Screening and Characterization of Novel Lipase Producing

Polymer Bulletin
https://doi.org/10.1007/s00289-021-03992-4
ORIGINAL PAPER
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
Aqsa kanwal1 · Min Zhang1 · Faisal Sharaf1 · Li Chengtao1
Received: 18 July 2021 / Revised: 22 November 2021 / Accepted: 24 November 2021

© The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2021
Abstract
The use of poly (butylene adipate-co-terephthalate) (PBAT) has increased widely
but PBAT-degrading bacteria have rarely been studied. During this study, we used
farm soil (Shaanxi (yuan Jia cun)) to isolate and identify PBAT-degrading bacte-
ria (Bacillus strains). We then accessed the effect of growth factors on PBAT deg-
radation as well as the lipase activity of PBAT-degrading bacteria. Most active
strains (SUST B1, SUST B2, and SUST B 3) were selected for degradation study. The
lipase activity under different pH, temperature, degradation products, and carbon
sources was studied. The degradation mechanism was investigated using attenu-
ated 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 condi-
tions, the lipase activity of strain SUST B
2 was 10.42 U/mL and degraded 10.5%
of PBAT films. Results of the study displayed a significant change in PBAT proper-
ties 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 bioremediation of PBAT in
the environment can be achieved using these strains.
* Min Zhang
minzhang628@gmail.com
1
School of Environmental Science & Engineering, Shaanxi University of Science & Technology,
Xi’an 710021, China
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Graphical abstract
Keywords Isolation · Identification · Enzyme extraction · Lipase · Polybutylene

