sharaf, Maxatao, Hanjunwen, Li yuru, Wan yu, Jia Hao, Zhu
mengzen, LisiHan, Song ge, for the memorable moments we shared. I am also highly thankful
to international office teachers especially Miss Salina and Han signorina for positive guidance
and support throughout my stay in China. I am very thankful to Shaanxi University of Science
and Technology for giving me chance to study in this healthy environment. Long Live Pak-
China friendship (中巴友谊万岁).

64
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 Directory of Achievements
1. Polymer pollution and its solutions with special emphasis on Poly (butylene adipate

terephthalate (PBAT)). (Polymer Bulletin) impact factor: 2.87; DOI : 10.1007/s00289-

021-04065-2

2. Screening and characterization of novel lipase producing Bacillus species from

agricultural soil with high hydrolytic activity against PBAT poly (butylene adipate co

terephthalate) copolyesters (Polymer Bulletin) impact factor: 2.87; DOI:

10.1007/s00289-021-03992-4

3. Enzymatic degradation of poly (butylene adipate co-terephthalate) (PBAT) copolymer

using lipase B from Candida Antarctica (CALB) and effect of PBAT on plant growth

(Polymer Bulletin) impact factor: 2.87; DOI: 10.1007/s00289-021-03946-w

4. Degradation of poly (butylene adipate-co-terephthalate) by Stenotrophomonas sp. YCJ1

isolated from farmland soil (Journal of Environmental Sciences) impact factor: 5.56;

DOI: 10.1016/j.jes.2020.10.001

5. Poly(butylene succinate- co -salicylic acid) copolymers and their effect on promoting

plant growth (Royal Society Open Science) impact factor: 2.51;

DOI: 10.1098/rsos.190504

6. A novel Poly(vinyl alcohol) / Carboxymethyl cellulose/yeast double degradable

hydrogel with yeast foaming and double degradable property (Ecotoxicology and

Environmental Safety) Impact factor: 4.87; DOI: 10.1016/j.ecoenv.2019.109765

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