Science of the Total Environment 704 (2020) 135931

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Science of the Total Environment

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Biodegradation of polyethylene microplastic particles by the fungus

Aspergillus flavus from the guts of wax moth Galleria mellonella
Junqing Zhang a,b,1, Danling Gao c,1, Quanhao Li b, Yixuan Zhao d, Li Li d, Hanfeng Lin d,
Qirui Bi e,⁎, Yucheng Zhao a,⁎
a
Department of Resources Science of Traditional Chinese Medicines and State Key Laboratory of Natural Medicines, School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nan-
jing, Jiangsu, PR China
b
School of Pharmacy, China Pharmaceutical University, Nanjing, Jiangsu, PR China
c
School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing, Jiangsu, PR China
d
School of Life Science and Technology, China Pharmaceutical University, Nanjing, Jiangsu, PR China
e
Department of TCMs Pharmaceuticals, School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing 211198, PR China

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

• Aspergillus flavus was isolated from the

guts of wax moth Galleria mellonella.
• The HT-GPC and FTIR results showed
the biodegradation of polyethylene
microplastics.
• Two laccase-like multicopper oxidases
(LMCOs) displayed up-regulated trends.
• The results highlight the potential of
LMCOs as tools for microplastic
remediation.

a r t i c l e i n f o a b s t r a c t

Article history: Polyethylene (PE) products are widely used in daily life, agriculture, and industry because of their convenience
Received 26 September 2019 and economic value. However, PE is one of the polymer materials remarkably resistant to degradation. Current
Received in revised form 20 November 2019 methods of plastic waste disposal pose a threat to the environment and produce microplastic particles (MPP),
Accepted 2 December 2019
which becomes a global environmental concern because of its accumulation. In this study, a PE-degrading fungus
Available online xxxx
Aspergillus flavus named PEDX3, was isolated from the gut contents of wax moth Galleria mellonella. The results
Editor: Frederic Coulon indicated that high-density polyethylene (HDPE) MPP was degraded into the MPP with a lower molecular weight
by strain PEDX3 after 28 days incubation. In addition, Fourier Transform - Infrared Spectroscopy (FT-IR) results
Keywords: showed the appearance of carbonyl groups and ether groups of MPP, which also validated the degradation of
Microplastic particles PE. Furthermore, the potential degradation enzymes were investigated by Reverse Transcription-Polymerase
Biodegradation Chain Reaction (RT-PCR). Finally, two laccase-like multicopper oxidases (LMCOs) genes, AFLA_006190 and
Aspergillus flavus AFLA_053930, displayed up-regulated expression during the degradation process, which may be the candidate
Galleria mellonella PE-degrading enzymes. These results have demonstrated that the A. flavus strain PEDX3 has an ability to degrade
Polyethylene degrading enzyme

⁎ Corresponding authors at: Department of Resources Science of Traditional Chinese Medicines and State Key Laboratory of Natural Medicines, School of Traditional Chinese Pharmacy,
China Pharmaceutical University, Nanjing, Jiangsu, PR China.
E-mail addresses: biqirui@cpu.edu.cn (Q. Bi), zhaoyucheng1986@126.com (Y. Zhao).
1
These authors have contributed equally to this paper.

