Science of the Total Environment 904 (2023) 166721

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Science of the Total Environment

journal homepage: www.elsevier.com/locate/scitotenv

Laccase driven biocatalytic oxidation to reduce polymeric surface

hydrophobicity: An effective pre-treatment strategy to enhance biofilm
mediated degradation of polyethylene and polycarbonate plastics
Anindya Shankar Ray, Muneeswari Rajasekaran, Maseed Uddin, Ramani Kandasamy *
Industrial and Environmental Sustainability Laboratory, Department of Biotechnology, School of Bioengineering, SRM Institute of science and technology, Kattankulathur-
603203, Chengalpattu District, Tamil Nadu, India

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• Enzymatic pre-treatment induced sur­

face oxidation of plastics.
• Reduced hydrophobicity improved bio­
film formation on plastics.
• Achieved enhanced biodegradation of
plastics with the increased microbial
activity.
• XPS and GPC confirmed the enhanced
biodegradation of plastics.
• Integrated enzyme and biofilm medi­
ated approach provides a sustainable
treatment technology.

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

Editor: Yi Yang Plastic pollution is a major global environmental issue due to its structural complexity and poor biodegradability.
Biological approaches are appropriate due to cost effectiveness and environmental friendliness, however effec­
Keywords: tive polymer degradation is still in its infancy. As biological treatments are slower than physical and chemical
Plastic wastes approaches, they could be applied in conjunction with pre-treatment techniques such as photo-oxidation, heat
Enzymatic pre-treatment
treatment, and chemical treatments. But these processes lead to high energy consumption and hazardous sec­
Surface oxidation
ondary pollution. To address these concerns, an enzymatic pre-treatment strategy has been proposed in this
Reduced hydrophobicity
Biofilm mediated degradation study, with an aim of promoting surface oxidation on the plastics leading to improved hydrophilicity. This in
turn, facilitates the surface attachment of microbes, ultimately, accelerating biodegradation. Scanning Electron
Microscopy (SEM) and Fourier Transform Infrared (FT-IR) spectroscopy analyses confirmed the surface oxidation
of the polyethylene (PE) and polycarbonate (PC) plastics mediated by the action of laccase enzyme. Contact angle
measurement witnessed the increased hydrophilicity of the treated plastics. Following, a potential biofilm
forming microbial consortium has been employed for the biodegradation of enzyme treated plastics. SEM
analysis indicated the increased formation of corrosive pits and surface aberrations on the enzymatically pre-
treated plastics and Confocal Laser Scanning microscopy (CLSM) analysis exhibited the enhanced biofilm for­
mation and exopolysaccharide deposition on the pre-treated PE and PC. In addition, X-ray photoelectron

* Corresponding author.
E-mail address: ramanik@srmist.edu.in (R. Kandasamy).

