Article

pubs.acs.org/est

Evidence of Polyethylene Biodegradation by Bacterial Strains from

the Guts of Plastic-Eating Waxworms
Jun Yang,*,†,∥ Yu Yang,†,∥ Wei-Min Wu,‡ Jiao Zhao,§ and Lei Jiang†
†
Key Laboratory of Bio-Inspired Smart Interfacial Science and Technology of Ministry of Education, School of Chemistry and
Environment, Beihang University, Beijing 100191, P. R. China
‡
Department of Civil and Environmental Engineering, William & Cloy Codiga Resource Recovery Research Center, Center for
Sustainable Development & Global Competitiveness, Stanford University, Stanford, California 94305-4020, United States
§
Shenzhen Key Laboratory of Bioenergy, BGI-Shenzhen, Shenzhen, Guangdong 518083, P. R. China
*
S Supporting Information

ABSTRACT: Polyethylene (PE) has been considered nonbiode-

gradable for decades. Although the biodegradation of PE by bacterial
cultures has been occasionally described, valid evidence of PE
biodegradation has remained limited in the literature. We found that
waxworms, or Indian mealmoths (the larvae of Plodia interpunctella),
were capable of chewing and eating PE films. Two bacterial strains
capable of degrading PE were isolated from this worm’s gut,
Enterobacter asburiae YT1 and Bacillus sp. YP1. Over a 28-day
incubation period of the two strains on PE films, viable biofilms
formed, and the PE films’ hydrophobicity decreased. Obvious
damage, including pits and cavities (0.3−0.4 μm in depth), was
observed on the surfaces of the PE films using scanning electron
microscopy (SEM) and atomic force microscopy (AFM). The
formation of carbonyl groups was verified using X-ray photoelectron
spectroscopy (XPS) and microattenuated total reflectance/Fourier transform infrared (micro-ATR/FTIR) imaging microscope.
Suspension cultures of YT1 and YP1 (108 cells/mL) were able to degrade approximately 6.1 ± 0.3% and 10.7 ± 0.2% of the PE
films (100 mg), respectively, over a 60-day incubation period. The molecular weights of the residual PE films were lower, and the
release of 12 water-soluble daughter products was also detected. The results demonstrated the presence of PE-degrading bacteria
in the guts of waxworms and provided promising evidence for the biodegradation of PE in the environment.

■ INTRODUCTION
Polyethylene (PE), the most common petroleum-based plastic,
oxidation and then through the biological activity of micro-
organisms.15,16 When PE samples are pretreated with UV light
is expressed as “[CH2−CH2]n” and is widely used in everyday or thermo-oxidation, depolymerization of the PE chains occurs
life, with an annual global production of approximately 140 and results in the formation of low molecular weight products,
million tons.1,2 However, the obvious contrast between the including alkanes, alkenes, ketones, aldehydes, various alcohols,
remarkable durability and the short service time of PE products and fatty acids, which are then further degraded by select
leads to the accumulation of PE waste in the environment, microorganisms.15−18 The pretreatments either decrease the
which has generated international concern.3−6 hydrophobicity of the polymer, making it more compatible with
Several features of PE make it resistant to biodegradation. microorganisms, or introduce groups such as CO or −OH,
Among these features are a) PE’s highly stable C−C and C−H which are more prone to microbial degradation.19 In contrast,
covalent bonds; b) its high molecular weight, which makes it almost no biodegradation of PE through the biological activities
too large to penetrate the cell walls of microbes; c) its lack of of select microorganisms can be observed without pretreat-
readily oxidizable and/or hydrolyzable groups; and d) its highly ments.15 The poor biodegradation of virgin PE has been
hydrophobic nature.7 Since the early 1970s, tests on the attributed to the lack of specific plastic-degrading micro-
biodegradation of virgin PE (unpretreated and without any organisms with long carbon chain oxidization and depolyme-
additives) have been performed under natural environmental rization capabilities. Certain researchers have assumed that the
conditions, including soils, seawater, sludge, and compost, time for microbial evolution, which is required for plastic
which harbor a multitude of diverse microbial communities.8−14
These studies have concluded that the biodegradation of virgin Received: August 18, 2014
PE is extremely limited in selected mixed microbial Revised: October 31, 2014
communities.2−7 It is believed that the environmental Accepted: November 10, 2014
degradation of PE can be achieved by photo- or thermo- Published: November 10, 2014

© 2014 American Chemical Society 13776 dx.doi.org/10.1021/es504038a | Environ. Sci. Technol. 2014, 48, 13776−13784
Environmental Science & Technology Article