https://doi.org/10.1016/j.scitotenv.2019.135931
0048-9697/© 2019 Elsevier B.V. All rights reserved.
2 J. Zhang et al. / Science of the Total Environment 704 (2020) 135931
microplastic particles and the two PE-degrading enzymes provide a promising application for the PE MPP
remediation.
1. Introduction
Plastic products are frequently used in daily life, agricultural field,
and packaging industry owing to the advantages of low cost, high
strength, and durability (Moharir and Kumar, 2019; Rajmohan et al.,
2019). The consumption of plastic products increases year by year and
the global production of plastic is about 150 million tons annually
(Verma et al., 2016). However, due to the slow degrading speed under
natural condition, the plastic wastes pose a serious threat to the envi-
ronment. Microplastic particles (MPP), are defined as plastic particles
with a diameter b 5 mm by NOAA (the National Oceanic and Atmo-
spheric Administration). Due to its small size, MPP can be easily
ingested by organisms (like fishes and birds) and accumulate in their
bodies (Andrady, 2011; Sharma and Chatterjee, 2017). Furthermore,
MPP has been found in drinking water, which may become a threat to
public health (Koelmans et al., 2019; Novotna et al., 2019). Polyethylene
(PE) is a widely used material as its ductility, portability and stable
chemical properties. However, as a result of its saturated linear hydro-
carbon chains, which can be expressed as -[CH2-CH2]n-, PE products
are difficult to be degraded by naturally (Kumar Sen and Raut, 2015;
Restrepo-Flórez et al., 2014).
The current methods for plastic wastes disposal include burning,
dumping into the ocean and burying in the landfill, all of which may
cause secondary pollution (Restrepo-Flórez et al., 2014). Thus, develop-
ing new biodegradable plastics or biodegradation methods are neces-
sary (Silva et al., 2018; Tang and Chen, 2019). Biodegradation is the
process during which organic polymer materials are decomposed into
smaller compounds, such as CO2 and H2O (Lucas et al., 2008; Shah
et al., 2008). Recently, several PE-degrading fungi have been reported,
such as Aspergillus, Acremonium, Fusarium, Penicillium, Phanerochaete
(Restrepo-Flórez et al., 2014). The degradation ability of A. flavus culture
to biodegradable PE plastic bags has been reported by El-Shafei et al. (El-
Shafei et al., 1998). Idowu et al. reported that evidences of LDPE biodeg-
radation by two fungal species (A. flavus MCP5 and A. flavus MMP10),
which revealed A. flavus can use LDPE as both nitrogen and carbon
source in the absence of additives (Idowu et al., 2019). Most of PE-
degrading microorganism strains were isolated from soil, buried PE
and seawater samples (Gajendiran, 2016; Harshvardhan and Jha,
2013; Mathur, 2011; Yoshida et al., 2016). Pramila et al. isolated an
A. flavus strain with the biodegradation activity of LDPE films from land-
fill soils (Pramila and Vijaya Ramesh, 2011). What's more, a LDPE
degrading potential Aspergillus species (A. flavus) isolated from munici-
pal landfill sites of Agra has been reported, which made 30.6% of weight
loss of LDPE film after 9 months degradation in soil (Nitesh and
Sharmita, 2019).
Some insects are able to chew and eat plastic packages or the bees-
wax, using them as the sole carbon source, and providing powerful
bio-resources for PE biodegradation (Kannan et al., 2019). Yang et al. re-
ported two PE-degrading bacterial strains isolated from the larvae of
Plodia interpunctella (Indian meal moths), Bacillus sp. YP1 and Entero-
bacter asburiae YT1 (Yang et al., 2014). Similarly, Bombelli et al. found
the fast mass loss of PE plastic bags caused by ~100 larvae of the wax
moth G. mellonella for 12 h (Bombelli et al., 2017). Subsequently, Ren
et al. isolated an Enterobacter sp. strain D1 with the ability of PE films
degradation from the guts of G. mellonella (Ren et al., 2019). These re-
sults suggested that guts of insects may be the potential resources for
selecting PE-degrading microorganisms.
Microbial enzymes play a crucial role in biodegradation process of PE
via microorganisms, especially fungi, which can promote the oxidation
or hydrolysis of PE by producing extracellular enzymes (Kumar et al.,
© 2019 Elsevier B.V. All rights reserved.
2013; Kumar Sen and Raut, 2015). According to Lucas et al., microorgan-
isms are involved in the depolymerization, assimilation and mineraliza-
tion processes (Lucas et al., 2008). For most microorganisms, however,
its hydrophilic surfaces make the initial colonization on PE surface really
difficult (Gilan et al., 2004). Microbial enzymes can promote the micro-
organism attachment to PE surface by improving the hydrophilicity of
PE (Tribedi and Sil, 2013). In recent years, several microbial enzymes ca-
pable of degrading PE have been reported to be involved in the PE bio-
degradation, including laccases, manganese peroxidase and lignin
peroxidases (Wei and Zimmermann, 2017). These enzymes take partic-
ipant in degradation process, including terminal oxidation, cleavage of
chains and fatty acid metabolization (Albertsson et al., 1987), although
the specific mechanism of PE-degrading enzymes has not been
reported.
The purposes of the present study were to isolate PE-degrading mi-
croorganisms from the enrichment of G. mellonella gut contents, and
identify their potential PE-degrading enzymes. As a result, a fungal
strain capable of degrading PE microplastic particles, A. flavus strain
PEDX3, was isolated. The PE-degrading activity was evaluated based
on the changes in mass loss, molecular weight, and chemical structure
(appearance of carbonyl groups) in a limited incubation period
(28 days). In addition, laccases and laccase-like multicopper oxidases
(LMCOs) were screened for potential PE-degrading enzymes. Finally,
we retrieved two potential PE-degrading enzymes, which would be
used to explore the mechanism of PE degradation by microbial in the fu-
ture and laid the foundations for screening strains with high laccase
content and activity.
2. Materials and methods
2.1. PE microplastic particles
Low-density polyethylene (LDPE) with density of 0.921 g·cm−3
(N210, China Petrochemical Corporation, Shanghai) and high-density
polyethylene (HDPE) with density of 0.955 g·cm−3 (5502LW, China Pet-