adipate terephthalate (PBAT) · Biodegradation
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 produc-
tion supplies. Synthetic polymers are being widely used in many fields including
agriculture, industry, national defense, transportation, etc. They exhibit excellent
properties like low density, high strength, good wear, and corrosion resistance;
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Polymer Bulletin
hence, they cannot be fully degraded. So they must be recycled to make the envi-
ronment clean. If not recycled properly, these plastics can be broken down into
smaller particles (< 5 mm) on exposure to sunlight. These microplastics can cause
many adverse effects on plants and animals and cause harm to the natural environ-
ment [1]. The "white pollution" caused by discarded plastics has become a public
hazard in the world, posing a potential hazard to the ecological environment [2, 3].
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 reduc-
tion. Animals that have eaten discarded plastic films are prone to intestinal obstruc-
tion. Synthetic fiber fishing nets and fishing lines that are lost or discarded into the
ocean have caused considerable harm to marine life. Managing white pollution and
seeking new environmentally friendly polymers have become a global problem to
be solved. Improper disposal of plastics can cause serious environmental pollution
[4]. This problem can be solved by treating conventional plastic waste and using
biodegradable plastics. Landfilling, incineration, blending with new materials after
granulation, chemical degradation, recycling are among the few methods used for
treating polymer waste [5]. Among them, some methods are very costly, while oth-
ers can cause secondary pollution problems. To overcome this problem, biodegrad-
able polymers can be the best solution. Using plastics that can degrade naturally in
the environment by the action of different microorganisms are termed biodegradable
[6]. Therefore, scientists started to design a plastic that possesses all the beneficial
properties like consistency and resilience as well as more vulnerability to micro-
bial degradation in nature without harming the environment. 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 uti-
lized by microbes as a carbon source [7]. Considering this, biodegradable plastics
have now been widely researched, manufactured, and used in almost every coun-
try [8]. Since the 1980s, biodegradable polymers have been widely manufactured
and used in every field of life. In 2020, biodegradable and non-biodegradable poly-
mers account for 941,000 metric tons and 1.17 million metric tons, respectively [9].
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
[10, 11]. Many fields of life including agriculture, medicine, tissue engineering, and
industry widely used biodegradable polymers. The biodegradable polymers have the
advantage materials of conserving fossil resources as well as reducing environmen-
tal pollution [12–14].
Poly (butylene adipate-co-terephthalate (PBAT) is among the most important ali-
phatic–aromatic copolymer with a wide range of importance. 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. High
elongation at break, good hydrophilic and processing properties make it a desira-
ble material for industry. Its flexibility can be enhanced by blending it with other
polymers. It can replace polyethylene which is non-biodegradable but has similar
properties to PBAT [15]. PBAT is biodegradable due to the presence of the butyl-
ene adipate group. Eco flex (trade name) is widely used for making bags, mulch-
ing films, laminated sheets, plastics for wrapping food, toothbrushes, and many
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more items like this [10, 17, 18]. It is environmentally friendly. Naturally occurring
enzymes consume it within weeks. Biodegradation of polymers requires microor-
ganisms to metabolize all organic components of the polymer [9]. 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 C O2 [19].
Microorganisms can degrade biodegradable polymers either aerobically or anaerobi-
cally and convert them to bio decomposed products including C H4, H2O, and some
inorganic compounds. It may also involve hydrolysis, photodegradation, oxidation,
etc. [20]. Degrading bacteria secrete lipase which cleaves the ester bonds present
in PBAT polymer and converts them to smaller particles [13]. Degradation prod-
ucts like terephthalic acid and adipic acid can change the pH of the environment
and can cause physiological toxicity in plants and animals. Many PBAT-degrading
microorganisms have been characterized in previous studies for their biodegrada-
tion behavior [21]. The pure aromatic polyesters are quite insensitive to any hydro-
lytic degradation [22]. It was also observed that direct microbial or enzymatic attack
of pure aromatic polyester is not significant [23] but other scientists claimed that
aromatic polyester could be disintegrated by microbial strains of Trichosporum and
Arthrobacter in a time scale of weeks. Some growth of Aspergillus Niger was also
reported on the surface of aromatic polyesters. On the contrary, aliphatic polyester
is considered to be more susceptible to microbial attack. Aliphatic polyester degra-
dation is seen as a two-step process: the first is polymerization, or surface erosion
[24]. The second is enzymatic hydrolysis, which produces water-soluble intermedi-
ates that can be assimilated by microbial cells [25]. PBAT is used as mulch film in
a natural environment, so understanding PBAT-degrading mesophiles is important
in the context of onsite degradation [23]. Studies showed that two PBAT hydrolases
from the anaerobic mesophilic bacteria Pelosinus fermentans and Clostridium bot-
ulinum are α/β hydrolases with a lid domain [14–19]. In compost, a thermophilic
actinomycete, Thermobifida fusca K13g, and Thermomonospora fusca DSM43793
play an important role in the degradation of PBAT [26]. Characterization of PBAT
hydrolase from T. fusca showed that the enzyme was cutinase [27], which degrades
the plant polyester cutin. Likewise, Thermobifida alba, Saccharomonospora viridis,
Thermobifida cellulosilytica, and Humicola insolens are all cutinases. Another study
showed that PBAT can be degraded and mineralized in the natural environment by
fungus Isaria fumosorosea strain NKCM1712 and Paraphoma-related fungus cuti-
nase-like enzyme (PCLE) and Cryptococcus flavus cutinase-like enzyme (CfCLE)
can degrade PBAT under mild conditions [23, 28–30]. It has been reported that mes-
ophilic 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 [3, 31]. A low degradation rate was among the main draw-
backs. 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 [3, 32, 33]
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In this study, PBAT-degrading bacteria (SUST B1, B2, and B3) were isolated and
identified from a cultivated field from Shaanxi. The colonies grew well at 25–45 °C
and did not require high nutrition. The colonies were yellow, round, raised, opaque,
neat, and sticky. Degradation characteristics and lipase activity of selected strains
were studied. Comparing the change in morphology of PBAT after degradation with
blank PBAT showed that significant degradation has occurred. These results provide
technical support for the highly efficient degradation of PBAT in the environment.
Material and Methods
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 [34] was used to
prepare a (200 mm × 100 mm × 0.7 mm) PBAT film for the degradation test, and the
PBAT (number-average molar mass of 1.0 × 105) was purchased from Zhuhai Wan-
tong Chemical Co., Ltd. (Guangdong, China) [15, 35].
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 follows: 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; Z nSO4.7H2O, 2.0; M nSO4.7H2O, 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, 0.10 g PBAT was dissolved in 10 mL of
chloroform. After attaining complete dissolution, 100 ml of SM medium and 0.5 ml
of tween80 (as an emulsifier for phacoemulsification) were added. Media was steri-
lized at 121ºC for 15 min before further process.