degradation, might be much longer than the history of PE molecular weight was analyzed using high-temperature gel
applications.20 To date, researchers are still attempting to permeation chromatography (GPC, PL220, Agilent, USA), and
discover PE-degrading microorganisms through tests involving the average molecular weight (Mw) and number-average
plastic-waste-contaminated soils, landfills, compost, and marine molecular weight (Mn) were found to be 88,200 and 27,700,
life and have isolated several bacterial strains (SI Table respectively.
S1).20−39 Some of these strains have shown a moderate ability The PE film was cut into 50 mm × 50 mm square sheets for
to use virgin PE as a carbon source based on the incubation in an agar medium plate and small 3 mm × 3 mm
characterization of biofilm formation on PE films, weight loss pieces for incubation in a liquid medium. The PE materials
of PE materials, surface deterioration, and changes in the were weighed, disinfected in 75% ethanol, and then air-dried in
mechanical and thermal properties of PE (SI Table S1). For a laminar-flow clean bench prior to use.
example, it has been reported that a Pseudomonas strain causes The liquid carbon-free basal medium (LCFBM) prepared
weight losses of up to 20% in the tested PE within 120 days.32 with deionized water contained (per 1,000 mL) 0.7 g of
Additionally, a Canadian Web site has reported the isolation of KH2PO4, 0.7 g of K2HPO4, 0.7 g of MgSO4·7H2O, 1.0 g of
two novel bacteria that rapidly degrade 22% or more of the NH4NO3, 0.005 g of NaCl, 0.002 g of FeSO4·7H2O, 0.002 g of
tested PE weight over a six week period.39 However, these ZnSO4·7H2O, and 0.001 g of MnSO4·H2O, according to the
promising reports of PE degradation based on weight loss have ASTM standard for determining the resistance of plastics to
not been confirmed due to a failure to provide additional bacteria (G22-76, 1996).41 The carbon-free basal agar medium
supporting evidence, such as changes in surface hydrophobicity, (CFBAM) was prepared by adding 15 g of agar to 1,000 mL of
the formation of oxidized carbonyl groups, the scission of long- LCFBM. These media were sterilized by autoclaving at 121 °C
chain molecules, or the release of low molecular weight for 20 min.
products. No report on the deposits of the aforementioned Enrichment and Isolation of PE-Degrading Bacteria.
bacteria in any culture collection center has been provided. Once the waxworms had chewed enough of the PE bags to
More studies are needed to confirm PE degradation by cause significant observable damage, approximately 200 of the
microorganisms via sequential two-step reactions, which have larvae were collected from the PE bags containing millet grains
been achieved by the synergistic actions of physicochemical (SI, Figure S1). To obtain the gut contents for the enrichment
oxidation and through the biological activity of microorganisms, of PE-degrading bacteria, the surfaces of the larvae were
as proposed by Albertsson et al.15 sterilized via immersion in 75% ethanol for 1 min and then
We found that the grain pest larvae of Plodia interpunctella rinsed 2 times with sterile saline water (SW). Next, the guts of
(Hübner), commonly known as waxworms or Indian meal- the larvae were drawn out and pooled into a 50 mL centrifuge
moths, can damage PE packing films by chewing and eating the tube containing 40 mL of SW. After being shaken on a vortex
film (SI, Figure S1). A similar observation stating that various mixer for 5 min, the gut tissues were carefully removed with a
species of stored-product insect pests like to chew and pipet. The remaining suspension, used as a microbial inoculum,
penetrate PE packaging films has been previously reported.40 was transferred into a 250 mL Erlenmeyer flask that contained
However, the focus of the previous report was to test for and 1 g of the small PE pieces and 80 mL of LCFBM. This flask was
discover a package film material that was resistant to the insects’ incubated on a rotary shaker (120 rpm) at 30 °C. After 60 days,
destructive tendencies. We were inspired by the insect used in the residual PE pieces were removed, and the cultures were
that test and began to wonder whether a microbial symbiont spread across plates with three different agar media containing
capable of digesting PE was present in the waxworm gut and complex organic carbon substrates: potato dextrose agar
whether the gut bacteria could act as a source for the isolation (PDA), bean sprout agar (BSA), and beef extract-peptone
of PE-degrading microorganisms. agar (BPA). After an incubation period of 24 h, the colonies
In this study, with PE film as a sole carbon source, we that formed were transferred to plates with fresh agar medium,
isolated two PE-degrading bacterial strains from the enrichment where they were kept until pure colonies of isolates (based on a
of waxworm gut contents and identified that these bacteria were microbial purity test) could be obtained. These isolates have
capable of degrading PE within a limited incubation period been deposited at the China General Microbiological Culture
based on the characterization of biofilm formation, changes in Collection Center (CGMCCC) in Beijing, China.
the PE’s physical properties (tensile strength and surface Preliminary Screening of Isolates for PE-Degrading
topography), chemical structure (hydrophobicity and appear- Ability. The bacterial isolates were grown in liquid nutrient
ance of carbonyl groups), molecular weight (accompanied by broth (NB) medium for 12 h. The cells that grew were
the formation of daughter products), and weight loss. Our collected via centrifugation (10,000 rpm) and rinsed with SW
results confirmed PE biodegradation by the two gut bacteria to remove the residual medium. This procedure was repeated
isolated from waxworms and indicated that the bacteria from three times. Next, the collected cells were resuspended in SW
these plastic-chewing insect larvae were a promising source of to obtain a cell suspension of approximately 108 cells per mL.
plastic-degrading microorganisms.

■
The melted sterile CFBAM (15 mL) was poured into a Petri
dish (90 mm diameter) and cooled to ambient temperature.
MATERIALS AND METHODS The cell suspension (0.5 mL) was spread homogeneously
PE Film and Medium. Linear low-density PE (LDPE) film across the surface of a CFBAM plate, which was subsequently
(DFDA-9020, 22.5 μm thickness) was purchased from covered with a PE sheet (50 mm × 50 mm). In the sterile
SINOPEC Beijing Yanshan Company in China. No catalysts controls, PE sheets were added without inoculation of the cell
or additives were added to this PE product, according to the suspension. CFBAM plates inoculated with the cell suspension
manufacturer’s standard (Q/SH3180 014). The chemical without added PE film were used as a control to check whether
composition was characterized by a thermogravimetric analyzer the isolates could grow on agar alone. All plates were then
(TGA, 209F1, NETZSCH, Germany) and Fourier transform sealed with Parafilm and incubated at 30 °C and 85% r.h. for 28
infrared spectroscopy (FTIR, iN10MX, Nicolet, USA). The days. Three plates were prepared for each isolated strain. The
13777 dx.doi.org/10.1021/es504038a | Environ. Sci. Technol. 2014, 48, 13776−13784
Environmental Science & Technology Article