rochemical Corporation, Maoming) were used. All the particles were
sieved through 120 mesh (125 μm) nylon sieve three times to prepare
MPP with a size below 200 μm. MPP was irradiated under UV light for
5 h as a sterilization treatment (more details in supporting information).
2.2. Medium and culture conditions
Medium A and medium B were used as acclimatization media. Sole
carbon source (SCS) medium was prepared according to ASTM standard
(ASTM G22–76), for the enrichment of PE-degrading microorganisms
and characterization of the degradation of PE MPP (ASTM, 1996). SCS
agar plates were prepared by adding 20 g of agar to 1000 mL of SCS me-
dium. Fungal cultures of the PE-degrading strains were maintained on
potato dextrose broth (PDB) medium (Solarbio), potato dextrose agar
(PDA) medium, and non‑carbon system with glucose added (NCS-Glu)
medium. More details about the media (medium A, medium B, SCS me-
dium and NCS-Glu medium) are provided in supporting information.
Liquid medium culture experiments were performed in 250 mL flasks,
each containing 100 mL medium, all the flasks were shake-cultured
(150 rpm) at 28 °C.
2.3. Preparation of G. mellonella guts contents
100 wax moth G. mellonella larvae (provided by Huiyude Biotech-
nology co. LTD, Tianjin, China) with same size were selected, and fed
 J. Zhang et al. / Science of the Total Environment 704 (2020) 135931
on PE films (sterilized with 75% alcohol) as a single carbon source for
2 weeks. All larvae were raised in a laboratory (room temperature at
24 ± 3 °C). All the G. mellonella larvae were soaked in 75% alcohol (v/
v) for 30 s to remove the microorganism on the surface and then
cleaned with sterilized water. Sterile dissection was carried out to ob-
tain the gut contents of the larvae, which was ground with a sterile mor-
tar. A series of gut content solutions with the concentration ranging
from 10−2 to 10−4 (v/v) were obtained by diluting with sterile water,
and then stored at −20 °C.
2.4. Isolation of the PE-degrading strains
Gut contents solution with three different concentrations (10−2,
10−3 and 10−4) were inoculated in 100 mL of medium A and medium
B for acclimatization, and each concentration was done in triplicates.
After 30 days incubation, the cultures were spread across PDA plates.
Then, the colonies were picked out and transferred to fresh PDA plates
repeatedly until pure colonies of isolates could be obtained.
2.5. Screening of the PE-degrading strains
In order to screen the PE-degrading strains for PE-degrading ability
in both LDPE and HDPE, the isolates were cultured in SCS-LDPE medium
and SCS-HDPE medium for 30 days. Their biodegradation ability was
preliminarily determined by the growth of microorganisms and the hy-
drophilicity of PE particles. To examine the growth of the microorgan-
ism and the hydrophilicity of PE particles, the turbidity of the culture
and the number of suspending PE particles were observed and scored
according to the following rules: ‘-’ represents the clear and transparent
culture, with non-suspended PE particles; ‘+’ to ‘++++’ represent
slightly turbid to extremely turbid medium combined with virtually
non-existent to voluminous PE particles suspended in the medium
(He et al., 2016; Luo, 2013). SCS-LDPE and SCS-HDPE plates were used
to verify whether strains could degrade LDPE and HDPE powers.
2.6. Phylogenetic analysis of PE-degrading strains
For phylogenetic analyses, total genomic DNA needed for calmodu-
lin (CaM) gene (Samson et al., 2014) amplification was extracted from
fresh hyphae after 4 days incubation on PDA plates by D3390 Fungal
DNA mini kit (E.Z.N.A Fungal DNA kit, D3390-02, Omega Bio-Tek). The
primers cmd5 (5′-CCGAGTACAAGGAGGCCTTC-3′) and cmd6 (5′-CCGA
TAGAGGTCATAACGTGG-3′) were used for gene amplification. The ho-
mologous sequences were retrieved from GenBank using the Blast data-
base on the NCBI website (http://www.ncbi.nlm.nih.gov/BLAST/), and
the phylogenetic tree was constructed according to the Neighbor-
Joining method by MEGA 7.0 software (Molecular Evolutionary Genet-
ics Analysis, USA).
2.7. Biodegradation assays
As its higher molecular weight and density, HDPE is much more dif-
ficult to be utilized as carbon source than LDPE in microorganisms
(Orhan et al., 2004; Restrepo-Flórez et al., 2014). Thus, HDPE MPP was
used to evaluate the biodegradation ability of the strains. The HDPE
MPP biodegradability was characterized by its weight loss, molecular
weight shift and the surface smoothness change of particles. Spores sus-
pensions (5 mL) were added to 100 mL of SCS-HDPE medium to make a
final concentration of approximate 108 cells per mL, with a blank flask as
sterile control. The surface of HDPE MPP was observed by optical mi-
croscopy after incubation, compared with the control group.
After incubation, residual HDPE MPP was collected, and incubated
with 2% (v/v) sodium dodecyl sulfate (SDS) overnight to remove the
biofilms (Sivan et al., 2006), then ultrasonicated for 30 min to separate
the powder from the hyphae, centrifuged at 12000 rpm for 10 min and
collected the suspended powder sample, washed with distilled water.
3
Finally, the washed particles samples were dried at 85 °C for the mea-
surement of residual weight.
The mass loss percentage (△m/m0) was calculated by the formula:
△m/m0 = (mbefore incubation-mafter incubation)/mbefore incubation × 100%.
The molecular weight (weight-average molecular weight (Mw) and
number-average molecular weight (Mn)) of the washed PE residues
were measured by high-temperature gel permeation chromatography
(HT-GPC) (PL220, Agilent, USA) according to the procedure reported
by Yamada et al. (Yamada-Onodera et al., 2001). The washed and
dried HDPE MPP was tested by FT-IR (Tensor 27, Bruker, Karlsruhe,
Germany) for characterizing the functional groups. The details of the
HT-GPC and FT-IR procedures are provided in supporting information.
2.8. Enzymes activity test
ABTS (2–2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)) was
used to measure the enzyme activity timely changes of laccases and
LMCOs in SCS-HDPE medium (Johannes and Majcherczyk, 2000; Reiss
et al., 2013). The enzyme activity was tested after incubation of 7, 14,
21 and 28 days by the procedure reported by Courty et al. (Courty
