metodologia da cgsm

Prévia do material em texto

Biodegradation of Polyethylene and Polystyrene by Greater Wax
Moth Larvae (Galleria mellonella L.) and the Effect of Co-diet
Supplementation on the Core Gut Microbiome
Yu Lou, Pererva Ekaterina, Shan-Shan Yang, Baiyun Lu, Bingfeng Liu, Nanqi Ren, Philippe F.-X. Corvini,
and Defeng Xing*
Cite This: Environ. Sci. Technol. 2020, 54, 2821−2831 Read Online
ACCESS Metrics & More Article Recommendations
ABSTRACT: Plastics waste and microplastics including poly-
ethylene (PE) and polystyrene (PS) have been an environmental
concern for years. Recent research has revealed that larvae of Galleria
mellonella are capable of biodegrading low density PE film. In this
study, we tested the feasibility of enhancing larval survival and the
effect of supplementing the co-diet on plastic degradation by feeding
the larvae beeswax or wheat bran as a co-diet. Significant mass loss of
plastic was observed over a 21-day period, i.e., with respective
consumption of 0.88 and 1.95 g by 150 larvae fed only either PS or
PE. The formation of CO and C−O containing functional groups
and long chain fatty acids as the metabolic intermediates of plastics
in the residual polymers indicated depolymerization and biode-
gradation. Supplementing beeswax and bran increased the survival
rates but decreased the consumption of plastic. The changes in the gut microbiome revealed that Bacillus and Serratia were
significantly associated with the PS and PE diets. Beeswax and bran showed different shaping effects on the core gut microbiome of
larvae fed the PE and PS. These results suggest that supplementing the co-diet affected the physiological properties of the larvae and
plastic biodegradation and shaped the core gut microbiome.
1. INTRODUCTION
Plastic accumulation and pollution in the ecosystem have
aroused attention worldwide. The world’s production of
thermoplastics and polyurethanes was 280 million tons in
2016, and this number increased to 335 million tons if
thermosets, adhesives, coatings, and sealants were counted.1
Among them, over 80 million tons of polyethylene (PE) were
produced, making it the world’s most important plastic, and
polystyrene (PS) is the third most important polymer in
human life and industry.2 Large production amounts with low
recycling rates have resulted in the accumulation of plastics in
the environment, further risking the survival of wildlife and
human health.3 Previous studies have been conducted on the
biodegradation of plastic, in which several bacteria and fungi
were found to be capable of degrading plastic materials,
including PS and PE, but at low degradation rates.4−6 Bacteria
from landfills and oceans have been reported to degrade PS,
PE, and their mixtures,7−12 and a recent study summarized
plastics and microplastics in the ocean, as well as the
colonization and possible biodegradation of plastic by marine
bacteria.13,14 The carbon isotope test was performed on some
invertebrates (earthworms and snails), and no 14CO2 was
produced.15 Mandibulate insects, such as Rhyzopertha dominica
and Lasioderma serricorne, are capable of chewing and ingesting
plastic bags, but little is known about the gut microbiome of
plastic-chewing insects.16
Recent research indicates that two invertebrates, yellow
mealworms (larva of Tenebrio molitor) and greater wax moth
larvae (Galleria mellonella), have the capacity to degrade
plastics.17−23 Mealworms chew and ingest PS and PE
foams.18−20,22,23 The plastic consumption rate of mealworm
is higher than that of bacteria and fungi isolated previously; up
to half of ingested PS or PE can be digested within a 12−15 h
retention time in the mealworm gut after a 1−3 week adaption
period.18−20 During biodegradation of PS by mealworms, the
gut microbiome plays a key role in plastic depolymerization
because PS depolymerization is inhibited when gut microbes
are depressed by feeding the antibiotic gentamycin.18,19,23
Although plastics can be ingested by mealworms, plastics as the
Received: November 20, 2019
Revised: January 19, 2020
Accepted: February 3, 2020
Published: February 3, 2020
Articlepubs.acs.org/est
© 2020 American Chemical Society
2821
https://dx.doi.org/10.1021/acs.est.9b07044
Environ. Sci. Technol. 2020, 54, 2821−2831
D
ow
nl
oa
de
d 
vi
a 
U
N
IV
 O
F 
E
X
E
T
E
R
 o
n 
Ju
ly
 1
7,
 2
02
0 
at
 1
3:
28
:1
1 
(U
T
C
).
Se
e 
ht
tp
s:
//p
ub
s.
ac
s.
or
g/
sh
ar
in
gg
ui
de
lin
es
 f
or
 o
pt
io
ns
 o
n 
ho
w
 to
 le
gi
tim
at
el
y 
sh
ar
e 
pu
bl
is
he
d 
ar
tic
le
s.