https://doi.org/10.1016/j.scitotenv.2023.166721
Received 26 June 2023; Received in revised form 21 August 2023; Accepted 29 August 2023
Available online 4 September 2023
0048-9697/© 2023 Elsevier B.V. All rights reserved.
A.S. Ray et al.
spectroscopy (XPS) revealed the reduction in the elemental composition of carbon with an increment in the
oxygen composition of plastics. Gel permeation chromatography (GPC) further confirmed the greater reduction
in the molecular weights of the plastics subjected to integrated enzymatic and biofilm treatment than only
biofilm treated plastics. This is the first report on the integration of enzymatic pre-treatment with the biofilm
mediated microbial degradation to achieve enhanced treatment of plastics which demonstrated to be a promising
technology for the effective mitigation of plastic pollution.
1. Introduction
Plastic pollution is a contentious issue on a global scale due to the
constant and unstoppable threat it poses to the entire natural environ­
ment. Despite increased recycling, a significant proportion of plastics
waste is disposed of in the open environment. Once there, plastics were
reported to undergo partial degradation, mainly cracking and fracturing
by physical, chemical, and biological processes, ultimately producing
small pieces called micro- and nano-plastics. Such produced micro and
nanoplastics may conjugate with the subsequently leached plastic ad­
ditives which are significantly recalcitrant, thus serve as “a trojan horse”
for bioaccumulation leading to catastrophic impact on aquatic envi­
ronments and human health (Sendra et al., 2017). The significant
detrimental impact of plastics in the environment implies that the pre­
sent strategies have failed to tackle the incessant pollution. Hence, the
exploration of green environmental friendly approach for the natural
bioremediation of plastic is an emerging field of assessment to eradicate
the limitations served by the conventional plastic curtailment processes
(Zeenat et al., 2021).
In the recent studies, bacterial biofilms, bacterial consortia, and pure
fungal cultures have been applied for plastic degradation (Oliveira et al.,
2020). Also, plastic consuming invertebrate mealworms and super
worms have been identified with the ability to digest the plastics into
simpler monomeric forms (Gao et al., 2018; Kim et al., 2020; Yang et al.,
2014). Yet, the major bottleneck of the these biological treatment stra­
tegies is the extended duration of degradation that ensues due to the
complex structural nature and surface hydrophobicity of the plastics
(Müller et al., 2001). The hydrophobic/hydrophilic balance and surface
roughness of a polymer are important parameters that can affect the
biodegradation rate. Incorporation of oxygen-containing functional
groups has been explored to increase the hydrophilicity of polymers
boosting the adhesion and activity of the microbial population on the
polymer surface and opening an opportunity to accelerate degradation
(Bher et al., 2023; Chamas et al., 2020). Hence, applicability of pre-
treatment strategy has been considered with the aim to promote sur­
face oxidation of the plastics resulting in the enhanced surface hydro­
philicity (Arutchelvi et al., 2008). So far, the notably employed pre-
treatment strategies to enhance surface hydrophilicity include UV-
oxidation, alkaline treatment (Aravinthan et al., 2016; Arkatkar et al.,
2010; Jeyakumar et al., 2013), photo-oxidation (Gewert et al., 2015),
thermo-oxidation (Jakubowicz, 2003; Khabbaz et al., 1999), ultrasound
(Pellis et al., 2016) and nitric acid treatment (Mahalakshmi and Andrew,
2018; Rajandas et al., 2012; Yamada-Onodera et al., 2002) and surface
coating with minerals (Jiang et al., 2022). Moreover, natural ageing
processes have been reported to enhance surface hydrophilicity of
plastics (Jiang et al., 2023). However, these strategies have stumbling
pitfalls owing to mechanical stress on the plastics, increased consump­
tion of energy, incorporation of chemical toxicity and longer duration.
Hence, the application of a sustainable pre-treatment strategy is
imperative to aid the bioremediation of plastics in an environmental-
friendly process.
Enzymatic biocatalysis has gained increasing attention as an eco-
friendly alternative technology to conventional plastic treatment and
recycling methods. To date, various microbial plastic-degrading en­
zymes have been discovered, representing promising biocatalyst can­
didates for plastic depolymerization (Zhu et al., 2022). In the previous
findings, production of several enzymes (esterase, cutinase, lipase,
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Science of the Total Environment 904 (2023) 166721
oxidoreductase, laccase, PETase, Polyamidase, etc.) during the course of
microbial degradation of the plastics, have been reported and the
application of such potential biocatalytic candidates could be a viable
pre-treatment technology due to its competent oxidative capabilities on
plastics (Zhu et al., 2022).
In this context, the application of purified laccase enzyme from
fungus Phanerochaete chrysosporium as a novel pre-treatment strategy
has been evaluated in the present study. In addition, efficient plastic
degrading microbial consortia has been constructed and employed for