et al., 2006), and triplicates for each sample. The enzyme activity unit
(U) is defined as the amount of the enzyme required to change absor-
bance (415 nm) of 0.001 per minute at 30 °C. The formula was: U =
106 × ΔOD 415/(V × Δt), and V was the volume of culture liquid (mL).
2.9. Total RNA extraction and RT-PCR
Fresh mycelium from two control groups (NCS-Glu medium and
PDB medium) and treated group (SCS-HDPE medium) were collected
after a 28-day incubation. Total RNA was extracted by using the Spin
Column Fungal Total RNA Purification Kit (Sangon Biotech, Shanghai,
China) and the cDNA was synthesized by HiScript® Q-RT SuperMix for
qPCR (+gDNA wiper) (Vazyme Biotech Co., Ltd., Nanjing, China) imme-
diately after RNA sample processed, which were performed using a
ChamQTM SYBR® qPCR Master Mix (Vazyme, China) with a total reac-
tion volume of 20 μl.
To test the regulate effect of PE on laccases and LMCOs gene expres-
sion at mRNA level, nine candidate genes (Table S1.) were retrieved
from the genome sequence of A. flavus NRRL3357 (no.
GCA_000006275.2). Actin was chosen as the internal reference gene
(Bohle et al., 2007), and the 2-ΔΔt method was used to calculate the can-
didate genes transcription levels (Schmittgen and Livak, 2008).
2.10. Statistical analysis
In this study, all assays were performed in triplicate and data was
presented as the mean ± standard deviation (x ± SD). All statistical
analyses were performed with t-test using GraphPad Prism version 7.0
(GraphPad Software, San Diego, California). p b .05 was considered to
be significant in the analyses.
3. Results and discussion
3.1. Isolation and screening of PE-degrading microorganisms
Twenty-five isolated strains were isolated during the enrichment of
the gut contents of the G. mellonella when using HDPE as the sole carbon
source. Studies has demonstrated that strong hydrophilicity of PE indi-
cates the efficiency of microorganisms for utilizing non-soluble sub-
strates, which was used to characterize the PE-degrading strains (Ren
et al., 2019; Sangeetha Devi et al., 2015; Yang et al., 2014). Thus, to
screen strains with potential PE (including both LDPE and HDPE) bio-
degradation activity, we characterized the growth of the microorganism
and hydrophilicity of PE particles in SCS-LDPE or SCS-HDPE medium by
observing the turbidity of the culture medium and the number of
suspending microplastic particles. Among the twenty-five isolates, a
4 J. Zhang et al. / Science of the Total Environment 704 (2020) 135931
fungal strain named PEDX3 was selected as the potential PE-degraders
as its better growth than other strains in both SCS-LDPE and SCS-
HDPE medium (Table S2). To further verify both HDPE and LDPE degra-
dation ability of strain PEDX3, SCS-LDPE and SCS-HDPE plates were
used. After 14 days, some colonies could be found on plates (Fig. 1.A-B).
3.2. Morphological and phylogenetic identification of PE degrading strain
Based on the characters that rapidly growing, yellow-green, radially
furrowed colony with villous aerial hyphae (Fig. 1.C), and radiating co-
nidial heads with the rough conidiophores under the microscope (Fig. 1.
D), strain PEDX3 was preliminarily identified as a mold (Frisvad et al.,
2019; Samson et al., 2014). Strain PEDX3 was further identified by Cal-
modulin (CaM) gene sequence analysis. Twenty-one CaM gene se-
quences were retrieved from GenBank, including four A. flavus
sequences, sixteen Aspergillus sequences, and the sequence of our iso-
late strain PEDX3 (accession no. MN271387). Finally, strain PEDX3
was identified as A. flavus as the 100% evolutionary homologous with
A. flavus by the Neighbor-Joining method (Fig. 2) and was named as
A. flavus strain PEDX3. The existence of A. flavus or even molds in the in-
testinal flora seems inconceivable, but it was reasonable in insect.
Gilliam et al. have reported that molds were the most numerous micro-
organisms isolated from the frass of G. mellonella larvae, such as
P. purpurogenum, A. flavus group, A. niger, and etc. (Gilliam et al.,
1989). Additionally, as its susceptibility, the G. mellonella larvae was rec-
ognized as the ideal host of the fungus A. flavus (Akhtar, 2014; Scully
and Bidochka, 2009). Caterpillars and other insects always have a
Fig. 1. Morphological features of strain PEDX3. Growth of strain PEDX3 on the SCS-HDPE plate (A) and the SCS-HDPE plate (B), with colonies indicated by red arrows. (C) The colony
morphology of strain PEDX3 after 4 days of incubation on the PDA plate. (D) Conidiophore of strain PEDX3 (from C). (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.)
dynamic but not a resident gut microbiome as the results of food or en-
vironment, which may explain the existence of A. flavus (Hammer et al.,
2017).
3.3. Biodegradation effect of strain PEDX3 on HDPE MPP
In order to assess the potential assimilation of microplastic particles
by strain PEDX3, a sample of HDPE MPP was subjected to visual analysis.
As depicted in Fig. 3, the fungus was found to adhere to the surface of
HDPE particles, which may cause the size changes of the particles. Com-
pared with the smooth surface of untreated MPP (Fig. 3.A), the irregular
surface appeared after a 28-day incubation (Fig. 3.B), which indicated
erosion at the surface of the pellets and corroborated previous reports
describing the growth of fungus at the surface of PE (Paço et al., 2017;
Park and Kim, 2019). However, to further observe the presence of bio-
logical material at the pellets' surface, scanning electron microscope
(SEM) results could provide more information (Paço et al., 2017;
Sangeetha Devi et al., 2015).
The weight loss is an important monitor index for biodegradation
of polymer, which includes mass loss and molecular weights loss
(Mw loss and Mn loss). After exposed to strain PEDX3 for 28 days,
the mass loss percentage (Δm/m0) was 3.9025 ± 1.18% (Fig. 4.A),
which was much higher than that elicited by Kocuria palustris M16
(Δm/m 0 = 1 ± 0.033%) over a 30-day incubation reported by
Harshvardhan et al. (Harshvardhan and Jha, 2013). Moreover, strain
PEDX3 performed much better in molecular weight change com-
pared with previously reported work (Kawai et al., 2002; Yang
 J. Zhang et al. / Science of the Total Environment 704 (2020) 135931 5