Isolation and identification of PBAT‑degrading microorganisms
For isolating PBAT-degrading microorganisms were used [23]. The soil was config-
ured as bacterial suspension and supernatant were inoculated in screening medium
using serial dilution-based plating method at 30 °C and 130 r/min. When the screen-
ing 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.
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Inoculum preparation
Strains were cultured for 12 h in 100 mL of LB medium on a shaker at 37 °C and
130 r/min.
Morphological observation
Selected strains were observed on the screening medium via scanning electronic
microscopy (SEM, FEI Q45, FEI) [36]. A morphological gram staining test was
done at 0.8–1 O.D (600 nm). 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 1 min 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 s 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.
Physiological and biochemical experiments
Strains were subjected to Gram’s staining, Methyl Red, Voges–Proskauer (V-P reac-
tion), starch hydrolysis tests, catalase reaction, and lipid hydrolysis [37].
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 min. The sample was centrifuged at 10,000 rpm for
2–5 min. The supernatant was discarded and the pellet was stored. 100uL of distill-
ing water was added to the pellet and boiled for 10–20 min. 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’AGA
GTTTGATCCTGGCTCAG-3’) and 16S-1492R (5’TACGGTTACCTTGTTACG
ACTT-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 °C for 3 min, denaturation at 95
°C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 30 s, 27 cycles and
finally extension at 72 °C for 10 min (PCR Amplifier: ABI Gene Amp® 9700 type)
[38].
Gel electrophoresis
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. After that, 10 uL of PCR
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products were run on the GE. Denaturing gel was made by adding 0.5% of agarose,
50-55 ml 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 solu-
tions. 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. The core was placed into the tank (containing 6
L of 1 × TAE buffer)[39]. 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. The experimental conditions used were
100–120 V, 400 mA, and 15 min for bacteria. 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 was then visualized on a gel documentation system
(UVI Tec Gel Doc). 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 [40]. MEGA X was used for constructing the
phylogenetic tree by the neighbor-joining method [41].
Lipase assay of selected strains and PBAT degradation ability.
The single-factor method was selected to study PBAT degradation and lipase pro-
duction. For measuring bacterial growth (O.D at 600 nm), isolated were picked up
from SM Petri plates and transferred to nutrient broth. It was then incubated at 30
ºC, 120 rpm for 24 h. Then, 1 ml of culture broth was collected from each culture
variant to record O.D values. To determine the utilization of the PBAT degradation
products, the lipase activity of selected strains was measured by the para-nitro phe-
nol (p-NP) method [9] [42]. 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 (30 mg) dissolved in isopropanol (10 ml)]; 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 stand-
ard and the pre-inactivated crude enzyme solution was used as a blank. The absorb-
ance 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. Following culture con-
ditions were examined (change in pH, change in temperature, change in inoculum
concentration, and degradation products). Firstly, the effect of pH on lipase activ-
ity was tested for each strain. For this purpose, SM medium (100 ml), 1,4-butan-
ediol (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 (1 ml inoculum) in the respective flask separately
and shaken at 30 °C at 130 r/min for 5 days. To check the effect of temperature at
pH 7.4, the cultures (1 ml inoculum) shook at different temperatures (25 °C, 32 °C,
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37 °C, 42 °C, and 47 °C) at 130 r/min for 5 days. Thirdly, we changed the concen-
tration 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. Fourthly, the effect of PBAT degrada-
tion products like terephthalate, adipic acid, glucose, and starch on lipase activity as
well as degradation of PBAT was also studied during the experiment. To study the
utilization of degradation products by SUST B1, SUST B2, and SUST B3, SM media
(100 ml), PBAT film, starch, glucose terephthalic acid, 1,4- butanediol, and adipic
acid (2 wt.% each) and inoculum (1.5 ml) of isolated strains were added separately
in 250 mL Erlenmeyer flask and shaken at 37 °C.
Microbial degradations of PBAT polymer.
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 chlo-
roform were mixed in a three-necked flask and shaken at 600 rpm for 24 h until
completely dissolved followed by subsequent purification using industrial alcohol.
Then weighed amount of purified PBAT and chloroform were added in a 3-necked
flask and shaken for 4–6 h 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 [22].
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 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 (100 ml), inoculum (1.5 ml) of respec-
tive 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
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where r, w0, and w1 are the degradation rates of PBAT (%), the weight before degra-
dation (g), and the weight after degradation (g), respectively [10].
Characterization of PBAT Degradation.
PBAT degradation was characterized by recording different parameters.
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(%) = × 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.
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) [43].
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.
in−1 with
Every scan was recorded in the range of 2θ = 5–50° at a scan speed of 2° m
an X-ray tube operated at 40 kV and 40 mA [44].
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 analy-
ses [25].
Scanning Electron microscope (SEM)
The surface topography of PBAT samples collected at different intervals was exam-
ined using a scanning electron microscope (SEM, S-4800, Rigaku Co. Ltd, Japan)
operated at 10 kV with a spot size of 10 nm [16].
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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 nitro-
gen atmosphere (60 mL min−1). About 5 mg of the sample was heated from 25 ºC to
500 °C at a heating rate of 10 ºC min−1 [45]
Results and discussion
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 B1, SUST B2 and SUST B3. Figure 1 shows phylogenetic trees of these iso-
lates 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. Isolated strains, SUST B 1 and
SUST B3 were gram-positive bacillus, while SUST B2 was gram-negative bacillus.
Based on grams staining and physicochemical tests (Table. 1), all strains secrete
lipase, which can degrade aliphatic–aromatic polyesters.
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 B 2, 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 Bacil-
lus sp. while SUST B2 resembled, Bacillus cereus, Bacillus sp. GZT, Bacterium
CWISO11, Bacillus thuringiensis, Bacillus paramycoides, Bacillus tropicus, Bacil-
lus paranthracis, Bacillus paramycoides, Bacillus sp. (in Bacteria) while B3 resem-
bles 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 showed that SUST B 1 resembles Bacillus thuringiensis
and SUST B 2 resembles Bacillus cereus while SUST B 3 resembles Bacillus paramy-
coides the most. (Fig. 1b–d).
For gel electrophoresis PCR samples were used to observe DNA bands of
microbes. Several DNA bands were observed along the increasing gradient of dena-
turing agent that separates DNA fragments according to their melting point and GC
content. The gel picture was observed under UV light (Fig. 1a). Compared with uni-
versal 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 B 1, 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. 2a–c).
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Fig. 1 a Image of a gel post electrophoresis. 2, 4, 9 and10 bands represent SUST B