https://pubs.acs.org/action/doSearch?field1=Contrib&text1="Yu+Lou"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdf
https://pubs.acs.org/action/doSearch?field1=Contrib&text1="Pererva+Ekaterina"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdf
https://pubs.acs.org/action/doSearch?field1=Contrib&text1="Shan-Shan+Yang"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdf
https://pubs.acs.org/action/doSearch?field1=Contrib&text1="Baiyun+Lu"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdf
https://pubs.acs.org/action/doSearch?field1=Contrib&text1="Bingfeng+Liu"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdf
https://pubs.acs.org/action/doSearch?field1=Contrib&text1="Nanqi+Ren"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdf
https://pubs.acs.org/action/doSearch?field1=Contrib&text1="Philippe+F.-X.+Corvini"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdf
https://pubs.acs.org/action/doSearch?field1=Contrib&text1="Defeng+Xing"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdf
https://pubs.acs.org/action/doSearch?field1=Contrib&text1="Defeng+Xing"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdf
https://pubs.acs.org/action/showCitFormats?doi=10.1021/acs.est.9b07044&ref=pdf
https://pubs.acs.org/doi/10.1021/acs.est.9b07044?ref=pdf
https://pubs.acs.org/doi/10.1021/acs.est.9b07044?goto=articleMetrics&ref=pdf
https://pubs.acs.org/doi/10.1021/acs.est.9b07044?goto=recommendations&?ref=pdf
https://pubs.acs.org/doi/10.1021/acs.est.9b07044?fig=tgr1&ref=pdf
pubs.acs.org/est?ref=pdf
https://pubs.acs.org?ref=pdf
https://pubs.acs.org?ref=pdf
https://dx.doi.org/10.1021/acs.est.9b07044?ref=pdf
https://pubs.acs.org/est?ref=pdf
https://pubs.acs.org/est?ref=pdf
only diet may cause a lower survival rate (SR) than bran-fed
mealworms.18−20
The PE degradation by greater wax moth larvae (G.
mellonella) was previously reported as a 92 mg weight loss of
a PE shopping bag by an average of 100 larvae in 12 h.21 This
previous study confirmed the degradation ability of PE, but
survival of the wax moth on a plastic diet was not investigated.
On the basis of mealworm research results, we assumed that
supplying plastic as the only diet over the long-term could
negatively affect larval survival, and possibly impact their
plastic biodegradation ability, as plastics do not contain the
necessary nutrients for cell growth and synthesis of enzymes.
Wheat bran and wax have been used as supplements to
improve the breeding of mealworms (T. molitor) and
waxworms (G. mellonella).24 Hence, we considered adding a
basic food source for the greater wax moth, such as beeswax or
wheat bran as cosubstrates, to shape the larvae gut microbiome
and improve larval survival and their ability to degrade plastics.
Previous mealworm research has illustrated the importance of
mealworm gut microbes in the biodegradation of PS.
Therefore, it is necessary to explore the response of the gut
microbiome to plastic diets and co-diets, to further illustrate
the roles of larval gut microbes in plastic metabolic
pathways.17,21
Additionally, only PE has been used to assess biodegradation
by the wax moth previously,21 and other polymer plastics with
more complex monomers and structures have not been
examined. We hypothesized that the enzymatic activities and
gut microbiome of G. mellonella larvaemay not be polymer-
specific and could degrade both PE and PS. In this study, we
tested this hypothesis and investigated the effect of co-diets
(beeswax and bran) on plastic degradation ability and survival
of larvae. High throughput 16S rRNA sequencing was
performed to detect changes in the core gut microbiome in
greater wax moth larvae supplied with beeswax and bran.
2. MATERIALS AND METHODS
2.1. Source of the Greater Wax Moth and the Plastic
Feedstocks. G. mellonella larvae (15−25 mm long) were
purchased from Huiyude Co. (Tianjin, China). PS foam
(density, 0.006 g/cm3) was purchased from Zhenchuang
Plastic Co. (Jiangsu, China). PE foam (density, 0.019 g/cm3)
was purchased from Baiyangchaoqian Packaging Factory
(Zhejiang, China). Beeswax and bran were utilized as the
control diets for the greater wax moth larvae. Yellow beeswax
in granules was purchased from Mumifang Co. (Hainan,
China). The bran was obtained from a farm in Baoji, Shanxi,
China.
2.2. Plastic Weight Loss and Survival of G. mellonella
Larvae. Prior to testing, the larvae were fed food containing
mixed nutrients, including honey, beeswax, and bran. The
larvae were starved for 36 h before the experiment to prevent
any effect of previously eaten food. There were nine different
feeding conditions: starvation, bran, beeswax, PS, PS + bran,
PS + beeswax, PE, PE + bran, and PE + beeswax. Each test diet
was assessed in triplicate. There were 150 larvae in each 25 ×
15 × 5 cm3 container. The containers were placed in an
incubator maintained at 27.5 ± 0.5 °C and with humidity of 75
± 5%. The SR of the larvae and the weight loss of the plastic
were measured every 3 days for 21 days. Dead larvae were
removed immediately to prevent any possible disease.
2.3. Chemical Property Changes of the Frass of
Plastic-Fed Larvae. After a 21-day of continuous feeding,
larvae fed the nine different diets were cleaned and transferred
to new containers to collect the frass. The frass was collected
every 24 h for 3 successive days under the starvation condition
to avoid the influence of any nutrients, and then stored at −20
°C for later chemical property tests. A thermogravimetric
analysis (TGA) was performed to detect the thermal
decomposition of the samples using a thermogravimetric
analyzer (Netzsch, Selb, Germany). Frass (1 mg for each diet
group), along with PS and PE foam as control samples, was
tested from 30 to 600 °C at a heating rate of 10 °C/min, with
nitrogen as the protective gas (purity >99%). Fourier
transform infrared spectroscopy (FTIR) was conducted to
determine the changes in chemical compounds and the
functional groups. The frass samples were placed in a drying
oven for 12 h to removal residual water before the test. The
FTIR analysis was performed using FTIR spectrometer
Spectrum One (PerkinElmer, Shelton, CT, U.S.A.). The
wavenumber of the spectra were in the range of 4000−500
cm−1, with spectral resolution of 4.00 cm−1 and a minimum
scanning number of 5.