the biofilm mediated degradation plastics. The efficiency of plastics
degradation has been investigated using two strategies include only
biofilm mediated approach and the integrated enzymatic and biofilm
mediated degradation. The surface morphology survey and chemical
composition changes by scanning electron microscopy (SEM) and
Fourier transform infrared spectroscopy (FT-IR) analysis. Biofilm ar­
chitecture on the plastics were visualized by scanning electron micro­
scopy (SEM) and confocal laser scanning microscopy (CLSM). The
hydrophobicity index of the plastics through the course of degradation
was determined by Water contact angle measurement. The process of
degradation was confirmed by X-ray photoelectron spectroscopy (XPS)
and gel permeation chromatography analyses. The study could provide a
sustainable biotechnological approach to degrade the plastics and would
be highly applicable to the real time mitigation of the plastic pollution in
the environment and contaminated site.
2. Materials and methodology
2.1. Collection of plastics
Plastic refuses were collected from various plastic contaminated
dumpsites and were cut into (5 cm × 5 cm) fragments, washed thor­
oughly via 70 % ethanol for 30 min, followed by deionized water. The
washed plastic samples were dried in oven at 50 ◦ C. The collected plastic
samples were determined to be polyethylene (PE) and polycarbonate
(PC) type and had molecular weight of 1774 Da and 267,341 Da
respectively.
2.2. Enzymatic pre-treatment
Pre-treatment of the plastics were carried out using purified laccase
enzyme. The pre-treatment of polymers was performed by incubating
the plastic samples in 250 mL conical flasks containing 100 mL of so­
dium acetate buffer (pH 4.5). Commercial laccase enzyme from fungus
Phanerochaete chrysosporium was added to the solution. The treatment
conditions were optimized to be pH 4.5, temperature of 30 ◦ C and 100
Units of enzyme concentration (Data not shown). The pre-treatment was
carried out for 4 days in the incubator with the agitation of 100 rpm.
2.3. Construction of biofilm forming potential microbial consortium
Soil samples heavily contaminated with plastic garbage were gath­
ered from areas near the SRMIST campus. Approximately 5 g of the
samples were cultivated in an Erlenmeyer flask containing 100 mL of
liquid enrichment media with PE and PC pieces. Liquid enrichment
media (LEM) per litre consists of: KH2PO4 0.7 g/L, K2HPO4 0.7 g/L,
MgSO4.7H2O 0.7 g/L, NH4NO3 1.0 g/L, NaCl 0.005 g/L, FeSO4.7H2O
2.0 mg/L, ZnSO4.7H2O 2.0 mg/L, and MnSO4.H2O 1.0 mg/L in sterilized
A.S. Ray et al.
mili-Q H2O and the pH of the media was about 7.0–7.2. The flasks were
shaken at 100 rpm in an incubator shaker with the temperature main­
tained at 30 ◦ C and were observed on a regular basis for the growth of
biofilm on the plastic pieces’ surfaces. Treated PE and PC pieces with
solutions were removed to fresh liquid enrichment media after 10 days
of incubation. After three cycles of enrichment, survived microbial
cultures were serially diluted and pure colonies were obtained.
2.3.1. Crystal violet staining assay for screening of efficient biofilm-forming
micro-organisms
The quantitative assay of biofilms with crystal violet staining was
performed as described (Nagaraj et al., 2017). The isolates from each
soil samples were grown for 24 h in test tubes containing 2 mL of
nutrient broth. After incubation of 24 h, the media was removed gently
and the wells of the microtiter plate were cleaned via 200 μL phosphate
buffer saline (pH 7.4) buffer and were kept for drying for a duration of
15 min. The wells of the microtiter plates were stained via 200 μl of
crystal violet (0.4 %) for 15 min in room temperature. The CV stain
(unbound) was then discarded and the wells were washed gently three
times with 200 μL of PBS buffer. For 15 min, the microtiter plate wells
were air-dried and the crystal violet present in the wells were fixed by
adding 200 μL of acetic acid (33 %). The OD was read at 620 nm via a
microtiter plate reader. Proficient biofilm forming isolates were sub­
jected to identification by 16S rRNA sequencing and were employed as
the effective microbial consortia for the plastic degradation.
2.4. Biofilm mediated degradation of plastics
The selected potential biofilm forming microbial strains were con­
structed as a microbial consortium and employed for the biofilm medi­
ated degradation of plastics. Biodegradation experiment was performed
individually for both untreated sterile PE and PC samples and enzy­
matically treated PE and PC samples in Erlenmeyer flasks (250 mL)
containing 100 mL liquid medium (mentioned in Section 2.3). Overnight
culture of microbial consortium grown in nutrient broth was washed
twice with saline water to remove residual medium then the cells were
collected by centrifugation at 8000 rpm for 5 min. Further, the microbial
cells were resuspended in saline water to maintain the absorbance of
approximately 1.0 OD at 600 nm for inoculation. An uninoculated flask
containing growth medium was used as control. Flasks were incubated
at 30 ◦ C with an agitation of 100 rpm. At the end of 30 days of incu­
bation, plastic samples were removed, washed and dried for further