Fig. 2. The phylogenetic dendrogram of strain PEDX3 showing the relationship between the Calmodulin (CaM) gene sequences retrieved from GenBank. The evolutionary history was
inferred using the Neighbor-Joining method. The optimal tree with the sum of branch length = 3.10615181 is shown. The percentage of replicate trees in which the associated taxa
clustered together in the bootstrap test (10,000 replicates) are shown next to the branches. The evolutionary distances were computed using the p-distance method and are in the
units of the number of base differences per site.

et al., 2015). Compared with the molecular weight of the control
sample (Mw = 222,003 Da and Mn = 55,135 Da), the Mw
(89,801 Da) and Mn (26,064 Da) of the strain PEDX3 treated sample
dropped significantly by 132,202 Da and 29,069 Da respectively
(Fig. 4.B). From the Mw distribution curve (Fig. 4.C), compared to un-
treated samples, more HDPE MPP was converted to smaller molecu-
lar weight, and more concentrated after exposed to strain PEDX3,
which suggested that the depolymerization of HDPE long chain
structure occurred and that lower molecular weight fragments
were formed in the presence of strain PEDX3.
3.4. Changes in chemical structure of HDPE MPP
After incubating with strain PEDX3 in SCS-HDPE medium for
30 days, HDPE MPP showed some changes by FT-IR analysis of the mi-
crobial treated samples of HDPE MPP showed some changes in the spec-
trum compared with the control samples (without exposure to strain
PEDX3). Obviously, the wide absorption peak appearing at the range
from 3500 to 3100 cm−1 was corresponded to hydroxyl groups (-OH)
(Fig. 5.A). Spectrum also showed an absorption peak at 1113 cm−1
and 1647–1716 cm−1 corresponding to ether groups (-C-O-C-) and