Gel permeation chromatography (GPC) analysis of PS
sample and the frass of larvae fed PS and supplementary
nutrients was performed to verify depolymerization of PS
digested by the larval gut. The samples for the GPC analysis
were pretreated following a method described previously.22
Briefly, the residual polymers were extracted from the frass
samples (0.8−1.0 g) with tetrahydrofuran (THF; 120 mL,
purity ≥99.9%, Merck KGaA, Darmstadt, Germany) as the
solvent for 12 h at 90 °C using a Soxhlet extractor. The
solvent-containing residual polymers were evaporated in a
rotary evaporator to obtain the residual polymers, which were
weighed and redissolved in THF to obtain a 1 mg/mL
solution. The PS sample treated as the control group
underwent the same extraction method to obtain an effective
average molecular weight comparison between the PS control
and the residual polymers extracted from the frass. GPC was
performed in duplicate for each group using a 1 mg/mL
solution, an injected volume of 50 μL, and a high performance
liquid chromatography system (Agilent 1100, Waldbronn,
Germany) equipped with a PLgel 5 μm MIXED-C (300 × 7.5
mm2) column at a flow rate of 1.0 mL/min and temperature of
30 °C.
Gas chromatography−mass spectrometry (GC−MS) was
performed to further investigate the intermediates and
products of plastics biodegradation. The samples were
pretreated based on a previous method with modifications.7,17
Briefly, 0.05 g of frass sample from the PE/PS-fed group was
extracted with 10 mL chloroform/THF and the guts of five
larvae fed PE/PS were extracted with 10 mL of chloroform/
THF: methanol (2:1). The samples were gently heated for 2 h
and immersed for 15 h. The solvents were evaporated, and the
residual polymers were redissolved in 100% hexane for the
GC−MS analysis. The PS and PE samples underwent the same
pretreatment and were treated as the control groups. The GC−
MS analysis was performed using an Agilent 7000 GC/MS
Triple Quad with an Agilent 7890A GC system (Agilent
Technologies, Palo Alto, CA, U.S.A.), equipped with an HP-5
(30 m × 250 μm × 0.25 μm) column and helium as the carrier
gas. The oven temperature programming started at 40 °C
followed by a hold for 4 min, an increased to 280 °C at a rate
of 10 °C/min, and a 5-min hold. The compounds were
identified based on the NIST17 database.
Environmental Science & Technology pubs.acs.org/est Article
https://dx.doi.org/10.1021/acs.est.9b07044
Environ. Sci. Technol. 2020, 54, 2821−2831
2822
pubs.acs.org/est?ref=pdf
https://dx.doi.org/10.1021/acs.est.9b07044?ref=pdf
2.4. Gut Microbiome Analysis Based on Illumina
Hiseq Sequencing of the 16S rRNA Gene. After the 21-
day experiment, 15 larvae from each diet were randomly
selected for DNA extraction of their gut microbiome to
eliminate possible individual variations among larvae fed the
same diet. Genomic DNA was extracted using the FastDNA
SPIN Kit for Soil (MP Biomedicals Co., Shanghai, China)
according to the manufacturer’s instructions. The fusion
primer with dual index and adapters was used for the
polymerase chain reaction (PCR) of the genomic DNA to
sequence the V4 region of the 16S rRNA gene.20 The raw
sequencing data were filtered to eliminate unqualified
sequences and adapters, and then further combined to form
pair-end reads using the Illumina Hiseq platform. The pair-end
reads were further merged to tags using FLASH (Fast Length
Adjustment of Short reads, v1.2.11), which were then clustered
to operational taxonomic units (OTUs) at the sequence
similarity of 97% using USEARCH software (v7.0.1090).
Alpha diversity, beta diversity, and a differential abundance
analysis were conducted based on the OTU sequences and
taxonomic ranks as described previously.25−27 Unassigned
OTUs were removed, and the remaining OTUs were used for
the taxonomic classification analysis. Ribosomal Database
Project (RDP) Classifier v2.2 was used to assign the taxonomic
ranks by comparing OTU representative sequences with the
Greengene database, using a 0.6 confidence value as the cutoff.
3. RESULTS
3.1. Survival and Plastic Consumption of G. mello-
nella Larvae. The G. mellonella larvae were confirmed to chew
and ingest Styrofoam (PS) and polyethylene foams (PE)
(Figure 1). With an initial 150 larvae in each group, the weight
loss of PE was 1.95 g for larvae fed PE only, and the weight loss
of PS was 0.88 g for PS-fed larvae after the 21-day experiment
(Figure 2a). After beeswax and bran were supplied as
additional nutrients, the PE + beeswax-fed and PE + bran-
fed larvae consumed 1.27 and 0.49 g of PE, and the PS +
Figure 1. Plastic and supplementary nutrients were ingested by the greater wax moth larvae fed a polystyrene (PS) diet (a), a polyethylene (PE)
diet (b), a PS + beeswax diet (c), a PE + beeswax diet (d), a PS + bran diet (e), and a PE + beeswax diet (f).
Environmental Science & Technology pubs.acs.org/est Article
https://dx.doi.org/10.1021/acs.est.9b07044
Environ. Sci. Technol. 2020, 54, 2821−2831
2823
https://pubs.acs.org/doi/10.1021/acs.est.9b07044?fig=fig1&ref=pdf
https://pubs.acs.org/doi/10.1021/acs.est.9b07044?fig=fig1&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.est.9b07044?fig=fig1&ref=pdf
https://pubs.acs.org/doi/10.1021/acs.est.9b07044?fig=fig1&ref=pdf
pubs.acs.org/est?ref=pdf
https://dx.doi.org/10.1021/acs.est.9b07044?ref=pdf
beeswax-fed and PS + bran-fed larvae consumed 0.64 and 0.32
g of PS, respectively. At the end of the 21-day experiment, the
SR of larvae fed PS only was 23.6% and that of the PE-fed
larvae was 35.3% (Figure 2b). The SR increased to 77.3% and
60% for the PS + beeswax and PS + bran groups, respectively.