1, B2, and B3, respec-
tively. Compared with universal ladder bp was found between 900–1200. While b, c, d represents the
phylogenetic tree of isolate SUST B1, B2, and B3, respectively
The evolutionary history was inferred by using the maximum likelihood method
and the Poisson correction model [46]. The bootstrap consensus tree inferred from
1000 replicates was taken to represent the evolutionary history of the taxa analyzed
[47]. 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
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Fig. 1 (continued)
Table 1 Physiological and Tests SUST B1 SUST B2 SUST B3

biochemical experiments
Gram staining + − +
Methyl red − − −
V-P reaction + − −
Starch hydrolysis test + − −
Catalase reaction + + +
Lipid hydrolysis test + + +
Where “ + ” = positive, and “−” = negative
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Fig. 2 (a1, a2, a3) represents the effect of degradation products (CK = control, terephthalate, adipic acid,
butanediol, and glucose) on degradation rate, and lipase activity of S USTB1, B2, and B3, respectively at
37 ºC and pH 7.5, respectively, (b1, b2, b3) 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 B 3, respectively, (c1, c2, c3)
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 and (d1. d2, d3) 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
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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 esti-
mated using a JTT model and then selecting the topology with a superior log-likeli-
hood value. This analysis involved 26 amino acid sequences. All positions contain-
ing 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 [46]. Phylogenetic relationships could be inferred through the alignment,
and the approximate phylogenetic position of the strains is shown in Fig. 1b–d.
Lipase assay of selected strains and PBAT degradation ability of selected strains
Lipases are extensively been used in different applications such as detergents, cos-
metics, food flavorings, diesel, papers, and pulps. Thus, measurement of lipase
activity is an important process for the quantification of lipases. The lipase activity
of isolated enzymes from SUST B1, B2, and B3 is presented in (Fig. 2a–d).
Effect of degradation products, temperature, pH, and inoculum concentration