Fig. 3. Visual analysis of the surface of HDPE MPP by optical microscopy. (A) Untreated HDPE MPP with the smooth and regular surface. (B) HDPE MPP exposed to strain PEDX3 after a 28-
day incubation, with adherent hyphae and the irregular surface.
6 J. Zhang et al. / Science of the Total Environment 704 (2020) 135931

Fig. 4. The weight loss and Mw distribution curve of HDPE MPP after 28 days of incubation with strain PEDX3. (A) The mass loss percentage of HDPE MPP was significantly increased
(P b .05) after incubation. (B) Molecular weight (Mn and Mw) changes of HDPE MPP. The Mw and Mn after degradation were significantly decreased (P b .0001) compared with that of
incubated before. (C) Mw distribution curves for HDPE MPP before and after incubation.

carbonyl groups (-C=O) respectively (Fig. 5.B). Based on previous stud-
ies (Arutchelvi et al., 2008; Cai et al., 2018; Kumar Sen and Raut, 2015),
the appearance of hydroxyl, carbonyl and ether groups provided the ev-
idence of bio-oxidation of HDPE MPP and ultimately promoted the bio-
degradation of PE.
3.5. Exploration of potential high-efficiency PE degrading enzymes
The extracellular enzymes secreted by the microorganisms are the
key for biodegradation, as they can make hydrophobic PE surface
more hydrophilic (Arutchelvi et al., 2008; Restrepo-Flórez et al., 2014).
Currently, there are few known examples of laccases, manganese perox-
ides or lignin peroxidases that are capable of hydrolyzing HDPE (Wei
and Zimmermann, 2017). Laccase-like multicopper oxidases (LMCOs)
has been reported to degrade organic polymeric dye and showed
broad substrate specificity (Reiss et al., 2013), which made PE being
substrate possible (Brander et al., 2014; Tamayo-Ramos et al., 2012).
To explore the genes that involved in HDPE degradation, we focused
on the laccases and LMCOs in strain PEDX3 as laccases have great poten-
tial ability catalyzing bio-oxidation of HDPE (Bilal et al., 2019; Santo
et al., 2013).
ABTS method was used for testing the activity of extracellular
laccases and LMCOs in SCS-HDPE medium supernatants (Fig. 6.A). The
results indicated that the laccases and LMCOs may be expressed by
strain PEDX3 and the enzymes activity reached the top at 21st day.
Nine candidate genes (Table S1.) including laccases and LMCOs were re-
trieved from GenBank based on genome sequence of A. flavus NRRL3357
(no. GCA_000006275.2). To eliminate the error caused by different cul-
ture mediums, two control groups were designed for the carbon-rich
environment, specifically, NCS-Glu medium represent for the synthetic
carbon-rich environment with glucose as the sole carbon source and
PDB medium represent for natural carbon-rich environment, while
SCS-HDPE as carbon-poor group. According to the Venn diagram of
up-regulated genes, AFLA_006190 and AFLA_053930 may become the
potential degrading enzymes (Fig. 6 B-D). However, the degradation
ability of the two enzymes should be validated. The future research
would focus on the degrading function of the enzymes, rational-
design to improve the enzyme activity, and elucidating the possible
mechanisms underlying the enzymatic degradation.
4. Conclusions
In the present study, a fungal strain, Aspergillus flavus PEDX3, was
isolated as the potential PE-degrading microorganisms from the gut
contents of G. mellonella larvae. Among the twenty-five isolates,
A. flavus strain was the most efficient PE-degrading strain that can de-
grade both LDPE and HDPE. Efficient biodegradation effect was con-
firmed by changes on the particle surface and reduction in mass, Mw
and Mn. It indicates that there is a great possibility of finding microor-
ganisms that can degrade MPP from the guts of insects. FT-IR analysis
demonstrated the formation of new functional groups such as hydroxyl
groups (3500–3100 cm−1), ether groups (1113 cm−1) and carbonyl
groups (1647–1716 cm−1), indicating the bio-oxidation effect of
A. flavus strain PEDX3, which lead to the exploration of PE-degrading
enzymes. Understanding the enzyme system of A. flavus would give an
insight to their role in biodegradation of PE, and two LMCOs were con-
sidered as the potential PE-degrading enzymes after preliminary screen.
As PE MPP accumulation in the environment is a serious threat, these
enzymes would provide a solution to minimize and abate the presence
of PE MPP in the environment.