analysis. All experiments were performed independently three times
(Chauhan et al., 2018).
2.5. Biochemical analyses during biofilm mediated degradation of plastics
Biofilm matrix is mainly composed of protein, exopolysaccharide
and lipid. The colorimetric method was used to determine the lipid
content, with cholesterol serving as the standard (Van Handel, 1985).
The total protein content was calculated by Bradford assay using bovine
serum albumin as the standard (Bradford, 1976) and the total carbo­
hydrate content was calculated using D-Glucose as the standard by
phenol sulphuric acid method (Liu et al., 1973).
NAD(PH)H: FMN oxidoreductases are commonly known to catalyze
the oxidation of long chain hydrocarbons (Jablonski and DeLuca, 1977).
Therefore, the production of such enzymes was monitored during
polymer degradation. For estimation of the oxidoreductase enzyme ac­
tivity, 50 mM Potassium Phosphate Buffer was prepared in deionized
water with 7.0 pH adjustment, 1.0 mM Flavin Mononucleotide Solution
(FMN) and β-Nicotinamide Adenine Dinucleotide Phosphate 2.0 mM,
Reduced Form were prepared and mixed well via inversion. Cell free
supernatant (crude enzyme solution) 0.10 mL, was added to the reaction
mixture and the decrease in optical density at 340 nm was recorded for
5 min by spectrophotometer (Cary 60 UV–Vis Spectrophotometer Agi­
lent technologies, USA) (Suganthi et al., 2018).
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Science of the Total Environment 904 (2023) 166721
2.6. Study of biofilm architecture
2.6.1. Scanning electron microscopy (SEM) analysis
The morphological investigation of the biofilm development on the
polymeric surfaces were conducted using High resolution-scanning
electron microscopy (Thermoscientific Apreo S). The plastics were
carefully sized into 1 × 1 cm pieces with the biofilm attachment and
were completely dried under 50 ◦ C temperature in a hot air oven.
Chromium was spread on top of the plastic samples with the help of ion
sputter-coater, and then the platform was fixed inside vacuum chamber
with 5 kV voltage acceleration (Huang et al., 2021).
2.6.2. Confocal laser scanning microscopy (CLSM) analysis
Biofilm colonization on the polymeric surfaces were also studied
using confocal laser scanning microscopy (CLSM). Polymer films with
biofilm attachment were dried completely prior to staining. The samples
were then stained by the fluorescence dyes in dark condition for dura­
tion of 30 min: 3.34 μmol/L of SYTO-9 (ThermoFisher, USA), live cells
staining as green and 20 μmol/L propidium iodide (ThermoFisher, USA),
dead cell staining as red and, 0.125 mg/mL Concanavalin A (Alexa
Flour™ 488 conjugate) (ThermoFisher, USA), exopolysaccharide stain­
ing as blue. The plastic samples were placed on coverslips for CLSM
analysis. Two separate beams were imparted for SYTO-9 and propidium
iodide excitation at 488 nm and 559 nm wavelengths respectively. The
area of scanning was 630 μm × 630 μm. Z-stack image scan was obtained
from the surface layer to the bottom layer of the plastic for the estima­
tion of the biofilm thickness on the plastic surface. Fluorescence
imposed images were obtained and were merged by the Zen 2010
software (Tu et al., 2020).
2.7. Change in surface hydrophobicity: Water contact-angle (WCA)
measurement
The change in hydrophobicity of the polymer surfaces were deter­
mined using the water contact-angle measurement (Jiang et al., 2021).
After treatment, the plastic samples were washed adequately with 70 %
ethanol following the sterile water then allowed for air drying. PE and
PC samples films without any treatment were also prepared in the same
way for analysis. The dried polymer samples were pasted on the surface
of glass slides and were placed in the path of light of the sessile goni­
ometer. Two microliters of water was placed on the polymer sample via
a micro-level syringe, which was held by contact to the sample for 10 s
after which the image was photographed. The contact angles were ob­
tained from three different contact positions on each plastic sample.
Static contact-angle of the water drop on the plastic surface was fitted
using SAMAS software.
2.8. Instrumental analyses of plastic degradation
2.8.1. Fourier transform infra-red spectroscopy analysis
Changes in the functional groups of the plastic surface after enzy­
matic and biofilm mediated degradation was investigated using ATR FT-
IR (IRTracer 100, Shimadzu, US). Each of the biofilm attached plastic
films were dehydrated in increasing concentrations of ethanol for 5 min
and in 100 % solution of ethanol twice for 10 min for total dehydration
and removal of the attached biofilm. The samples were then completely
dried in hot air oven at a fixed temperature of 50 ◦ C. Untreated PE pieces
were analysed and observed for control. Dried plastic substrates were
analysed by FTIR having 4 cm− 1 resolution and a mid-IR range of
400–4000 cm− 1 with the rate of 32 scans for each spectrum (De Geyter
et al., 2008).
2.8.2. X-ray photoelectron spectroscopy analysis
The surface chemical composition of the plastics surface was quan­
tified using PHI 5000 VersaProbe III x-ray photoelectron spectrometer
(ULVAC-PHI, Inc. Japan) fitted with a monochromatic x-ray source.
A.S. Ray et al. Science of the Total Environment 904 (2023) 166721