on degradation rate, and lipase activity of SUSTB1, 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 car-
bon source) Fig. 2 (a1, a2, a3). The PBAT degradation rate of SUST B1, 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 tere-
phthalate, 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.
The temperature had a considerable influence on the PBAT degradation rate and
lipase activity of the strain SUST B1, B2, and B3 Fig. 2 (b1, b2, b3). At 27 °C, the
PBAT degradation rate of SUST B 1, 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 B1, B2, and B 3 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, respec-
tively, for PBAT degrading strains SUST B1, B2, and B3, respectively. At 42 °C and
45 °C, 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 degra-
dation 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.
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Polymer Bulletin
The effect of pH (6, 6.5, 7, 7.5, and 8) Fig. 2 (c1, c2, c3) and inoculum (1, 1.5, 2,
and 2.5 ml) Fig. 2 (d1, d2, d3). was the same as temperature. In summary, the best
conditions required for the growth of these organisms were observed 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. 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 [48]. 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., Brevi-
bacterium 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. [49] 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 lipo-
lytic activity [50]. 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) [20].
Characterization
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. 3a. 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, respec-
tively, 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 [51]. 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 bio-
degradation was mainly caused by microbial degradation and reported that the
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Polymer Bulletin
(a) (b)
12 B1 7.20 B1
B2 10.5 B2
10 7.15
B3 9 B3
8.1
Mass loss (%)
8 7.10
pH
7.05
6
7.00
4
6.95
6.95
2 6.93
6.90
6.91
0
0 2 4 6 8 10 12 14 2 4 6 8 10 12 14
Time (d) Time (d)
Fig. 3 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
biodegradation of the non-crystalline portion was faster than that of the crystal-
line portion. Likewise, Weng et al. [52] also worked on biodegradation of poly-
butylene adipate-terephthalate (PBAT). The results of the study showed visible
signs of PBAT biodegradation.
Change in pH
Change in pH during PBAT degradation at different time intervals is presented in

Fig. 3b. 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 B2
strain treated PBAT, i.e., 6.91 that was significantly lower than blank PBAT treat-
ment. 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 [53]. 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 differ-
ent modifications. Some simple and complex metabolites were excreted and reached
the extracellular surroundings (e.g., organic acids, aldehydes, terpenes, antibiot-
ics, CO2, N2, CH4, H2O, and different salts, etc.). This might be the reason for the
change in pH that was observed throughout our study [54].
X‑rays diffraction (XRD)
The XRD diffractograms of PBAT polymers are shown in Fig. 4a–c. Blank PBAT
has characteristic peaks at 011, 010, 101 100, and 111 correspondings to diffrac-
tion 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
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Polymer Bulletin
Fig. 4 a, b, c Represents XRD diffractograms of the PBAT biodegradation at different time intervals
using isolated strains SUST B 1, B2, and B3, respectively, while 5 d, e, f represents infrared (FTIR) spec-
tra of PBAT (polybutylene adipate terephthalate) biodegradation at different time interval using isolated
strains SUST B 1, B2 and B3, respectively
their low crystallinity. As the degradation time increased, the locations of diffrac-
tion peaks were almost the same, and it suggested that their crystal structures were
unchanged. The intensities of PBAT diffraction peaks increased slightly compared
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Polymer Bulletin
with that of blank PBAT. This indicated that the amorphous phase of PBAT
decreased due to degradation by enzymes (Fig. 4a–c).
Fourier transform‑infrared spectroscopy (FTIR) Analysis of PBAT
FTIR is a useful tool to detect the changes after degradation (Fig. 4d–f). The width,
intensity, and peak position of the vibrational band are sensitive to molecular con-
ception and molecular environment changes. Before degradation PBAT FTIR infor-
mation showed the following: 2975 cm−1 represented the asymmetric stretching
vibration of methylene (-CH2-), 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 rep-
resented 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, corresponded 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 molec-
ular chain, respectively.
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. 4d).
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, 1475 cm−1,
respectively. Further, after PBAT degradation, ester carbonyl (-C = O) groups
stretching vibration position shifted from 1750 cm−1 to 1710 cm−1 (Fig. 4e). Using
SUST B3, the PBAT peaks became weaker and the peak at 2975 cm−1 almost disap-
peared 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. 4f), these all supported our results that
SUST B1, B2, and B3 successfully degraded PBAT polymer (Fig. 5 (d, e, f). Weng
et al. [19] also reported similar results of PBAT degradation.
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. 5a–d and the 50% and 95%
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Polymer Bulletin
(a)100 (b) 100