Similarly, the SR of the PE + beeswax and PE + bran groups
were about 66%.
3.2. Characterization of Plastic Biodegradation. The
TGA indicated an obvious mass loss in the PS control that
occurred from 370 to 430 °C, which sharply dropped around
85% of total PS weight (Figure 3a), and the maximum
decomposition rate occurred at 405 °C. In contrast, no
obvious mass loss was observed in the TGA curves for the frass
of PS-fed larvae. The mass loss of PS-fed larvae frass decreased
gradually from 30 to 600 °C with an 74.1% mass loss, in which
two stages of mass loss were relatively distinct with peak
decomposition rates at 302 and 425 °C. The mass loss of the
PS + beeswax-fed and PS + bran-fed larvae exhibited similar
trends with those of the PS-fed larvae, including two stages of
relatively obvious mass loss. Similarly, mass loss of the PE
control mainly occurred in one stage at 400 to 480 °C, with the
maximum decomposition rate at 463 °C (Figure 3b). PE-fed
larval frass showed a gradual decrease in mass loss with two
obvious stages, and the peak decomposition rates were at 307
and 380 °C.
The FTIR spectra of frass from the PS-fed larvae exhibited
an obvious peak at around 1700 cm−1 and peaks at 1050−1150
cm−1, which represented CO and C−O stretching,
respectively (Figure 4). The peak at 2500−3500 cm−1
represented the hydrogen bonds of the hydrophobic and
carboxylic acid groups, and the FTIR spectrum of PS-fed larvae
frass in this range was broader than that of the untreated PS.
The frass of larvae fed PS + beeswax and PS + bran produced
similar FTIR spectra as that of PS-fed larvae frass. The FTIR
spectra of PE-fed, PE + bran-fed, and PE + beeswax-fed larvae
frass also exhibited CO and C−O stretching peaks and a
broader hydrogen bonds peak compared to the untreated PE.
The GPC analysis of frass from PS-fed larvae indicated a
shift in the average molecular weight distribution in the
direction of a higher molecular weight compared to the curve
for untreated PS in the range of log Mw 4.5−6.5, representing a
slight increase in the average molecular weight from untreated
PS to the frass of PS-fed larvae (Figure 5). The number-
averaged molecular weight (Mn) of PS-fed diet increased from
1.321 × 105 g/mol (PS sample) to 1.469 × 105 g/mol (PS-
fed), and the weight-averaged molecular weight (Mw)
increased from 3.617 × 105 g/mol (PS sample) to 3.778 ×
105 g/mol (PS-fed). Similarly, the frass of the PS + bran-fed
larvae exhibited an increase of average molecular weight
compared to the untreated PS, along with another peak
obtained closely to the peak of the bran sample, which shifted
toward a lower average molecular weight. The peak for the PS
+ beeswax diet was detected near the peak of the beeswax diet.
The GC−MS analysis revealed the formation of long chain
fatty acids, such as oleic acid (C18H34O2) and octadecanoic
acid (C18H36O2), in the frass of PS-fed larvae, and the relative
abundance of n-hexadecanoic acid (C16H32O2) also increased
(Figure 6a). The peaks possibly representing more complex
compounds with the benzene ring structure of phthalate
(C24H38O4; phthalic acid, di(2-propylpentyl) ester), cyclo-
hexane,1,3,5-triphenyl- (C24H24), and β-hydroxyphenethylben-
zoate (C15H14O3) decreased in the frass and intestinal samples
of PS-fed larvae. Similarly, long chain fatty acids, such as n-
hexadecanoic acid and oleic acid, were also observed in the
intestine of PE-fed larvae, along with the long chain carboxylic
acid esters, such as ethyl hexadecanoate (C18H36O2), linoleic
Figure 2. Plastic consumption by greater wax moth larvae fed
polystyrene (PS) or polyethylene (PE) (a) and larvae survival rates
for the nine different diets (b).
Figure 3. Thermogravimetric analysis (TGA) curve for plastic and
frass of plastic-fed larvae. (a) TGA curve for polystyrene (PS) sample
and frass of larvae fed PS. (b) TGA curve for polyethylene (PE)
sample and frass of larvae fed PE.
Environmental Science & Technology pubs.acs.org/est Article
https://dx.doi.org/10.1021/acs.est.9b07044
Environ. Sci. Technol. 2020, 54, 2821−2831
2824
https://pubs.acs.org/doi/10.1021/acs.est.9b07044?fig=fig2&ref=pdf
https://pubs.acs.org/doi/10.1021/acs.est.9b07044?fig=fig2&ref=pdf
https://pubs.acs.org/doi/10.1021/acs.est.9b07044?fig=fig2&ref=pdf
https://pubs.acs.org/doi/10.1021/acs.est.9b07044?fig=fig2&ref=pdf
https://pubs.acs.org/doi/10.1021/acs.est.9b07044?fig=fig3&ref=pdf
https://pubs.acs.org/doi/10.1021/acs.est.9b07044?fig=fig3&ref=pdf
https://pubs.acs.org/doi/10.1021/acs.est.9b07044?fig=fig3&ref=pdf
https://pubs.acs.org/doi/10.1021/acs.est.9b07044?fig=fig3&ref=pdf
pubs.acs.org/est?ref=pdf
https://dx.doi.org/10.1021/acs.est.9b07044?ref=pdf
acid ethyl ester (C20H36O2), and (E)-9-octadecenoic acid ethyl
ester (C20H38O2) (Figure 6b). Weak signals of methyl 9,12-
octadecadienoate (C19H34O2) and methyl 9-octadecenoate
(C19H36O2) were also exhibited in the intestines of the PE-fed
larvae. These peaks likely represented 2-hexadecanoyl glycerol
(C19H38O4) and glycerol 1-monostearate (C21H42O4) which
obviously decreased in the frass and intestinal samples
compared to the PE control. The formation of long chain
fatty acids and the decrease of more complex long chain
carboxylic acid esters with hydroxide radical or benzene ring
structures indicated digestion and biodegradation of PS and
PE.