(a) (b)

(c) (d)

(e) (f)

Fig. 1. FT-IR spectral analysis of (a) PE (control and enzymatically pretreated) (b) PC (control and enzymatically pretreated) and SEM Micrographs of (c) PE control
and (d) enzymatically pretreated PE (e) PC control and (f) enzymatically pretreated PC.

Survey scanning was obtained from zero to 1400 eV, after which the
atomic composition of the elements detected were evaluated from the
areas which were present under the peaks of the spectrum. Scans of high
resolution were then obtained across the peaks with correspondence to
C1s and O1s energies of binding via range of scanning of about 20 eV.
Peaks that were detected in the higher-resolution spectra were attached
with Gaussian-Lorentzian components with linear background subtrac­
tion (Stark and Matuana, 2007).
2.8.3. Gel permeation chromatography analysis
Variation in the molecular weight distribution in the plastics upon
biodegradation processes were determined using gel Permeation Chro­
matography. Ten milligrams of dried plastic samples were dissolved in
10 mL of tetrahydrofuran. The solution was filtered through a 0.22 μm
syringe filter prior to the injection. The analysis was carried out with
Turbo matrix - 40 series GPC equipment. An aliquot of 100 μl of sample
was injected and the column flow rate was maintained at 1.0 mL min− 1.
The calibration was made using the polystyrene standards (Sigma

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A.S. Ray et al.
Fig. 2. Water contact angle measurement of (a) untreated and (b-d) treated PE and (e) untreated and (f-h) treated PC at different treatments.
Aldrich). The sample measurement time was 30 min per sample (Miao
et al., 2020; Satti et al., 2017).
3. Results
3.1. Development of efficient microbial consortium for the biofilm
mediated treatment of plastics
Enriched soil microbial cultures showed thick biofilm formation on
PE and PC plastic surfaces. Thirty morphologically different microbial
colonies were isolated and screened for efficient biofilm forming strains.
Upon the quantitative assessment of biofilm development using crystal
violet staining, three isolates namely SC31, SC420, SC51 achieved
higher OD value of >0.200 and constructed as an efficient biofilm
forming consortia for the plastic degradation studies (Supplementary
Fig. S1(a)).
The three selected isolates were subjected to bacterial identification
by 16S rRNA sequencing. The nucleotide sequences obtained were
compared with reference sequences by using BLAST similarity search
and the closely related sequences were procured from Genbank. The
gene sequences were aligned- using CLUSTALW software. The isolates
were identified to be Enterobacter brevis, Citrobacter werkmanii and
Enterobacter hormachaei with similarity index of 99.06 %, 97.3 % and
98.6 % respectively. The phylogenetic tree was structured using
neighbour joining method (Supplementary Fig. S1(b-d)).
3.2. Laccase driven enzymatic pre-treatment of polyethylene and
polycarbonate plastics
A class of multi‑copper enzymes, known as laccase enzymes have
been widely reported to play an important role in biodegradation of
plastics including polyethylene and polystyrene (Mohanan et al., 2020).
A recent studies report the laccase mediated oxidative degradation of PE
(Zampolli et al., 2023) and UV-irradiated PE (Yao et al., 2022). In the
present study, laccase enzyme in purified form (fungal origin,
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Science of the Total Environment 904 (2023) 166721
Phanerochaete chrysosporium) was chosen for the pre-treatment study,
specifically to analyse its role in the oxidation of C–C backbone of
plastics. Samples were collected on a daily basis and were subjected to
ATR-FT-IR analysis which revealed gradual development of hydroxyl
stretching signifying increased hydrophilicity of the plastic surfaces.
Fig. 1(a and b) shows the ATR-FT-IR spectra of untreated and enzy­
matically pre-treated PE and PC respectively. The spectrum of untreated
PE has two large characteristic absorption peaks at ~2920 and ~2852
cm− 1 which indicates C–H asymmetric and C–H symmetric stretching
vibrations in -CH2- respectively. The spectrum also exhibited two
smaller absorption peaks at ~1466 and ~723 cm− 1 which can be
attributed to C–H deformation vibrations in -(CH2)n- and C–C rocking
vibrations in -(CH2)n- respectively. In the spectra of PC, three charac­
teristic peaks near ~1740, ~1280 and ~1010 cm− 1 corresponding to
C–– O, O-C-O and O-C-C stretches respectively were observed. The
enzymatic treatment of PE leads to the formation of shoulder peak near
~1260 cm− 1 due to C–H rocking vibrations of aldehydes, C–H bending
vibrations of ketones and O–H deformation vibrations and also showed
significant reduction in the peak intensities of characteristics bands of
PE (Fig. 1a). In spectrum of treated PC (Fig. 1b), reduction in peak in­
tensity was observed for ~1723 cm− 1 of C– – O stretching, ~1425 cm− 1
of C–H bending and peaks near ~1126 and ~1074 cm− 1 attribute to
C–O stretching. In addition, the broad band between ~3600 and 3350
cm− 1 attributing to O–H stretching vibrations appeared along the
treatment for both PE and PC plastics (De Geyter et al., 2008; Jung et al.,
2018; Zampolli et al., 2023). Overall, FT-IR spectral inferences signify
the oxidation of polymer surface due to the activity of the laccase
enzyme. Similarly, SEM analysis also revealed the development of pores
and abrasions on the polymer surfaces indicated enzyme-imposed
oxidation of the C–C backbone. From the above results, it can be
considered that the laccase mediated enzymatic treatment leads to the
formation of oxygen containing functional groups on the PE (Fig. 1(c, d))
and PC (Fig. 1(e, f)) surface. These results are in agreement with the
study where copper-binding laccase enzyme enacts a significant role in
the degradation of PE plastic with the potential of polymer hydrocarbon
A.S. Ray et al. Science of the Total Environment 904 (2023) 166721