evidence of PE biodegradation was preliminarily determined by ATR/FTIR microscope was 400 × 400 μm, and the scanning
characterizing the changes in the tensile strength of the was performed at a step size of 10 μm under a contact pressure
incubated PE sheets on a universal test machine (AGS-X-1KN, of 3 MPa.
Shimadzu, Japan), according to the standard method.42 Biodegradation Assays. The PE biodegradation was
To examine changes in the PE film’s hydrophobicity over 28 characterized by the timing of its weight loss, molecular weight
days, the sole carbon source of the isolated strains was a PE shift, and the formation of soluble daughter products in the
sample in the LCFBM. Afterward, the biofilm on the PE sheet LCFBM. PE pieces (100 mg) and bacterial suspensions (10
was completely removed by mixing it with 2% w/v aqueous mL) were added to a 150 mL Erlenmeyer flask with 40 mL of
sodium dodecyl sulfate (SDS) for 4 h and then rinsing it with LCFBM. The final bacterial concentration was approximately
deionized water, according to the washing procedure reported 108 cells per mL, and a flask without inoculation served as a
by Sivan A et al.21,22 Following the treatment, no cells were sterile control. Three samples were prepared for each
observed on the surface of the PE film. The change in the PE incubation period of 7, 14, 21, 28, and 60 days, and the flasks
surface’s hydrophobicity was determined by measuring the were incubated on a rotary shaker (120 rpm) at 30 °C. For
water contact angle (WCA) using a contact angle measuring each incubation period, the residual PE pieces were collected,
device (OCA40, Dataphysics, Germany). washed using the procedure reported by Sivan et al.,21,22 and
Characterization of Biofilm Formation and Cell dried at ambient temperature for the measurement of residual
Viability. The isolates with positive results obtained during weight.
preliminary screen tests were further tested for their ability to The washed PE residues collected at the end of the test were
form biofilms on PE film as a sole carbon source and for the cell randomly sampled for molecular weight distribution analysis
viability in the biofilm. A cell suspension (108 cells per mL) was using the HT-GPC (PL220, Agilent, USA).23 The liquid culture
spread across the CFBAM plate, which was subsequently was centrifuged at 10,000 rpm for 15 min, and the supernatant
covered with the PE film sheet (50 mm × 50 mm), as described was filtered through a 0.22-μm membrane filter. The soluble
above. Three samples were tested at incubation periods of 0, 3, daughter products in the filtrate were analyzed using electro-
7, 14, 21, and 28 days at 30 °C and 85% r.h. For the incubation spray ionization-mass spectrometry (ESI-MS, LCQ, Finnigan,
of zero days, samples were collected after 3 h of inoculation. USA) in the positive ion mode.44
The plates without inoculation served as a sterile control. Sequencing and Phylogenetic Analysis. The genomic
After a biofilm formed on the PE surface, the cell number of DNA needed for 16S rDNA amplification of the isolated strains
bacteria that had grown on the PE film surface was counted was extracted from cells grown in the late log phase using a
using the following procedure. The PE film sheet containing a conventional proteinase K treatment and phenol-chloroform
biofilm was transferred into a 50 mL centrifuge tube with 40 extraction. Amplification of the 5′ end of the gene was
mL of SW, and the tube was shaken on a vortex mixer for 5 performed with the universal primers 8-F (5′-AGAGTTTGA-
min. The PE film was transferred into another centrifuge tube TYMTGGCTCAG-3′) and 1942-R (5′-GGTTACCTTGT-
with 40 mL of SW, and this tube was also shaken on the vortex TACGACTT-3′). The obtained sequences were aligned with
mixer for 5 min. The suspensions of the two tubes were then organisms present on the GenBank database using the Basic
pooled in a centrifuge tube, which was centrifuged at 10,000 Local Alignment Search Tool (BLAST) created by the National
rpm for 15 min. The supernatant was discarded, and the pellet Center for Biotechnology Information, USA.

■
was resuspended in 5 mL of SW. The cell number of the
suspension was counted using the series dilution method of
RESULTS AND DISCUSSION
plate counting. The biofilm on the PE film sheet was examined
using a scanning electron microscopy (SEM, Quanta FEG250, Preliminary Screen of PE-Degrading Isolates. Eight
FEI, USA),21 and the viability of the bacterial cells was bacterial strains were isolated during the enrichment of the gut
characterized in situ using a fluorescence microscope (BX51, contents of the PE-eating waxworms when using PE as a sole
Olympus, Japan) after staining with the LIVE/DEAD BacLight carbon source (SI, Figure S1). Screenings for PE-degrading
Bacterial Viability Kit.22 microorganisms among these isolates were subsequently
Microscale Analysis of Surface Topography and performed in terms of the colonization on the PE film sheets
Chemistry. After a 28-day incubation period, the biofilm on in the CFBAM plates, the changes in tensile strength of the PE
the PE sheet was completely removed using a washing sheets, the floating behavior of the PE pieces in the LCFBM,
procedure with 2% w/v aqueous sodium dodecyl sulfate and the WCA of the PE surfaces.
(SDS) solution for 4 h.21,22 The PE sheets in the sterile control The Gram-positive strain YP1, identified by 16S rDNA
were also treated using the same procedure. Consequently, the sequencing as Bacillus sp., grew as relatively thick, opaque
surface topography of all PE sheets was characterized by SEM colonies visible to the naked eye on the PE film sheet. The
observation (Quanta FEG250, FEI, USA) and with an atomic Gram-negative strain YT1, identified as Enterobacter asburiae,
force microscope (AFM, Dimension Icon, Veeco, USA).21,26 grew as visible, thin, translucent colonies (Figure 1a and Table
The surface chemical components were investigated using X- 1). The other six isolated bacterial strains did not develop
ray photoelectron spectroscopy (XPS, Thermo Scientific, USA) visible colonies on the PE film sheets. In addition, no visible
and a microattenuated total reflectance/Fourier transform colonization occurred on the two control plates: a) the CFBAM
infrared imaging microscope (Nicolet iN10 MX, Thermo with a PE film sheet but without inoculation or b) the CFBAM
Scientific, USA).43 During the XPS spectra analysis, scanning without a PE sheet but with inoculation.
was carried out over a broad-band energy range (0−1,200 eV) After a 28-day incubation period, all of the incubated PE film
at an electron takeoff angle of 90° from the sample areas less sheets were collected to test their tensile strength. The samples
than 1 mm in diameter. The overlapping peaks in the C 1s spread with the culture of Bacillus sp. YP1 or E. asburiae YT1
region were resolved into their individual components using a exhibited tensile strength decreases of over 50% compared to
peak-fitting program (XPSPEAK v3.0). The view field of the that of the sterile controls, while the samples with the other six
13778 dx.doi.org/10.1021/es504038a | Environ. Sci. Technol. 2014, 48, 13776−13784
Environmental Science & Technology Article