3.3. Bacterial Community Diversity of the Larval Gut
Supplemented with Different Diets. Illumina sequencing
of the 16S rRNA gene amplicons showed that 82 000−84 000
clean reads were obtained from all samples, with good’s
coverage of 99% (Table 1). The OTUs of PE-fed, PS-fed, and
bran-fed larvae were relatively lower than those of larvae fed a
co-diet.
The Shannon and Simpson indices indicated that species
richness increased substantially after adding bran and beeswax.
In contrast, the PS-fed and PE-fed larval gut microbiomes
exhibited much lower community diversity. The gut micro-
biomes of larvae fed the co-diet, bran, or beeswax showed a
higher evenness than those fed the PS or PE due to and a
similar number of OTUs.
A principal coordinate analysis (PCoA) and heatmap based
on Bray−Curtis and weighted UniFrac distances indicated that
the gut microbiome of larvae fed PE was distinct from the
others (Figure 7). The microbiomes of the larvae fed PS and
PS + beeswax were similar, as were the larvae fed bran and PE
+ bran (Figure 7b and d). The alpha and beta diversity analysis
indicated that the gut microbiome of larvae fed PE could be
shaped by the beeswax diet, as the gut microbiome of larvae
fed PS was shaped by bran.
3.4. Core Gut Microbiome in Larvae Supplemented
with Different Diets. Firmicutes and Proteobacteria were the
predominant phyla in the guts of larvae under the nine
different feeding conditions (Figure 8a). An obvious difference
in predominant phyla was detected between the gut micro-
biomes of larvae fed only PE or PS. The majority of
predominant phyla belonged to Proteobacteria in larvae fed
PE and Firmicutes in larvae fed PS, respectively. The core
microbiome was markedly different between the larval gut fed
PS and PE. The predominant population in the gut of PE-fed
larvae was affiliated with Serratia sp. (relative abundance
94.1%), whereas Enterococcus sp. (relative abundance 95.5%)
dominated the core gut microbiome of PS-fed larvae (Figure
8b and Figure 9). Enterococcus sp. also occurred in the gut
microbiome of the larvae fed the nine different diets, and was
the major population in most of the diets (Figure 9).
Enterococcussp. was the dominant population in the core gut
microbiome of larvae fed PS + beeswax, with 83.6% relative
abundance, which was an 89.0% increase than that from larvae
fed beeswax only (relative abundance 44.2%). In contrast, the
dominant populations in the guts of starved larvae belonged to
Enterococcus sp. and Bacillus sp.. Larvae fed bran (including
bran, PS + bran, and PE + bran) maintained a certain
abundance of Okibacterium in the gut microbiome, suggesting
that the genus Okibacterium is related to the digestion of bran
(Figure 8b).
The differential abundance analysis exhibited that Bacillus
sp. and Serratia sp. populations in the PE-fed group were much
higher than those in the bran-fed and beeswax-fed groups
(Figure 10a and Figure 10b), indicating that these two genera
were significantly related to PE-fed diet. Bacillus and Serratia
also exhibited significant differences associated with the PS-fed
group when bran was used as the control diet, along with
Pseudomonas sp., Geobacillus sp., and Propionibacterium sp.. As
Figure 4. Fourier transform infrared spectroscopy analysis (FTIR) for
plastic and frass of plastic-fed larvae. FTIR spectra for plastic samples
and frass of larvae fed plastics ranging from 4000 cm−1 to 500 cm−1
(a) and ranging from 1150 cm−1 to 1050 cm−1 for the characteristic
C−O stretching peak (b).
Figure 5. Gel permeation chromatography (GPC) analysis for
polystyrene (PS) sample (as control) and frass of larvae fed PS and
supplementary nutrients.
Environmental Science & Technology pubs.acs.org/est Article
https://dx.doi.org/10.1021/acs.est.9b07044
Environ. Sci. Technol. 2020, 54, 2821−2831
2825
https://pubs.acs.org/doi/10.1021/acs.est.9b07044?fig=fig4&ref=pdf
https://pubs.acs.org/doi/10.1021/acs.est.9b07044?fig=fig4&ref=pdf
https://pubs.acs.org/doi/10.1021/acs.est.9b07044?fig=fig4&ref=pdf
https://pubs.acs.org/doi/10.1021/acs.est.9b07044?fig=fig4&ref=pdf
https://pubs.acs.org/doi/10.1021/acs.est.9b07044?fig=fig5&ref=pdf
https://pubs.acs.org/doi/10.1021/acs.est.9b07044?fig=fig5&ref=pdf
https://pubs.acs.org/doi/10.1021/acs.est.9b07044?fig=fig5&ref=pdf
https://pubs.acs.org/doi/10.1021/acs.est.9b07044?fig=fig5&ref=pdf
pubs.acs.org/est?ref=pdf
https://dx.doi.org/10.1021/acs.est.9b07044?ref=pdf
only PE or PS was supplied as the carbon source to support the
growth of gut microbes Serratia and Bacillus in PE-fed or PS-
fed group, these two genera should have contributed in PE and
PS depolymerization.