(a) (b)

(c) (d)

(e) (f)

(g) (h)

Fig. 3. CLSM micrographs of biofilm and EPS composition on the surface of (a, c) biofilm only treated PE; (b, d) integrated enzyme-biofilm treated PE; (e, g) biofilm
only treated PC and (f, h) integrated enzyme-biofilm treated PC respectively.

backbone oxidation (Santo et al., 2013).
3.3. Comparison of efficiency of biofilm only treatment and integrated
enzyme-biofilm mediated treatment of polyethylene and polycarbonate
plastics
E. hormachaei was employed for the biofilm mediated treatment of PE
and PC plastics. Two different approaches viz. only biofilm mediated
treatment and integrated enzymatic and biofilm treatment were con­
ducted for the treatment of PE and PC plastics. Microbial activity, bio­
film formation and plastic degradation were evaluated and compared
between both the treatments.

The microbial consortium comprising E. brevis, C. werkmanii and

6
A.S. Ray et al.
(a)
(b)
(d)
Fig. 4. SEM Micrographs of (a) untreated and treated PE plastic subjected to biofilm only treatment and integrated enzyme-biofilm treatment before (b and c) and
after biofilm removal (d and e) respectively.
3.3.1. Total protein, lipid, exopolysaccharide and oxidoreductase enzyme
activity during biofilm only treatment and integrated enzyme-biofilm
mediated treatment of plastics
Profile of total protein, lipid, exopolysaccharide and also, total
oxidoreductase enzyme activity were investigated to monitor the mi­
crobial activity during the course of plastic degradation.
In case of PE, total protein concentration was observed to be 4.5 ±
1.103 μg/mL in only biofilm treated medium whereas concentration
increased to 5.46 ± 0.92 μg/mL in the integrated enzymatic and biofilm
treatment approach. Similarly, in case of PC, total protein concentration
was observed to be 4 ± 0.199 μg/mL in only biofilm treated medium
whereas it increased to 9.55 ± 1.126 μg/mL in case of integrated
enzymatic and biofilm treatment approach (Supplementary Fig. S2(a)
and S3(a)).
During treatment of PE, lipid concentration was observed to be 7.09
± 0.218 μg/mL in case of biofilm treatment alone. In case of integrated
enzymatic and biofilm treatment medium, concentration increased to
8.38 ± 0.577 μg/mL. During biofilm treatment of PC treatment, lipid
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Science of the Total Environment 904 (2023) 166721
(c)
(e)
concentration of 3.57 ± 0.495 was observed whereas in case of inte­
grated approach, 6.86 ± 0.286 μg/mL concentration was estimated
(Supplementary Fig. S2(b) and S3(b)).
Total EPS concentration, in case of PE, was observed to be 31.59 ±
0.092 μg/mL in only biofilm treated medium whereas it increased to
50.827 ± 2.24 μg/mL in case of integrated enzymatic and biofilm
treatment approach. Similarly, in case of PC, total EPS concentration
was observed to be 33.63 ± 0.16 μg/mL in only biofilm treated medium
whereas it increased to 37.47 ± 1.043 μg/mL in case of integrated
enzymatic and biofilm treatment approach (Supplementary Figs. S2(c)
and S3(c)).
Total oxidoreductase enzyme activity was observed to be higher with
0.220 ± 0.037 U/mL after 2 weeks of biofilm treatment, whereas 0.283
± 0.046 U/mL enzyme activity was recorded in integrated enzymatic
and biofilm treatment approach. Similarly in PC treatment maximum
enzyme activity of 0.330 ± 0.04 U/mL was estimated after 2 weeks of
biofilm treatment and, 0.504 ± 0.026 U/mL enzyme activity was
recorded in integrated enzymatic and biofilm treatment approach. The
A.S. Ray et al.
(a)
(b)
(d)
Fig. 5. SEM Micrographs of (a) untreated and treated PC plastic subjected to biofilm only treatment and integrated enzyme-biofilm treatment before (b and c) and
after biofilm removal (d and e) respectively.
increase in the enzyme activity during treatment of enzymatically
treated plastics signify the enhanced colonization of the microorganisms
on the plastics where the increased production of enzyme leads to
enhanced degradation of the plastics (Supplementary Figs. S2(d) and S3
(d)).
From all the results, the biofilm related structural components were
observed to be significantly increased in case of integrated enzymatic
biofilm treatment approach, which indicates enhanced microbial ac­
tivity and biofilm growth on the enzymatically pre-treated plastics as a
result of increased porosity on polymer surfaces due to the enzyme
induced plastic oxidation.
3.3.2. Change in hydrophobicity of biofilm only treated and integrated
enzyme-biofilm treated plastics through water contact angle measurement
Oxidation as the crucial step in the biodegradation of plastics which
transformed the hydrophobic plastic surface to hydrophilic surface,
which can be qualitatively confirmed by the changes in the contact
angles between water droplets and the plastic surface. The reduced
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Science of the Total Environment 904 (2023) 166721
(c)
(e)
contact angle between the water droplets and the plastic surface rep­
resents the surface tension of water decreasing due to more abundant
and stronger polar interactions resulted from oxygen insertion on the
plastic surface upon oxidation (Kim et al., 2020). Contact angle of >100◦
denotes hydrophobic surface, whereas below 90◦ denotes hydrophilic
surface (Zhang et al., 2018). Therefore, water contact angle measure­
ment was used as an indicator to investigate the change in the hydro­
phobicity of the PE and PC plastics during enzymatic and biofilm
treatment processes (Fig. 2). The enzyme treated plastics, biofilm
treated plastics and, integrated enzymatic and biofilm treated plastics of
PE and PC were analysed for water contact angle. Virgin plastic films
were analysed as control (Tu et al., 2020). The angle of untreated PE and
PC films were observed to be 107.4◦ and 120.1◦ respectively. After
enzymatic pre-treatment, the angle reduced to 103.3◦ (PE) and 102.2◦
(PC). In case biofilm treated plastics, the angle reduced to 99.4◦ (PE) and
95.7◦ (PC). Further reduction in angle was recorded in integrated
enzymatic and biofilm treatment of the plastics (76.3◦ for PE; 91.0◦ for
PC).
A.S. Ray et al.
(a)
Fig. 6. FT-IR spectra of (a) untreated and treated PE and (b) untreated and treated PC at different treatments.
Above WCA results reveal the gradual decrease in the contact angle
between water droplet and plastic surfaces which might be due to the
change in the hydrophobic nature of plastic surface by the action of
laccase enzyme oxidation and surface depolymerization due to the in­
fluence of the microbial biofilm. Substantial change in plastic surface
during the integrated application of both enzyme and biofilm mediated
treatment approach, suggesting the influential role of enzyme in the
surface oxidation and making it more susceptible towards the microbial
attack thereby the enhanced degradation of the plastics. Interestingly,
these results are in corroboration with the study on the biodegradation
of bisphenol A -polycarbonate plastic by Pseudoxanthomonas sp. strain
NyZ600 (Yue et al., 2021).
3.3.3. Measurement of biofilm formation on the biofilm only treated and
integrated enzyme-biofilm treated plastics through CLSM analysis
Visualization of biofilm architecture was performed with the help of
CLSM analysis. Biofilm colonized on the polymer surfaces were stained
with fluorescent dyes, SYTO-9 for live cells staining and concanavalin A