Fig. 5. FT-IR analysis of the untreated and PEDX3-treated HDPE MPP. (A) Absorbance spectrum with wavelengths from 4000 cm−1 to 2500 cm−1, and the peaks of 2919 cm−1 and
2851 cm−1 were corresponded to C\ \H groups. (B) Transmittance spectrum with wavelengths from 2000 cm−1 to 500 cm−1. The peaks of 719 cm−1 are the characteristics of the
C\\H groups.
 J. Zhang et al. / Science of the Total Environment 704 (2020) 135931
Fig. 6. Screening of PE degrading enzyme. (A) The enzyme activity of laccases and LMCOs in culture supernatant. (B) Venn diagram of the up-regulated genes induced by HDPE. (C) and
(D) were the relative expression of candidate genes with NCS-Glu medium and PDB medium as control groups respectively.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgements
This project is funded by National Natural Science Foundation of
China (81703637) and Natural Science Foundation of Jiangsu Province
(BK20170736).
Appendix A. Supplementary data
Additional information on ultraviolet irradiation of MPP, media,
morphology examination, HT-GPC procedure, FT-IR procedure, total
RNA extraction and RT-PCR. The information of candidate degrading
genes and RT-PCR primers (Table S1). Growth of the microorganism
and the hydrophilicity of PE among 25 isolated strain (Table S2). Sup-
plementary data to this article can be found online at doi:https://doi.
org/10.1016/j.scitotenv.2019.135931.
References
Akhtar, N., 2014. In vivo pathogenicity studies of Aspergilli in lepidopteran model host
Galleria mellonella. APCBEE. Procedia. 8, 293–298. https://doi.org/10.1016/j.
apcbee.2014.03.043.
Albertsson, A.-C., Andersson, S.O., Karlsson, S., 1987. The mechanism of biodegradation of
polyethylene. Polym. Degrad. Stabil. 18, 73–87. https://doi.org/10.1016/0141-3910
(87)90084-X.
Andrady, A.L., 2011. Microplastics in the marine environment. Mar. Pollut. Bull. 62,
1596–1605. https://doi.org/10.1016/j.marpolbul.2011.05.030.
Arutchelvi, J., Sudhakar, M., Arkatkar, A., Doble, M., Bhaduri, S., Uppara, P.V., 2008. Biodeg-
radation of polyethylene and polypropylene. Indian J. Biotechnol. 7, 9–22.
ASTM, 1996. Standard Practice for Determining Resistance of Plastics to Bacteria G22–76,
West Conshohocken, Pennsylvania.
7
Bilal, M., Rasheed, T., Nabeel, F., Iqbal, H.M.N., Zhao, Y., 2019. Hazardous contaminants in
the environment and their laccase-assisted degradation – a review. J. Environ. Manag.
234, 253–264. https://doi.org/10.1016/j.jenvman.2019.01.001.
Bohle, K., Jungebloud, A., Göcke, Y., Dalpiaz, A., Cordes, C., Horn, H., 2007. Selection of ref-
erence genes for normalisation of specific gene quantification data of Aspergillus
niger. J. Biotechnol. 132, 353–358. https://doi.org/10.1016/j.jbiotec.2007.08.005.
Bombelli, P., Howe, C.J., Bertocchini, F., 2017. Polyethylene bio-degradation by caterpillars
of the wax moth Galleria mellonella. Curr. Biol. 27, R292–R293. https://doi.org/
10.1016/j.cub.2017.02.060.
Brander, S., Mikkelsen, J.D., Kepp, K., 2014. TtMCO: a highly thermostable laccase-like
multicopper oxidase from the thermophilic Thermobaculum terrenum. J. Mol. Catal.
B-Enzym. 112, 59–65. https://doi.org/10.1016/j.molcatb.2014.12.002.
Cai, L., Wang, J., Peng, J., Wu, Z., Tan, X., 2018. Observation of the degradation of three
types of plastic pellets exposed to UV irradiation in three different environments.
Sci. Total Environ. 628–629, 740–747. https://doi.org/10.1016/j.
scitotenv.2018.02.079.
Courty, P.-E., Pouysegur, R., Buée, M., Garbaye, J., 2006. Laccase and phosphatase activities
of the dominant ectomycorrhizal types in a lowland oak forest. Soil Biol. Biochem. 38,
1219–1222. https://doi.org/10.1016/j.soilbio.2005.10.005.
El-Shafei, H.A., El-Nasser, N.H.A., Kansoh, A.L., Ali, A.M., 1998. Biodegradation of dispos-
able polyethylene by fungi and Streptomyces species. Polym. Degrad. Stabil. 62,
361–365. https://doi.org/10.1016/S0141-3910(98)00019-6.
Frisvad, J.C., Hubka, V., Ezekiel, C.N., Hong, S.B., Nováková, A., Chen, A.J., 2019. Taxon-
omy of Aspergillus section Flavi and their production of aflatoxins, ochratoxins
and other mycotoxins. Stud. Mycol. 93, 1–63. https://doi.org/10.1016/j.
simyco.2018.06.001.
Gajendiran, A., 2016. Microbial degradation of low-density polyethylene (LDPE) by Asper-
gillus clavatus strain JASK1 isolated from landfill soil. J. 3 Biotech. 6, 52. https://doi.
org/10.1007/s13205-016-0394-x.
Gilan, I., Hadar, Y., Sivan, A., 2004. Colonization, biofilm formation and biodegradation of
polyethylene by a strain of Rhodococcus ruber. Appl. Microbiol. Biotechnol. 65,
97e104. https://doi.org/10.1007/s00253-004-1584-8.
Gilliam, M., Prest, D.B., Lorenz, B.J., 1989. Microbes from apiarian sources: molds in frass
from larvae of the greater wax moth, Galleria mellonella. J. Invertebr. Pathol. 54,
406–408. https://doi.org/10.1016/0022-2011(89)90126-2.
Hammer, T.J., Janzen, D.H., Hallwachs, W., Jaffe, S.P., Fierer, N., 2017. Caterpillars lack a res-
ident gut microbiome. P. Natl. Acad. Sci. USA. 114, 9641. https://doi.org/10.1073/
pnas.1707186114.
Harshvardhan, K., Jha, B., 2013. Biodegradation of low-density polyethylene by marine
bacteria from pelagic waters, Arabian Sea, India. Mar. Pollut. Bull. 77, 100–106.
https://doi.org/10.1016/j.marpolbul.2013.10.025.
He, H., Liu, P., Luo, B.X., 2016. Isolation and complex mutagenesis of an actinomycetes
with degraded starch/polyethylene powder. J. Sichuan. Normal. Univ. 39, 408–413.
https://doi.org/10.3969/j.issn.1001-8395.2016.03.019.
8 J. Zhang et al. / Science of the Total Environment 704 (2020) 135931
Idowu, O.K., Obasola, E.F., Blessing, I.N., 2019. Biodegradation of low density polyethylene
(LDPE) by certain indigenous bacteria and fungi. Int. J. Environ. Stud. 76, 428–440.
https://doi.org/10.1080/00207233.2019.1579586.
Johannes, C., Majcherczyk, A., 2000. Laccase activity tests and laccase inhibitors.
J. Biotechnol. 78, 193–199. https://doi.org/10.1016/s0168-1656(00)00208-x.
Kannan, M., Mubarakali, D., Thiyonila, B., Krishnan, M., Padmanaban, B., Shantkriti, S.,
2019. Insect gut as a bioresource for potential enzymes - an unexploited area for in-
dustrial biotechnology. Biocatal. Agr. Biotechnol. 18, 101010. https://doi.org/10.1016/
j.bcab.2019.01.048.
Kawai, F., Watanabe, M., Shibata, M., Yokoyama, S., Sudate, Y., 2002. Experimental analysis
and numerical simulation for biodegradability of polyethylene. Polym. Degrad. Stabil.
76, 129–135. https://doi.org/10.1016/S0141-3910(02)00006-X.
Koelmans, A.A., Mohamed Nor, N.H., Hermsen, E., Kooi, M., Mintenig, S.M., De France, J.,
2019. Microplastics in freshwaters and drinking water: critical review and assess-
ment of data quality. Water Res. 155, 410–422. https://doi.org/10.1016/j.
watres.2019.02.054.
Kumar Sen, S., Raut, S., 2015. Microbial degradation of low density polyethylene (LDPE): a
review. J. Environ. Chem. Eng. 3, 462–473. https://doi.org/10.1016/j.jece.2015.01.003.
Kumar, S., Das, M., Rebecca, J., Kripanand, S.S., 2013. Isolation and identification of LDPE
degrading fungi from municipal solid waste. J. Chem. Pharm. Res. 5, 78–81.