4. DISCUSSION
4.1. Effect of Co-diets on Plastic Consumption and
Survival of G. mellonella Larvae. Our results verified that
G. mellonella larvae are capable of chewing and ingesting
Styrofoam (PS) and PE foam. Larvae fed only PS or PE
consumed a large amount of plastic, which was greater than
previous studies reporting on plastic consumption of meal-
worms.20,22 The SRs of PS-fed and PE-fed larvae were low and
similar to that of the starvation condition, indicating that PE
and PS were not toxic for short-term growth of the larvae.
Maximum survival rate occurred when larvae were fed PS +
beeswax (77.3%), which was about 26% and 228% higher than
the beeswax-fed (61.3%) and the PS-fed (23.6%) conditions,
respectively. Similar results were observed for the PE +
beeswax diet. These results suggest that the synergistic effect of
beeswax and PS/PE facilitated larval survival, as the survival of
PS/PE + beeswax group was higher than that of the beeswax or
PS/PE alone diets. The SRs of larvae fed PS and PE
supplemented with beeswax or bran increased while plastic
consumption decreased. Supplying beeswax substantially
increased larval SR from 23.6% in the PS-fed to 77.3% in
the PS + beeswax fed larvae. PS consumption only decreased
from 0.88 g in the PS-fed to 0.64 g in the PS + beeswax fed
groups. Adding bran increased SR to 60% in the PS + bran fed
group, with only a 0.32 g PS weight loss. In comparison,
adding beeswax significantly enhanced the SR of larvae, which
also maintained a relative high plastic consumption. The
results for larvae fed PE support this inference. Larvae of the
PE-fed group consumed 1.95 g PE with a SR of 35.3%, in
which PE consumption decreased to 1.27 g in the PE +
beeswax group and 0.49 g in the PE + bran group, and larval
SR increased to 66% in both groups. These results suggest that
Figure 6. Gas chromatography−mass spectrometer (GC−MS) analysis of the plastic samples (as control), frass and intestines of larvae fed
plastics. Compounds in figure (a): 1. β-hydroxyphenethylbenzoate (C15H14O3); 2. cyclohexane,1,3,5-triphenyl- (C24H24); 3. phthalic acid, di(2-
propylpentyl) ester (C24H38O4); 4. n-hexadecanoic acid (C16H32O2); 5. oleic acid (C18H34O2); 6. octadecanoic acid (C18H36O2). Compounds in
figure (b): 1. hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl)ethyl ester (C19H38O4); 2. octadecanoic acid, 2,3-dihydroxypropyl ester (C21H42O4);
3. n-hexadecanoic acid (C16H32O2); 4. hexadecanoic acid, ethyl ester (C18H36O2); 5. oleic acid (C18H34O2); 6. linoleic acid ethyl ester (C20H36O2);
7. (E)-9-octadecenoic acid ethyl ester (C20H38O2); 8. 9,12-octadecadienoic acid, methyl ester (C19H34O2); 9. 9-octadecenoic acid, methyl ester
(C19H36O2). Compound 7 in figure (a) and 10 in figure (b) are antioxidant of plastic foam of C23H32O2.
Table 1. Quality of the Reads and Bacterial Diversity Analyses Based on Illumina Sequencing of the 16S rRNA Gene
Amplicons
diet clean reads OTUs Chao 1 ACE Shannon Simpson good’s coverage
bran 82 320 121 164.5882 167.099 1.160286 0.422722 0.99902
beeswax 82 360 378 396.3667 406.9377 1.958532 0.273272 0.998411
PS 82 640 171 190.5682 205.1292 0.358646 0.91254 0.998944
PS + bran 82 760 346 352.759 362.9419 2.25026 0.213469 0.999031
PS + beeswax 83 400 198 199.4737 200.208 1.098632 0.700372 0.99979
PE 82 240 60 87 114.9692 0.276952 0.887945 0.99932
PE + bran 83 240 196 218.7778 220.9047 1.398873 0.368113 0.998901
PE + beeswax 83 280 377 381.7736 384.7692 2.254075 0.234408 0.99939
starvation 82 960 111 156.3158 152.4786 0.8089 0.5015 0.9989
Environmental Science & Technology pubs.acs.org/est Article
https://dx.doi.org/10.1021/acs.est.9b07044
Environ. Sci. Technol. 2020, 54, 2821−2831
2826
https://pubs.acs.org/doi/10.1021/acs.est.9b07044?fig=fig6&ref=pdf
https://pubs.acs.org/doi/10.1021/acs.est.9b07044?fig=fig6&ref=pdf
https://pubs.acs.org/doi/10.1021/acs.est.9b07044?fig=fig6&ref=pdf
https://pubs.acs.org/doi/10.1021/acs.est.9b07044?fig=fig6&ref=pdf
pubs.acs.org/est?ref=pdf
https://dx.doi.org/10.1021/acs.est.9b07044?ref=pdf
optimizing the ratio of nutrients to plastic is important to
enhance biodegradation and survival of G. mellonella larvae.