study. Sequences of the two strains were deposited in GenBank

under references KJ466896 to KJ466897.
Biofilm Formation Process on PE Film. The biofilm
formation on PE film determines its potential biodegradation
because a biofilm enables the microorganisms to efficiently
utilize the nonsoluble substrate.21,22 The biofilm formation
processes by the two select strains on the PE film in this study
were characterized by cell number counting, morphotypes, and
the biofilm’s viability.
The biofilm development on the PE film sheet in a CFBAM
plate was monitored by counting the number of cells colonizing
on the PE film sheet via series dilution during the 28-day
incubation. The results showed that both strains almost
immediately adhered to the PE film sheets and built detectable
biofilms within the first 3 h of incubation (Figures 2a-c). The
cell density of the initial biofilm of strain YT1 was 3.8 × 107
CFU/cm2. The cell density of strain YP1 was 1.4 × 106 CFU/
cm2, which was approximately 1 order of magnitude lower than
that of strain YT1. The biofilm densities of strains YT1 and YP1
increased from day 3 to day 21, reaching mature biofilms of
Figure 1. Assay of the strains YT1 and YP1 isolated for their PE- 16.0 and 9.4 × 107 CFU/cm2, respectively, on day 28 (Figures
degrading activity. (a) Formation of colonies on the PE film sheets in 2b and c).
CFBAM plates. No colony was present on the sterile control. (b) The At the end of the 28-day incubation, the biofilm morphotype
PE pieces became suspended in the inoculated liquid medium, but the was observed using SEM. The cells of strain YT1 were rods
control remained unchanged. (c) Decreased water contact angles measuring 0.2 × 0.8 μm. This strain developed a denser biofilm
(WCA) of PE samples after inoculation. but produced less extracellular polymeric substance (EPS) than
the cells of strain YP1 (Figures 2e-f). The cells of strain YP1
isolated bacterial strains exhibited no reduction in tensile were mainly short irregular rods measuring 0.5 × 3 μm, and
strength (SI, Figure S2) fewer spore-like cells were observed in its biofilm (Figure 2f).
At the beginning of incubation, the PE pieces floated in the The viability of the biofilms’ bacterial cells grown on the PE
upper portion of the liquid medium because its density (0.915− film was estimated in situ using a fluorescent microscope after
0.940 g/cm3) was lower than that of water (1 g/cm3) (Figure staining with the LIVE/DEAD BacLight Bacterial Viability
1b). After a 28-day incubation period with strains YT1 and Kit.21 Under fluorescence (Figure 2g-i), the live cells (green)
YP1, the PE pieces did not float and became suspended in the dominated in the biofilms of the two strains, and only a limited
LCFBM, whereas the PE pieces in the sterile controls number of dead cells (red) was observed. The predominance of
continued to float (Figure 1b). The PE film pieces incubated live cells in the biofilm indicated that these cells received
with the other six strains also continued to float in the liquid enough growth substrate,21,22,26 which likely came from the
medium (data not shown). This result was likely because the metabolism of the PE material and its daughter products.
cells in strains YT1 and YP1 were able to adhesively grow on Changes in Physicochemical Surface Properties. After
the PE species, and this cell adhesion increased the weight of a 28-day incubation period, the changes in the physicochemical
the PE species, resulting in the formation of a suspension. surface properties of the incubated PE film sheets on the
An analysis of the water contact angles of the PE pieces CFBAM plates were observed after the biofilm was completely
showed that the WCAs of the samples inoculated with strains removed from the PE samples. The change in surface
YT1 and YP1 were 69.3 ± 3.8° and 67.1 ± 1.6° (n = 5), topography was examined using SEM and AFM, and the
respectively, which were much lower than the control WCA of surface deterioration was estimated based on the formation of
97.2 ± 1.6° (n = 5) (Figure 1c). The other six isolated strains pits and cavities observed on the surfaces of the PE samples
did not significantly change the WCA of the PE pieces (data with bacterial inoculation (Figures 3c-f). The surfaces of the
not shown). The results indicated that the inoculation of controls remained smooth, without any defects (Figures 3a-b).
bacterial strains YT1 and YP1 decreased the hydrophobicity of The cavities on the surfaces of the PE sheets had maximum
the tested PE materials. As the PE materials became less depths of approximately 0.3 and 0.4 μm after inoculation with
hydrophobic, they became less resistant to subsequent strains YT1 and YP1, respectively (Figures 3d and f). This type
degradation by the bacterial cells.26 of deep cavity has not been reported by other researchers
Based on these results, we selected the E. asburiae strain YT1 working on the characterization of PE-degrading micro-
and Bacillus sp. strain YP1 as potential PE-degraders for further organisms.26,31 The results obtained from the microscale

Table 1. Isolation and Identification of PE-Degrading Bacterial Strains

straina highest-homology organism maximum identity, % Gram color size, μm isolation mediumb
YT1 Enterobacter asburiae 99 − yellow 0.2 × 0.8 BPA
YP1 Bacillus sp. 99 + yellow 0.5 × 3 PDA

a
The two strains have been preserved at the China General Microbiological Culture Collection Center (CGMCCC) in Beijing, China as patented
cultures. bBPA = beef extract-peptone agar; PDA = potato dextrose agar.