for exopolysaccharide staining. Samples from both the treatment setups
i.e., biofilm treatment alone and integrated enzymatic and biofilm
treatment were analysed. Multiple images were obtained by variation of
the area and angle, and Z-stack images (3D images) were generated with
the help of Zeiss VSM 3 software which was further used for the eval­
uation of the thickness of the biofilm. Thick lush green clusters/patches
of live cells were visible due to SYTO-9 fluorescence which proves the
massed colonization of organisms attached on the polymer surface.
Significant blue stretches of biofilm exopolysaccharide were visualized
by concanavalin A staining confirming the greater EPS deposition due to
enhanced microbial activity. The biofilm thickness of biofilm only
treated PE and PC was estimated to be 50 μm in both the plastics (Fig. 3
(a) and (e)), whereas the thickness of biofilm formed on the PE and PC
plastics undergone integrated enzyme- biofilm treatment approach was
estimated to be 60 μm and 80 μm, as shown in Fig. 3 (b) and (f)
respectively. Similarly, EPS aggregation was estimated to be <2 μm and
25 μm in case of biofilm only treated PE and PC respectively (Fig. 3 (c)
and (g)). In case of integrated enzyme- biofilm treated PE and PC, EPS
aggregation increased significantly to 12 μm and 50 μm as shown in
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Science of the Total Environment 904 (2023) 166721
(b)
Fig. 3 (d) and (h) respectively. Enhanced biofilm thickness and EPS
deposition on the pre-treated plastics conveys that the laccase mediated
surface oxidation increased hydrophilicity of the polymeric surface
thereby led to enhanced microbial colonization on the surface and
therefore again prove to play significant role in the enhanced degrada­
tion of the polymers.
3.3.4. Change in surface morphology of biofilm only treated and integrated
enzyme-biofilm treated plastics through SEM analysis
The adhesion of bacterial film to the polymer surface is one of the
most significant mechanisms leading to biodegradation. First, biofilm
attachment and microbial colonization on the plastic samples were
analysed by SEM analysis. After the treatment for 30 days, round ag­
gregations of microbial colonies on the surface of the plastics PE and PC
polymers were observed. Remarkably, the biofilm formation on the
enzymatically pre-treated plastics were denser and completely packed
when compared to the only biofilm treated PE and PC surfaces (Fig. 4 (b
and c) and Fig. 5 (b and c)).
PE and PC surfaces were again examined after the removal of biofilm
for which the microbial colonization on the plastic surfaces were
removed by rigorous ethanol washing (Huang et al., 2020). The plastics
were observed under high resolution SEM which revealed the formation
of pits and abrasions on the surface of both PE and PC plastics. SEM
micrographs of the untreated plastics were taken as control which did
not exhibit any changes on surface morphology and had smooth surface
upon comparison with treated plastics films. SEM micrographs of bio­
film treated plastic samples were compared to that of integrated enzy­
matic and biofilm treated plastic samples. Increased pore formation in
close vicinity were observed in integrated enzymatic and biofilm treated
PE and PC plastic films (Fig. 4 (d and e) and Fig. 5 (d and e) respectively.
The morphological changes on polymeric surfaces confirms that laccase
mediated enzymatic pre-treatment had the most important role in the
surface oxidation of plastics by enhancing the hydrophilicity of the
plastics which lead to the increased colonization of the plastics on the
plastic surfaces thereby enhanced the degradation of PE and PC.
A.S. Ray et al.
(a)
(c)
Fig. 7. XPS C1s spectra of (a) untreated PE, (b) biofilm only treated PE after 30 days, (c) Integrated enzyme-biofilm treated PE after 30 days.
3.3.5. Change in functional groups of the biofilm only treated and
integrated enzyme-biofilm treated plastics through FT-IR analysis
The FTIR analysis was performed to determine the extent of degra­
dation by the biofilm approach and the integrated approach based on
the alteration of the functional groups of the plastics (Fig. 6). The
characteristic peaks of the untreated PE and PC films were obtained and
the corresponding spectra was compared to that of the treated plastic
films to locate the change in the functional groups for the determination
of plastic degradation (Jung et al., 2018; Kim et al., 2020). The in­
tensities of the characteristic peaks at 2917 cm− 1 and 2847 cm− 1 assign
to C–H asymmetric and symmetric stretching from CH2 were found to
be reduced in the spectrum of PE subjected to integrated treatment than
the biofilm treatment. In addition, intensities of the peaks at 1466 cm− 1
and 721 cm− 1 which attributed to C–H deformation vibrations in
–(CH2)n – and C–C rocking vibrations in –(CH2)n – respectively, were
also found to be reduced significantly, in the spectrum of PE treated
using integrated approach when compared to the biofilm only treat­
ment. Similarly, in case of PC, notable decrease in the intensities of
characteristics peaks at 2926,1721, 1428, 1264, and 1126 and 621 cm− 1
attributing to –(CH2)n, C– – O stretching, C–– C stretching, C–O stretch­
ing and aromatic CH bend respectively, were observed for the integrated
enzyme-biofilm treatment, whereas reduction in the peak intensities
was comparatively less in the biofilm only treatment. These results
corroborate the enhanced depolymerization of the plastic surface due to
laccase mediated surface oxidation.
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Science of the Total Environment 904 (2023) 166721
(b)
3.3.6. Change in surface chemical composition of the biofilm only treated
and integrated enzyme-biofilm treated plastics through XPS analysis
Surface chemical composition analysis of the control and treated
plastic samples was performed by XPS spectral analysis. The high res­
olution C1s spectrum for the untreated and treated PE and PC were
deconvoluted to identify the atomic surface concentrations of different
carbon oxidation states (Bonyadinejad et al., 2022). As shown in the
Fig. 7a, a dominant peak at the binding energy of around 284 eV asso­
ciated with -C-C- related to the structural backbone of untreated PE was
detected. Although, the peak at the binding energy of ~284 eV is still
dominant for PE subjected to biofilm only treatment, the new peak at
~286 eV corresponding to -C-O- was detected indicating the oxidation
of PE (Fig. 7b). Whereas, in the spectrum of PE subjected to integrated
enzyme-biofilm treatment (Fig. 7c), an additional peak at ~288 related
to -C=O- along with a decline in -C-C- peak was found suggesting the
enhanced surface oxidation of PE. Accordingly, ratio of oxygenated
carbon to the carbon unoxygenated (Coxygenated/Cunoxygenated) was found
to higher for PE subjected to integrated treatment (0.414) than the
biofilm only treatment (0.1213).
In case PC, C1s spectrum of untreated showed dominant peak at
~284 (-C-C-), and peaks at ~286 (-C-O-) and ~288 (-C=O-) eV with
lower abundance (Fig. 8a). In the biofilm treated PC (Fig. 8b), there was
an increase in the surface concentration of -C-O- and -C=O- functional
groups with a decline in the concentration of -C-C- group, indicating the
surface oxidation of PC. While in the C1s spectrum of PC subjected to
A.S. Ray et al. Science of the Total Environment 904 (2023) 166721