Lucas, N., Bienaime, C., Belloy, C., Queneudec, M., Silvestre, F., Nava-Saucedo, J.-E., 2008.
Polymer biodegradation: mechanisms and estimation techniques – a review.
Chemosphere 73, 429–442. https://doi.org/10.1016/j.chemosphere.2008.06.064.
Luo, B., 2013. Screening and Identification of Strains Degrading Polyethylene (PE) and Re-
searches on Degradation Characteristics of LBX-2 Strain. Master’s Thesis. Univ. Si-
chuan. Normal.
Mathur, G., 2011. Colonization and degradation of thermally oxidized high-density poly-
ethylene by Aspergillus niger (ITCC no. 6052) isolated from plastic waste dumpsite.
Bioremediat. J. 15, 69–76. https://doi.org/10.1080/10889868.2011.570281.
Moharir, R.V., Kumar, S., 2019. Challenges associated with plastic waste disposal and al-
lied microbial routes for its effective degradation: a comprehensive review. J. Clean.
Prod. 208, 65–76. https://doi.org/10.1016/j.jclepro.2018.10.059.
Nitesh, V., Sharmita, G., 2019. Assessment of LDPE degrading potential Aspergillus species
isolated from municipal landfill sites of Agra. SN. Appl. Sci. 1, 701. https://doi.org/
10.1007/s42452-019-0746-3.
Novotna, K., Cermakova, L., Pivokonska, L., Cajthaml, T., Pivokonsky, M., 2019.
Microplastics in drinking water treatment – current knowledge and research needs.
Sci. Total Environ. 667, 730–740. https://doi.org/10.1016/j.scitotenv.2019.02.431.
Orhan, Y., Hrenovic, J., Buyukgungor, H., 2004. Biodegradation of plastic compost bags
under controlled soil conditions. Acta Chim. Slov. 51, 579–588.
Paço, A., Duarte, K., da Costa, J.P., Santos, P.S.M., Pereira, R., Pereira, M.E., 2017. Biodegra-
dation of polyethylene microplastics by the marine fungus Zalerion maritimum. Sci.
Total Environ. 586, 10–15. https://doi.org/10.1016/j.scitotenv.2017.02.017.
Park, S.Y., Kim, C.G., 2019. Biodegradation of micro-polyethylene particles by bacterial col-
onization of a mixed microbial consortium isolated from a landfill site. Chemosphere
222, 527–533. https://doi.org/10.1016/j.chemosphere.2019.01.159.
Pramila, R., Vijaya Ramesh, K., 2011. Biodegradation of low density polyethylene (LDPE)
by fungi isolated from municipal landfill area. J. Microbiol. Biotechnol. Res. 1,
131–136.
Rajmohan, K.S., C, R., Varjani, S., 2019. Plastic pollutants: waste management for pollution
control and abatement. Curr. Opin. Environ. Sci. Health. In Press. https://doi.org/
10.1016/j.coesh.2019.08.006.
Reiss, R., Ihssen, J., Richter, M., Eichhorn, E., Schilling, B., Thöny-Meyer, L., 2013. Laccase
versus laccase-like multi-copper oxidase: a comparative study of similar enzymes
with diverse substrate spectra. PLoS One 8, e65633. https://doi.org/10.1371/journal.
pone.0065633.
Ren, L., Men, L., Zhang, Z., Guan, F., Tian, J., Wang, B., 2019. Biodegradation of polyethylene
by sp. D1 from the guts of wax moth. Int. J. Environ. Res. Public Health 16, 1941.
https://doi.org/10.3390/ijerph16111941.
Restrepo-Flórez, J.-M., Bassi, A., Thompson, M.R., 2014. 2013. Microbial degradation and
deterioration of polyethylene – a review. Int. Biodeterior. Biodegradation 88, 83–90.
https://doi.org/10.1016/j.ibiod.2013.12.014.
Samson, R.A., Visagie, C.M., Houbraken, J., Hong, S.B., Hubka, V., Klaassen, C.H.W., 2014.
Phylogeny, identification and nomenclature of the genus Aspergillus. Stud. Mycol.
78, 141–173. https://doi.org/10.1016/j.simyco.2014.07.004.
Sangeetha Devi, R., Rajesh Kannan, V., Nivas, D., Kannan, K., Chandru, S., Robert Antony, A.,
2015. Biodegradation of HDPE by Aspergillus spp. from marine ecosystem of gulf of
Mannar, India. Mar. Pollut. Bull. 96, 32–40. https://doi.org/10.1016/j.
marpolbul.2015.05.050.
Santo, M., Weitsman, R., Sivan, A., 2013. The role of the copper-binding enzyme – laccase
– in the biodegradation of polyethylene by the actinomycete Rhodococcus ruber. Int.
Biodeter. Biodegrad. 84, 204–210. https://doi.org/10.1016/j.ibiod.2012.03.001.
Schmittgen, T.D., Livak, K.J., 2008. Analyzing real-time PCR data by the comparative C
(T) method. Nat. Protoc. 3, 1101–1108. https://doi.org/10.1038/nprot.2008.73.
Scully, L.R., Bidochka, M.J., 2009. An alternative insect pathogenic strategy in an Aspergillus
flavus auxotroph. Mycol. Res. 113, 230–239. https://doi.org/10.1016/j.
mycres.2008.10.007.
Shah, A.A., Hasan, F., Hameed, A., Ahmed, S., 2008. Biological degradation of plastics: a
comprehensive review. Biotechnol. Adv. 26, 246–265. https://doi.org/10.1016/j.
biotechadv.2007.12.005.
Sharma, S., Chatterjee, S., 2017. Microplastic pollution, a threat to marine ecosystem and
human health: a short review. Environ. Sci. Pollut. Res. Int. 24, 21530–21547. https://
doi.org/10.1007/s11356-017-9910-8.
Silva, A.B., Costa, M.F., Duarte, A.C., 2018. Biotechnology advances for dealing with envi-
ronmental pollution by micro(nano)plastics: lessons on theory and practices. Curr.
Opin. Environ. Sci. Health. 1, 30–35. https://doi.org/10.1016/j.coesh.2017.10.005.
Sivan, A., Szanto, M., Pavlov, V., 2006. Biofilm development of the polyethylene-degrading
bacterium Rhodococcus ruber. Appl. Microbiol. Biot. 72, 346–352. https://doi.org/
10.1007/s00253-005-0259-4.
Tamayo-Ramos, J., van Berkel, W.J.H., de Graaff, L.H., 2012. Biocatalytic potential of
laccase-like multicopper oxidases from Aspergillus niger. Microb. Cell Factories 11,
165. https://doi.org/10.1186/1475-2859-11-165.
Tang, X., Chen, E.Y.X., 2019. Toward infinitely recyclable plastics derived from renewable
cyclic esters. Chem 5, 284–312. https://doi.org/10.1016/j.chempr.2018.10.011.
Tribedi, P., Sil, A.K., 2013. Low-density polyethylene degradation by Pseudomonas sp. AKS2
biofilm. Environ. Sci. Pollut. Res. Int. 20, 4146e4153. https://doi.org/10.1007/s11356-
012-1378-y.
Verma, R., Vinoda, K.S., Papireddy, M., Gowda, A.N.S., 2016. Toxic pollutants from plastic
waste- a review. Procedia Environ. Sci. 35, 701–708. https://doi.org/10.1016/j.
proenv.2016.07.069.
Wei, R., Zimmermann, W., 2017. Microbial enzymes for the recycling of recalcitrant
petroleum-based plastics: how far are we? Microb. Biotechnol. 10, 1308–1322.
https://doi.org/10.1111/1751-7915.12710.
Yamada-Onodera, K., Mukumoto, H., Katsuyaya, Y., Saiganji, A., Tani, Y., 2001. Degradation
of polyethylene by a fungus, Penicillium simplicissimum YK. Polym. Degrad. Stabil. 72,
323–327. https://doi.org/10.1016/S0141-3910(01)00027-1.
Yang, J., Yang, Y., Wu, W.M., Zhao, J., Jiang, L., 2014. Evidence of polyethylene biodegrada-
tion by bacterial strains from the guts of plastic-eating waxworms. Environ. Sci.
Technol. 48, 13776–13784. https://doi.org/10.1021/es504038a.
Yang, Y., Yang, J., Wu, W.-M., Zhao, J., Song, Y., Gao, L., 2015. Biodegradation and mineral-
ization of polystyrene by plastic-eating mealworms: part 2. Role of gut microorgan-
isms. Environ. Sci. Technol. 49, 12087–12093. https://doi.org/10.1021/acs.
est.5b02663.
Yoshida, S., Hiraga, K., Takehana, T., Taniguchi, I., Yamaji, H., Maeda, Y., 2016. A bacterium
that degrades and assimilates poly(ethylene terephthalate). Science 351, 1196–1199.
https://doi.org/10.1126/science.aad6359.