13779 dx.doi.org/10.1021/es504038a | Environ. Sci. Technol. 2014, 48, 13776−13784

Environmental Science & Technology Article

Figure 2. Biofilm formation and viability. (a-c) Changes in bacterial cell number on the PE film sheets. (d-f) Morphotypes of the cells in the mature
biofilm on the PE sheet. (g-i) Fluorescent microscopic images of biofilms, which show cell viability after the 28-day incubation. Live cells are green
and dead cells are red. The PE sheets were covered in CFBAM.

surface topography indicated that the two strains did cause (Figures 4c-e) were constructed based on the transmittance
damage to the PE’s physical integrity. of carbonyl bands (1715 cm−1), with pixels ranging in color
XPS and micro-ATR/FTIR imaging were used to analyze the from blue to red (transmittance: 100−96%). Compared to the
changes to the surface chemical components and function controls, the surfaces of the inoculated samples showed
groups. Figure 4a shows the XPS spectra of C 1s in the increased amounts of color ranging from light blue to red,
inoculated PE sheet surface versus the sterile control. The indicating heterogeneous oxidation by the bacterial strains.
peaking-fitting result of C 1s for the control showed only one A previous study on PE degradation after pretreatment has
peak at 284.8 eV, which was attributed to a −C−C− group in demonstrated that the appearance of carbonyl groups is an
the long chain skeleton of PE (Figure 4a). In the incubated essential sign of PE biodegradation.19 Wasserbauer et al. have
samples, in addition to the peak at 284.8 eV, another peak indicated that PE foils, which were exposed to bacterial
appeared at 288.2 eV and was assigned to the carbonyl bands (Bacillus brevis and Pseudomonas putida) and liver homogenates,
(−CO−), which is generally considered suggestive of PE
showed oxidation structures and signs of carbonyl-like groups
oxidation (Figure 4a).17 Accordingly, the O/C ratios and
in FTIR spectra.45 However, these researchers have not
relative abundance of −CO− group peaks in the incubated
samples are remarkably higher than those of the control (Table reported follow-up work on PE degradation. To the best of
2). our knowledge, no other researchers have reported the
The ATR/FTIR spectra of the inoculated samples’ surfaces appearance of carbonyl groups in the XPS and FTIR spectra
were different from that of the control (Figure 4b). All samples of PE foils or films inoculated with microorganisms. In this
had a common peak at 1450 cm−1, which was attributed to the study, we observed an increasing number of carbonyl group
C−H bending vibration in the PE’s long chain skeleton. peaks in the samples inoculated with strains YT1 and YP1 using
However, the inoculated samples had a peak at 1715 cm−1, XPS spectra and micro-ATR/FTIR image analysis, indicating
which was assigned to the carbonyl bands (−CO−). This that the isolated strains were capable of attacking or oxidizing
observation was consistent with the XPS spectra results PE structures to produce carbonyl groups without any
described above (Figure 4a). Micro-ATR/FTIR images pretreatment, such as photo- or thermo-oxidation.
13780 dx.doi.org/10.1021/es504038a | Environ. Sci. Technol. 2014, 48, 13776−13784
Environmental Science & Technology Article

Figure 3. SEM and AFM observations of the physical surface topography of the sterile control versus the PE sheets incubated with strains YT1 and
YP1 after 28 days.

Weight Loss and Molecular Weight Decrease of PE results in a net loss in the samples of only up to 5%, based on
Samples. PE’s degradation efficiency can be directly measured our estimate of their 60-day removal efficiency.26,28 In this
by the weight loss of samples, while the material’s study, the incubation with YT1 resulted in a net weight loss of
depolymerization can be documented by the shift of its 6.1 ± 0.3%. To date, this is the first reported Enterobacter strain
molecular weight and the release of lower molecular weight with the ability to degrade PE in the literature. The growth
compounds. yields of YP1 and YT1 on PE as a sole carbon source were
The weight loss over time of the PE samples inoculated with estimated to be 0.82 ± 0.05 and 0.66 ± 0.04 g cells per g PE,
the two strains is presented in Figure 5a. During a 60-day respectively.
incubation period in LCFBM, the weight loss of the PE The molecular weight and molecular weight distribution
specimens with inoculation increased consistently, whereas that (MWD) of the PE samples after a 60-day incubation were
of the controls did not (Figure 5a). On day 60, the incubation determined using HT-GPC. The MWDs of the PE samples
with strain YP1 resulted in a net loss in the PE sample of 10.7 ± incubated with strains YT1 and YP1 showed a clear negative
0.2%, which was higher than the 7.5% weight loss reported over trend compared to the controls (Figure 5b). The molecular
a 56-day period for Rhodococcus rubber C208, a previously weights (Mw/Mn) of the incubated PE samples containing
studied PE-degrading strain.21−23 Other researchers have strains YT1 and YP1 were 82,500/24,700 and 78,200/23,900,
reported that incubation with four PE-degrading Bacillus strains respectively, which represents a ∼6−13% reduction from that
13781 dx.doi.org/10.1021/es504038a | Environ. Sci. Technol. 2014, 48, 13776−13784
Environmental Science & Technology Article

Figure 4. Surface chemical analysis of the sterile control and PE samples after the 28-day incubation with strains YP1 and YT1. Samples with
bacterial strains had −CO− bonds, but the control did not. (a) XPS spectra of the C 1s of PE samples. (b) ATR-FTIR spectra of PE samples. (c-
e) Distribution of carbonyl bands (1715 cm−1) imaged with a micro-FTIR microscope with pixels of 1 point per 10 μm. The changes in color
indicate the heterogeneous oxidation of PE samples by the bacterial strains.