(a) (b)

(c)

Fig. 8. XPS C1s spectra of (a) untreated PC, (b) biofilm only treated PC after 30 days, (c) Integrated enzyme-biofilm treated PC surface after 30 days.

due to enzymatic pre-treatment. Moreover, increased oxygen percentage

Table 1 indicates the enhanced incorporation of oxygen in the backbone of
Molecular weight analysis of polyethylene and polycarbonate under different polymers due to the action of laccase enzyme and further depolymer­
treatments.
ization of the plastic due to the enhanced microbial activity.
Treatment Polyethylene (PE) Polycarbonate (PC)

Mw Mn Mz Mw Mn Mz 3.3.7. Change in molecular weight of the biofilm only treated and integrated
enzyme-biofilm treated plastics through gel permeation chromatography
Untreated 1774 984 3273 267,341 6095 558,164
Biofilm only treatment 1703 958 3147 235,007 3352 529,756 GPC analysis was performed at the end of the 30 days for the
Integrated enzyme- 1604 941 2844 228,398 3005 487,993 confirmation of plastic biodegradation and depolymerization by the
biofilm treatment biofilm only and integrated enzyme-biofilm treatment processes. Weight
# Mw: weight average molecular weight; Mn: number average molecular weight; average molecular weight (Mw), number average molecular weight
Mz: size average molecular weight. (Mn), and size average molecular weight (Mz) of untreated and treated
plastics were compared and presented in Table 1. The lowest reported
integrated approach (Fig. 8c), significant increase in the peak area was molecular weight of PE is 198 Da and the highest is 150,000 Da (McLain,
observed for -C-O- and -C=O- functional groups in addition to the new 2007) and the molecular weight of PC varies from 15,000 to 300,000 Da
peak formation at ~289 corresponding to -O-C=O- functional group (Tanaka et al., 2014). Weight average molecular weight (Mw) of both the
along with a greater decline in the area of -C-C- peak. Again, the Coxy­ untreated PE and PC used in the present study were 1774 Da and
267,341 Da respectively. Mw, Mn and Mz of the treated PE and PC were
genated/Cunoxygenated ratio was found to higher for the integrated
approach (0.873) than the biofilm only treatment (0.248). found to be reduced noticeably in comparison to untreated PE and PC.
Therefore, the reduction in the surface carbon percentage signifies Biofilm only treatment reduced the molecular weight of PE from 1774 to
that the microorganisms were able to utilize the PE and PC as carbon 1703 Da and PC from 267,341 to 235,007 Da. In the case of integrated
source in the carbon deprived environment, whereas the increase in the enzyme-biofilm treatment, further decline in the molecular weights of
surface oxygen percentage denotes the surface oxidation of polymers the both plastics were achieved in which Mw of PE reduced to 1604 Da
during the course of degradation. Greater reduction of carbon in the and PC reduced to 228,398 Da. Similarly, Mn and Mz of both PE and PC
integrated enzymatic and biofilm treated samples indicates the were reduced significantly by integrated enzyme-biofilm treatment
improved microbial activity on the polymer surface which might be due when compared to biofilm only treatment. Hence, these results clearly
to the changes in porosity and hydrophilicity of the polymeric surfaces demonstrated that the laccase driven surface oxidation as the pre-

11
A.S. Ray et al. Science of the Total Environment 904 (2023) 166721

Fig. 9. Hypothetical mechanism behind the biofilm mediated degradation of enzymatically pre-treated and untreated polyethylene and polycarbonate plastics.

treatment strategy accelerated the biofilm mediated depolymerization
and biodegradation of the plastics than biofilm treatment alone.
Based on the above results, the hypothetical mechanism behind the
biodegradation of PE and PC plastics via integrated enzymatic and
biofilm mediated treatment and only biofilm mediated treatment
approached is schematically represented in the Fig. 9.
4. Conclusion
The present study offered valuable insights on the application of
laccase enzyme from Phanerochaete chrysosporium to oxidize the surface
of the polymeric substrates thereby improves the surface hydrophilicity
of the PE and PC plastics. The enzymatic treatment increased the
accessibility of plastic surface to the biofilm forming microbes and
leading to a successful assimilation of plastics as a sole carbon and en­
ergy source. The current research also corroborates with the earlier
studies, suggesting that the surface oxidation treatment establishes the
polymeric surface hydrophilicity which fosters enhanced microbial
attachment, subsequently hastening the process of biodegradation.
Moreover, laccase is extensively studied for its wide-ranging industrial
and environmental applications with its well-established commercial
production, which would facilitate its application in large scale treat­
ment processes. Hence, it could be suggested that laccase mediated
surface oxidation must be integrated as a pre-treatment approach to
develop a sustainable biofilm-based bioremediation technology for the
effective management of plastic solid wastes. Future researches should
be focussed on harnessing its catalytic efficiency and stability via various
protein engineering strategies to develop a successful laccase mediated
pre-treatment strategy to achieve accelerated biodegradation of plastics.
CRediT authorship contribution statement
Anindhya Shankar Ray: Writing – original draft, Investigation,
Formal analysis, Data curation, Visualization, R. Muneeswari:
Conceptualization, Investigation, Formal analysis, Data curation, Visu­
alization, Writing – original draft, Writing – review & editing. Maseed
Uddin: Formal analysis, Methods development, Training, Data curation.
K. Ramani: Conceptualization, Methods development, Training,
Writing – review & editing, Investigation, Data Curation, Project
administration, Funding acquisition, Supervision.
Declaration of competing interest
All the authors declare that there is no conflict of interest.
Data availability
The data that has been used is confidential.
Acknowledgement
The authors are highly thankful to the Department of Science and
Technology, Ministry of Science and Technology, New Delhi, for funding
the project under the “ Scheme for Young Scientist and Technologists
(SYST-SEED)” Project No: SP/YO/2019/1360(G). Authors are sincerely
acknowledging the Dept. of Biotechnology, School of Bioengineering,

12
A.S. Ray et al. Science of the Total Environment 904 (2023) 166721

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