Blank Blank
80 B1 80 B1
Mass loss (%)

M a s s lo s s (% )
B2
60 B3 60
100 100
95 Blank 95
40
Blank
Mass loss (%)

40
B1
90
Mass loss (%)

B1
90
85
85
80
80
75
20 20 70
300 320 340 360 380 400 420
75
70
Temperature (°C ) 360 380 400 420
Temperature (°C )
0 0
50 100 150 200 250 300 350 400 450 500 100 200 300 400 500
Temperature (°C ) Temperature (°C )
(c) (d)
100 100
Blank Blank
80 B2
M a s s lo s s (% )
80 B3
M a s s lo s s (% )
60 100 100 60 100

100
90 90
Mass loss (%)
Mass loss (%)

Mass loss (%)
Mass loss (%)

80 80 80 80
40 70 70 40
60 60 60 60
50 50
300 320 340 360 380 400 420 440 460
20
360 380 400 420 300 320 340 360 380 400 420 440 460 360 380 400 420
20 Temperature (°C ) Temperature (°C ) Temperature (°C ) Temperature (°C )
0 0
100 200 300 400 500 100 200 300 400 500
Temperature (°C ) Temperature (°C )
Fig. 5 a,b,c,d Represents the TGA of the PBAT biodegradation at different time intervals using isolated
strains SUST B 1, B2, and B3, respectively
thermogravimetric temperatures are given in Table 2. It showed the quickest decom-

position 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 tempera-
tures 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 iso-
lated strain B3 were 395ºC and 361ºC, respectively. It means that with the increase
Table 2 50% and 95% Samples 50% 95%

thermogravimetric temperatures
PBAT0 409 372
PBAT1 400 368
PBAT2 394 361
PBAT3 395 361
PBAT0: Before degradation; P BAT1: After degradation with SUST

1; PBAT2: After degradation with SUST B2; PBAT3: After degrada-
B
tion with SUST B3
13
Polymer Bulletin
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 basi-
cally without major change.
As can be seen from the TG curve, all degraded PBAT samples shift 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 [19].
Morphology Analysis of Composites
Scanning electron microscopic (SEM) analysis was conducted to investigate the deg-
radation of PBAT during different times using isolated strains SUST B1, B2, and B3,
respectively (Fig. 6A–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 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 × are shown in
Fig. 6a–c from Fig. 6A (a-e), are the micrographs of PBAT using isolated strain
SUST B1. The surface of the PBAT was clean and no fractures were present ini-
tially (Fig. 6A-C(a)). But as the degradation time increased, fractures appeared and
became more obvious at the end of the 12-day experiment (Fig. 6A-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 degrada-
tion time further increased. The PBAT sample surface had become very rough after
12 days of degradation, and numerous cracks were observed (Fig. 6A-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, micro-
organisms 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 microor-
ganisms. Due to this process, microbial metabolic products such as water, carbon
dioxide, and methane (anaerobic degradation) are produced [55].
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Polymer Bulletin
Fig. 6 A, B, C. SEM image of PBAT (polybutylene adipate terephthalate) biodegradation at different