Table 2. O/C Atomic Ratios and Relative C 1s Peak Areas of other 12 compounds were more abundant in the PE samples
−C−C− and −CO− Groups inoculated with strains YT1 and YP1 (SI, Table S2). Among
these 12 compounds, four with an m/z range between 400 and
relative area (%)
500 were ten times more abundant in all incubated samples
material O/C −C−C− −CO− than in the control (SI, Table S2). These potential daughter
control 0.02 100 0 products occurred in the media containing an inoculation but
treated by YT1 0.10 97 3 were absent in the control. Although the bacterial cells could
treated by YP1 0.11 96 4 secrete a diverse mixture of secondary metabolic products in
the medium, causing peaks, the ESI-MS results still provide
additional information on the occurrence of PE’s heterotrophic
of the control (Figure 5b internal figure). The decrease in
molecular weight suggested that depolymerization/cleavage of microbial metabolism.
the PE’s long chain structure occurred, and lower molecular In summary, the weight loss and molecular weight decrease
weight fragments were formed in the presence of these bacterial of the PE samples and the appearance of daughter products
strains. indicate that the bacterial strains isolated from waxworm guts
The formation of water-soluble daughter products was were capable of degrading PE.
characterized by ESI-MS.44 The control exhibited a simple Implications. To date, photo- and thermal-based pretreat-
spectrum with only two peaks at m/z 110.1 and 130.1. In the ments have been considered a critical step in the
samples inoculated with the two strains, 12 new peaks biodegradation of PE, and the studies of PE-degrading
appeared, covering an m/z range from 100 to 600 (Figure microorganisms have focused on plastic-contaminated environ-
5c). Most of these compounds contained elements of C, H, and ments, such as soils, compost, landfills, and marine environ-
O in their assumed formulas (SI, Table S2). Fold-change ments.2 This study is the first to report the presence of PE-
analysis was used to identify these 14 compounds with degrading bacteria in the guts of waxworms, which eat PE films.
differential abundances in each of the incubated samples versus PE degradation was confirmed not only by the bacterial growth
the products released from the controls (SI, Table S2). In on PE films, which acted as sole carbon source and the PE
addition to the two compounds at m/z 110.1 and 130.1, the substrate’s weight loss, but also by identification of key
13782 dx.doi.org/10.1021/es504038a | Environ. Sci. Technol. 2014, 48, 13776−13784
Environmental Science & Technology Article

Figure 5. Characterization of PE biodegradation by strains YT1 and YP1 in LCFBM. (a) Changes in the dry weight of the PE specimens over the 60-
day period (mean value ± SD, n = 3). (b) Molecular weight distribution (MWD) and molecular weight (weight-averaged molecular weight (Mw)/
number-averaged molecular weight (Mn)) shift (internal figure) of the PE residues versus the controls after 60 days. (c) ESI-MS analysis showing
degraded oligomeric intermediates in the PE residues after 60 days of incubation versus the controls.

reactions similar to those in photo- and thermal-based

pretreatments, such as changes in surface topography, decreases
■ AUTHOR INFORMATION
Corresponding Author
in hydrophobicity, the formation of carbonyl groups, decrease *Phone/Fax: 86-10-8233-8552. E-mail: yangjun@buaa.edu.cn.
in molecular weight, and release of soluble daughter products. Corresponding author address: School of Chemistry and
The soluble daughter products were not identified in this study, Environment, Beihang University (BUAA), 37 Xueyuan Road,
and future study will be needed to identify the daughter and/or Beijing 100191, P. R. China.
intermediate products of PE degradation and the associated Author Contributions
metabolic pathways. Further research should also focus on the ∥
J.Y. and Y.Y. contributed equally to this work.
ecophysiological relationship between the PE-degrading bac- Notes
teria and waxworms, which may be mutually beneficial, similar The authors declare no competing financial interest.

■
to the microbial symbionts in cow rumens. The discovery of the
presence of PE-degrading microorganisms in waxworm guts has ACKNOWLEDGMENTS
provided a promising direction for further study of the
This research was supported by the National Natural Science
microbial degradation of both PE and other plastics, along
Foundation of China (51373006, 20477002), the State Basic
with new insights into the life cycle of synthetic plastics in Research Program of China (2014CB931800), and the
nature. In addition, the isolation and characterization of more Shenzhen Key Laboratory of Bioenergy (grant
PE-degrading microorganisms from this source and a better CXB201005240001A). The authors thank Ms. Andria Wu for
understanding of the enzymatic system involved in PE her help with manuscript preparation.

■
degradation could be helpful in the development of
remediation approaches for plastic wastes, which could REFERENCES
eliminate plastic pollution concerns. (1) Yang, J.; Song, Y. L.; Qin, X. Y. Biodegradation of polyethylene.