4.2. Plastic Biodegradation and Depolymerization.
The TGA of untreated plastic and frass of plastic-fed larvae
illustrated changes in chemical composition. Under the same
heating process, the weight loss of the plastics was rapid and
concentrated in one stage, while the weight loss of frass from
Figure 7. Gut microbial community structure analysis of the nine different diets. Principal coordinate analysis based on weighted-UniFrac (a) and
Bray−Curtis (c) distances and community diversity distance analysis based on weighted-UniFrac (b) and Bray−Curtis (d) distances.
Figure 8. Relative abundance of dominant populations (top 10) in
the gut microbiomes fed the different diets at the phylum (a) and the
genus levels (b).
Figure 9. Hierarchical cluster analysis with a heatmap of predominant
genera (top 18) in the gut microbiome of larvae fed the nine different
diets.
Environmental Science & Technology pubs.acs.org/est Article
https://dx.doi.org/10.1021/acs.est.9b07044
Environ. Sci. Technol. 2020, 54, 2821−2831
2827
https://pubs.acs.org/doi/10.1021/acs.est.9b07044?fig=fig7&ref=pdf
https://pubs.acs.org/doi/10.1021/acs.est.9b07044?fig=fig7&ref=pdf
https://pubs.acs.org/doi/10.1021/acs.est.9b07044?fig=fig7&ref=pdf
https://pubs.acs.org/doi/10.1021/acs.est.9b07044?fig=fig7&ref=pdf
https://pubs.acs.org/doi/10.1021/acs.est.9b07044?fig=fig8&ref=pdf
https://pubs.acs.org/doi/10.1021/acs.est.9b07044?fig=fig8&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.est.9b07044?fig=fig8&ref=pdf
https://pubs.acs.org/doi/10.1021/acs.est.9b07044?fig=fig8&ref=pdf
https://pubs.acs.org/doi/10.1021/acs.est.9b07044?fig=fig9&ref=pdf
https://pubs.acs.org/doi/10.1021/acs.est.9b07044?fig=fig9&ref=pdf
https://pubs.acs.org/doi/10.1021/acs.est.9b07044?fig=fig9&ref=pdf
https://pubs.acs.org/doi/10.1021/acs.est.9b07044?fig=fig9&ref=pdf
pubs.acs.org/est?ref=pdf
https://dx.doi.org/10.1021/acs.est.9b07044?ref=pdf
the PS-fed and PE-fed larvae was slower with additional stages.
This difference indicates that the frass contained compounds
other than PS or PE, formed through digestion in the larval
gut. The frass of PE lost more than 85% of its weight at 400−
480 °C where the frass of PE-fed larvae lost only 15% of the
weight, as almost 60% of the PE-fed groups frass weight loss
occurred before 400 °C. Similar results were observed for the
weight loss of PS and the frass of PS-fed larvae. This is visual
evidence of chemical compositional changes between the
plastic and frass of plastic-fed larvae.
Changes in the chemical functional groups between the
plastic and frass of plastic-fed larvae were determined by FTIR
analysis. The peaks of CO stretching and C−O stretching
(1700 cm−1 and 1050−1150 cm−1) appeared in the FTIR
spectra of the frass from PE-fed larvae, indicating oxidation of
plastic in the larval gut. The appearance of oxygen was also
revealed in the PS-fed larvae. Broadening peaks were observed
at 2500−3500 cm−1 in the FTIR spectra of the frass from PS-
fed and PE-fed larvae, illustrating that the frass of plastic-fed
larvae was more hydrophobic than that of the PS and PE
controls. The obvious increase in oxygen-containing structures
by analyzing the egested original frass was solid evidence of
oxidization and depolymerization of PS and PE in the larval
gut. Biodegradation of high density PE by waxworms needs
further investigation.
The GPC analysis showed the increase in average molecular
weight of frass from PS-fed larvae compared to untreated PS.
The Mn and Mw of PS-fed diet increased by 11.20% and 4.45%
than that of the PS sample (Student’s t-test, p > 0.05). These
results illustrate that biodegradation of PS by greater wax moth
larvae cannot be verified precisely. The increase in average
molecular weight of frass from the PS-fed group might
represent selective degradation of PS; that is, the larvae
consumed the partial polymer with relatively a lower molecular
weight, to produce smaller molecules or CO2 and left the
higher molecular weight polymer, resulting in an increased
average molecular weight. The induction of enzymes related to
PS biodegradation or the syntrophic interaction between
plastic-degrading microbes in the gut microbiota may require
more time. The sampling time of frass from PS-fed larvae and
the sample amount may have affected the GPC analysis. In
addition, differences in their molecular weights exist between
frass and plastics, representing the transformation of PS into
other compounds, which could also be regarded as a positive
signal of PS disposal by the greater wax moth larvae.
The GC−MS analysis revealed the formation of inter-
mediates in PS and PE metabolism (Figure 6). The results
were similar with previous research, which obtained methyl 9-
octadecenoate and methyl hexadecanoate as intermediates of
PE metabolism.17 The more complex long chain carboxylic
acid esters with hydroxide radicals decreased dramatically.
These results may represent digestion and biodegradation of
PE by the larvae. Compounds with oxygen were detected in
the PS and PE samples, which may have been plasticizers,
Figure 10. Differential relative abundance of the predominant populations in the guts of larvae fed the plastic and control diets (beeswax and bran).
(a) Bran-fed vs polyethylene (PE)-fed (b) Beeswax-fed vs PE-fed (c) Bran-fed vs polystyrene (PS)-fed (d) Beeswax-fed vs PS-fed. Genus only
presented in one of the two diets are expressed with log2 fold change of 10 or −10 and marked with *.