time intervals using isolated strains SUST B
1, B2, and B
3, respectively. a: PBAT (without degradation) b:
PBAT (blank) c:PBAT (4 days degradation) d: PBAT 8 days degradation) e: PBAT (12 days degradation)
Conclusion
The use of biodegradable films in agriculture can promote sustainability and reduce
soil contamination. 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 degra-
dation products, temperature, pH, and inoculum concentration on degradation rate,
and lipase a ctivity, the PBAT degradation of SUST B 1, B2, and B
3 under labora-
tory conditions. 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 intensi-
ties of PBAT diffraction peaks slightly increased with time as compared to blank
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Polymer Bulletin
PBAT. It showed that the amorphous phase of PBAT decreased due to degradation
by enzymes. The results of FTIR demonstrated that there was possible degrada-
tion 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 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 toward the lower decomposition temperature.
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Screening and Characterization of Novel Lipase Producing

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Screening and Characterization of Novel Lipase Producing

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Polymer Bulletin

Screening and characterization of novel lipase producing

Aqsa kanwal1 · Min Zhang1 · Faisal Sharaf1 · Li Chengtao1

Received: 18 July 2021 / Revised: 22 November 2021 / Accepted: 24 November 2021

Keywords Isolation · Identification · Enzyme extraction · Lipase · Polybutylene

Soil collection and Sample preparation

Media used for bacterial isolation

Isolation and identification of PBAT‑degrading microorganisms

Physiological and biochemical experiments

Sample preparation and analysis of 16S rRNA gene sequence

A loop full of bacterial culture was transferred to 100 µl of filter-sterilized distilled

Lipase assay of selected strains and PBAT degradation ability.

Microbial degradations of PBAT polymer.

Preparation of PBAT film for degradation study

PBAT degradation using isolated strains microbes.

Degradation analysis of the samples was carried out in phosphate-buffered saline

Characterization of PBAT Degradation.

PBAT degradation was characterized by recording different parameters.

Degradation rate of PBAT by mass loss method (%)

X‑ray diffractometer (XRD) analysis

Fourier transforms infrared (FT‑IR) spectroscopy

Fourier transforms infrared (FT-IR) spectroscopy analysis was also performed

Scanning Electron microscope (SEM)

Thermogravimetric Analysis (TGA)

Thermal measurements were carried out using a TA instruments Auto-MTGA Q500

Isolation and identification of PBAT degrading microorganisms

Fig. 1 a Image of a gel post electrophoresis. 2, 4, 9 and10 bands represent SUST B

Table 1 Physiological and Tests SUST ­B1 SUST ­B2 SUST ­B3

Where “ + ” = positive, and “−” = negative

Lipase assay of selected strains and PBAT degradation ability of selected strains

Effect of degradation products, temperature, pH, and inoculum concentration

Effect of degradation products showed a significant increase in degradation rate,

Mass loss (%)

The progress of enzymatic degradation was monitored at regular intervals. The

Change in pH during PBAT degradation at different time intervals is presented in

X‑rays diffraction (XRD)

Fourier transform‑infrared spectroscopy (FTIR) Analysis of PBAT

Thermal Gravimetric Analysis of PBAT (TGA)

(a)100 (b) 100

Mass loss (%)

Mass loss (%)

Mass loss (%)

60 100 100 60 100

Mass loss (%)

Mass loss (%)

Temperature (°C ) Temperature (°C )

thermogravimetric temperatures are given in Table 2. It showed the quickest decom-

Table 2 50% and 95% Samples 50% 95%

PBAT0: Before degradation; P ­ BAT1: After degradation with SUST

in degradation time, the initial decomposition temperature of PBAT also decreased.

Morphology Analysis of Composites

Fig. 6 A, B, C. SEM image of PBAT (polybutylene adipate terephthalate) biodegradation at different

17. Li P, Wang X, Su M, Zou X, Duan L, Zhang H (2020) Characteristics of Plastic Pollution in

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Fig. 1 a Image of a gel post electrophoresis. 2, 4, 9 and10 bands represent SUST B

Table 1 Physiological and Tests SUST B1 SUST B2 SUST B3

Table 2 50% and 95% Samples 50% 95%

PBAT0: Before degradation; P BAT1: After degradation with SUST

Fig. 6 A, B, C. SEM image of PBAT (polybutylene adipate terephthalate) biodegradation at different