■
*
ASSOCIATED CONTENT
S Supporting Information
Environ. Sci. (in Chinese) 2007, 28 (5), 1165−1168.
(2) Restrepo-Flórez, J. M.; Bassi, A.; Thompson, M. R. Microbial
degradation and deterioration of polyethylene-A review. Int.
Biodeterior. Biodegrad. 2014, 88, 83−90.
Summary of the updated reported virgin PE-degrading (3) Roy, P. K.; Hakkarainen, M.; Varma, I. K.; Albertsson, A. C.
microorganisms (Table S1). Fold-change analysis of the Degradable polyethylene: fantasy or reality. Environ. Sci. Technol. 2011,
compounds released from the biodegradation of PE (Table 45 (10), 4217−4227.
(4) Zettler, E. R.; Mincer, T. J.; Amaral-Zettler, L. A. Life in the
S2). Photograph of waxworms eating PE film (Figure S1). “Plastisphere”: microbial communities on plastic marine debris.
Changes in tensile strength of the PE film samples incubated Environ. Sci. Technol. 2013, 47 (13), 7137−7146.
with eight bacterial strains (Figure S2). This material is (5) Ammala, A.; Bateman, S.; Dean, K.; Petinakis, E.; Sangwan, P.;
available free of charge via the Internet at http://pubs.acs.org. Wong, S.; Yuan, Q.; Yu, L.; Patrick, C.; Leong, K. H. An overview of

13783 dx.doi.org/10.1021/es504038a | Environ. Sci. Technol. 2014, 48, 13776−13784

Environmental Science & Technology Article

degradable and biodegradable polyolefins. Prog. Polym. Sci. 2011, 36 (26) Sudhakar, M.; Doble, M.; Murthy, P. S.; Venkatesan, R. Marine
(8), 1015−1049. microbe-mediated biodegradation of low-and high-density poly-
(6) Shah, A. A.; Hasan, F.; Hameed, A.; Ahmed, S. Biological ethylenes. Int. Biodeterior. Biodegrad. 2008, 61 (3), 203−213.
degradation of plastics: a comprehensive review. Biotechnol. Adv. 2008, (27) Nowak, B.; Pająk, J.; Drozd-Bratkowicz, M.; Rymarz, G.
26 (3), 246−265. Microorganisms participating in the biodegradation of modified
(7) Gautam, R.; Bassi, A. S.; Yanful, E. K. A review of biodegradation polyethylene films in different soils under laboratory conditions. Int.
of synthetic plastic and foams. Appl. Biochem. Biotechnol. 2007, 141 Biodeterior. Biodegrad. 2011, 65 (6), 757−767.
(1), 85−108. (28) Harshvardhan, K.; Jha, B. Biodegradation of low-density
(8) Jones, P. H.; Prasad, D.; Heskins, M.; Morgan, M. H.; Guillet, J. polyethylene by marine bacteria from pelagic waters, Arabian Sea,
E. Biodegradability of photodegraded polymers. I. Development of India. Mar. Pollut. Bull. 2013, 77 (1), 100−106.
experimental procedures. Environ. Sci. Technol. 1974, 8 (10), 919−923. (29) Balasubramanian, V.; Natarajan, K.; Hemambika, B.; Ramesh,
(9) Albertsson, A. C.; Bánhidi, Z. G.; Beyer-Ericsson, L. L. N.; Sumathi, C. S.; Kottaimuthu, R.; Rajesh Kannan, V. High-density
polyethylene (HDPE)-degrading potential bacteria from marine
Biodegradation of synthetic polymers. III. The liberation of 14CO2
ecosystem of Gulf of Mannar, India. Lett. Appl. Microbiol. 2010, 51
by molds like Fusarium redolens from 14C labeled pulverized high-
(2), 205−211.
density polyethylene. J. Appl. Polym. Sci. 1978, 22 (12), 3435−3447. (30) Yoon, M. G.; Jeon, H. J.; Kim, M. N. Biodegradation of
(10) Albertsson, A. C.; Karlsson, S. The three stages in degradation polyethylene by a soil bacterium and AlkB cloned recombinant cell. J.
of polymers−polyethylene as a model substance. J. Appl. Polym. Sci. Biorem. Biodegrad. 2012, 3 (4), 145.
1988, 35 (5), 1289−1302. (31) Tribedi, P.; Sil, A. K. Low-density polyethylene degradation by
(11) Pegram, J. E.; Andrady, A. L. Outdoor weathering of selected Pseudomonas sp. AKS2 biofilm. Environ. Sci. Pollut. Res. 2013, 20 (6),
polymeric materials under marine exposure conditions. Polym. Degrad. 4146−4153.
Stab. 1989, 26 (4), 333−345. (32) Kyaw, B. M.; Champakalakshmi, R.; Sakharkar, M. K.; Lim, C.
(12) Ohtake, Y.; Kobayashi, T.; Asabe, H.; Murakami, N.; Ono, K. S.; Sakharkar, K. R. Biodegradation of low density polythene (LDPE)
Biodegradation of low−density polyethylene, polystyrene, polyvinyl by Pseudomonas species. Indian J. Microbiol. 2012, 52 (3), 411−419.
chloride, and urea formaldehyde resin buried under soil for over 32 (33) Jeon, H. J.; Kim, M. N. Isolation of a thermophilic bacterium
years. J. Appl. Polym. Sci. 1995, 56 (13), 1789−1796. capable of low-molecular-weight polyethylene degradation. Biodegra-
(13) Ohtake, Y.; Kobayashi, T.; Asabe, H.; Murakami, N.; Ono, K. dation 2013, 24 (1), 89−98.
Oxidative degradation and molecular weight change of LDPE buried (34) Albertsson, A. C. Biodegradation of synthetic polymers. II. A
under bioactive soil for 32−37 years. J. Appl. Polym. Sci. 1998, 70 (9), limited microbial conversion of 14C in polyethylene to 14CO2 by some
1643−1648. soil fungi. J. Appl. Polym. Sci. 1978, 22 (12), 3419−3433.
(14) Artham, T.; Sudhakar, M.; Venkatesan, R.; Madhavan Nair, C.; (35) Raghavan, D.; Torma, A. E. DSC and FTIR characterization of
Murty, K. V. G. K.; Doble, M. Biofouling and stability of synthetic biodegradation of polyethylene. Polym. Eng. Sci. 1992, 32 (6), 438−
polymers in sea water. Int. Biodeterior. Biodegrad. 2009, 63 (7), 884− 442.
890. (36) Esmaeili, A.; Pourbabaee, A. A.; Alikhani, H. A.; Shabani, F.;
(15) Albertsson, A. C.; Andersson, S. O.; Karlsson, S. The Esmaeili, E. Biodegradation of low-density polyethylene (LDPE) by
mechanism of biodegradation of polyethylene. Polym. Degrad. Stab. mixed culture of Lysinibacillus xylanilyticus and Aspergillus niger in soil.
1987, 18 (1), 73−87. PloS One 2013, 8 (9), e71720.
(16) Tokiwa, Y.; Calabia, B. P.; Ugwu, C. U.; Aiba, S. (37) Iiyoshi, Y.; Tsutsumi, Y.; Nishida, T. Polyethylene degradation
by lignin-degrading fungi and manganese peroxidase. J. Wood Sci.
Biodegradability of plastics. Int. J. Mol. Sci. 2009, 10 (9), 3722−3742.
1998, 44 (3), 222−229.
(17) Albertsson, A. C.; Bánhidi, Z. G. Microbial and oxidative effects
(38) Yamada-Onodera, K.; Mukumoto, H.; Katsuyaya, Y.; Saiganji,
in degradation of polyethylene. J. Appl. Polym. Sci. 1980, 25 (8), 1655−
A.; Tani, Y. Degradation of polyethylene by a fungus, Penicillium
1671. simplicissimum YK. Polym. Degrad. Stab. 2001, 72 (2), 323−327.
(18) Albertsson, A. C.; Erlandsson, B.; Hakkarainen, M.; Karlsson, S. (39) Burd, D. Plastic Not Fantastic. 2008. wwsef.uwaterloo.ca/
Molecular weight changes and polymeric matrix changes correlated archives/2008/08BurdReport.pdf (accessed Oct 20, 2014).
with the formation of degradation products in biodegraded poly- (40) Riudavets, J.; Salas, I.; Pons, M. J. Damage characteristics
ethylene. J. Environ. Polym. Degrad. 1998, 6 (4), 187−195. produced by insect pests in packaging film. J. Stored Prod. Res. 2007, 43
(19) Arutchelvi, J.; Sudhakar, M.; Arkatkar, A.; Dolbe, M.; Bhaduri, (4), 564−570.
S.; Uppara, P. V. Biodegradation of polyethylene and polypropylene. (41) ASTM G 22-76. Standard practice for determining resistance of
Indian J. Biotechnol. 2008, 7 (1), 9−22. plastics to bacteria; 1996.
(20) Andrady, A. L. Assessment of environmental biodegradation of (42) ASTM D 3826-98. Standard practice for determining
synthetic polymers. J. Macromol. Sci., Polym. Rev. 1994, 34 (1), 25−76. degradation end point in degradable polyethylene and polypropylene
(21) Gilan, I.; Hadar, Y.; Sivan, A. Colonization, biofilm formation using a tensile test; 1998.
and biodegradation of polyethylene by a strain of Rhodococcus ruber. (43) Nagle, D. J.; George, G. A.; Rintoul, L.; Fredericks, P. M. Use of
Appl. Microbiol. Biotechnol. 2004, 65 (1), 97−104. micro-ATR/FTIR imaging to study heterogeneous polymer oxidation
(22) Sivan, A.; Szanto, M.; Pavlov, V. Biofilm development of the by direct solvent casting onto the ATR IRE. Vib. Spectrosc. 2010, 53
polyethylene-degrading bacterium Rhodococcus ruber. Appl. Microbiol. (1), 24−27.
Biotechnol. 2006, 72 (2), 346−352. (44) Odelius, K.; Höglund, A.; Kumar, S.; Hakkarainen, M.; Ghosh,
(23) Santo, M.; Weitsman, R.; Sivan, A. The role of the copper- A. K.; Bhatnagar, N.; Albertsson, A. C. Porosity and pore size regulate
binding enzyme−laccase−in the biodegradation of polyethylene by the the degradation product profile of polylactide. Biomacromolecules 2011,
actinomycete Rhodococcus ruber. Int. Biodeterior. Biodegrad. 2013, 84, 12 (4), 1250−1258.
204−210. (45) Wasserbauer, R.; Beranova, M.; Vancurova, D.; Doležel, B.
(24) Satlewal, A.; Soni, R.; Zaidi, M. G. H.; Shouche, Y.; Goel, R. Biodegradation of polyethylene foils by bacterial and liver homoge-
Comparative biodegradation of HDPE and LDPE using an nates. Biomaterials 1990, 11 (1), 36−40.
indigenously developed microbial consortium. J. Microbiol. Biotechnol.
2008, 18 (3), 477−482.
(25) Watanabe, T.; Ohtake, Y.; Asabe, H.; Murakami, N.; Furukawa,
M. Biodegradability and degrading microbes of low-density poly-
ethylene. J. Appl. Polym. Sci. 2009, 111 (1), 551−559.

13784 dx.doi.org/10.1021/es504038a | Environ. Sci. Technol. 2014, 48, 13776−13784