Environmental Science & Technology pubs.acs.org/est Article
https://dx.doi.org/10.1021/acs.est.9b07044
Environ. Sci. Technol. 2020, 54, 2821−2831
2828
https://pubs.acs.org/doi/10.1021/acs.est.9b07044?fig=fig10&ref=pdf
https://pubs.acs.org/doi/10.1021/acs.est.9b07044?fig=fig10&ref=pdf
https://pubs.acs.org/doi/10.1021/acs.est.9b07044?fig=fig10&ref=pdf
https://pubs.acs.org/doi/10.1021/acs.est.9b07044?fig=fig10&ref=pdf
pubs.acs.org/est?ref=pdf
https://dx.doi.org/10.1021/acs.est.9b07044?ref=pdf
antioxidants, and other additives in the plastic foam. Similarly,
long chain fatty acids, such as n-hexadecanoic acid, oleic acid,
and octadecanoic acid, were detected in the frass of larvae fed
PS, whereas compounds with a benzene ring structure
decreased, representing possible metabolism of the benzene
structure and biodegradation of PS.
The GPC and GC−MS extraction methods in our study
followed those of previous reports.7,17,22 Most of the extraction
methods have limitations, and the extracts collected in our
experiment may not have included all of the intermediates
formed during biodegradation. The differences in molecular
weight and the intermediates between raw plastic and frass of
larvae fed plastic also suggested the possibility of plastic
biodegradation. Different from the GPC and GC−MS
analyses, TGA and FTIR were conducted with the original
and unextracted frass samples. Hence, the results of TGA and
FTIR may exclude the negative effects induced by the
extraction methods and provided solid evidence of plastic
biodegradation. However, the slight increase in average
molecular weight of the extracted intermediates compared to
the PS control represented the formation of compounds other
than PS. Long chain fatty acids and long chain carboxylic esters
have been reported previously as metabolic intermediates of
plastics,17 which were also extracted and detected by the GC−
MS analysis in our study. The decreased compounds in plastic
samples had either higher or lower molecular weights
compared to the intermediates produced in frass. Therefore,
the slight increase in average molecular weight was a coeffect of
the formation of intermediates, and the destruction of
compounds with both higher and lower molecular weights
than those of the intermediates.
4.3. Shaping the Larval Core Gut Microbiome Using
the Co-diets. This study is the first to explore the response of
the wax moth larvae gut microbiome to plastic diets and co-
diets. Illumina sequencing of the 16S rRNA gene demonstrated
that the gut microbiomes were shaped by different diets,
resulting in a significant difference in community structure.
The supplements of additional nutrients changed the microbial
community structure, and the species richness and evenness of
the PE-fed larval gut microbiome increased by supplying
beeswax (Table 1). Fifteen larvae with different diets were
randomly selected to eliminate the possible influence of
variations among individuals based on statistical principles.
Whether the variations exist among the larvae is not clear yet,
and an investigation of individual gut microbiomes should be
performed in the future to help understand individual
differences in gut microbiome function during the degradation
process.
Adding beeswax to the larval diet significantly increased SR
(88.5%) with a decrease in PE consumption (34.87%)
compared to the PE-fed group (Figure 2). Although the
decrease in PE consumption was not negligible, PE
consumption was considerable at 1.27 g in the PE + beeswax
group, which was much higher than PS consumption of larvae
fed the PS + beeswax diet (0.64 g). The PCoA based on
weighted UniFrac distances indicated that the microbial
community structure of the PE + beeswax diet was distinct
from that of the other diets (Figure 7). The SR increased
dramatically by 227% and plastic consumption decreased by
27.27% in the PS + beeswax-fed larvae compared to PS-fed
larvae. Adding beeswax adjusted the core gut microbiome and
improved microbial diversity,which increased the SR of larvae
and further improved the degradation ability of PE and PS.
Adding bran as a nutrient resulted in a weaker increase in
microbial diversity compared with adding beeswax. The SR of
larvae fed the PE + bran diet was similar to that of larvae fed
the PE + beeswax, but plastic consumption by the PE + bran
group (0.49 g) was lower than that of the PE + beeswax group
(1.27 g). These results reveal that beeswax may be more
efficient than bran to maintain a high larval SR and improve PE
degradation. PS produced similar results. Although adding
beeswax revealed a relatively lower microbial diversity than
adding bran in the gut of larvae fed PS, it exhibited an
obviously higher SR and plastic consumption rate. The core
gut microbiome of larvae markedly changed when beeswax and
bran were added to the diet, except that of the PS + beeswax-
fed larvae. However, beeswax and bran had different shaping
effects on the core gut microbiome of larvae fed PE and PS.
The core gut microbiome of PE-fed larvae was shaped by
supplying beeswax, and it was obviously shaped by supplying
bran to PS-fed larvae.
4.4. Putative Plastic-Biodegrading Species in the
Larval Gut. The gut bacterial communities of larvae fed PE
and PS revealed simple core microbiomes that were affiliated
with Serratia marcescens (relative abundance 94.1% in PE-fed
group) and Enterococcus sp. (relative abundance 95.5% in PS-
fed group) (Figures 8b and 9). S. marcescens and B. cereus were
significantly related to the PE-fed larvae, in which S. marcescens
showed overwhelming superiority in the PE-fed gut micro-
biome (Figure 10a and b). Serratia belongs to the family
Enterobacteriaceae, in which another Enterobacter is capable of
PE degradation.28 Bacillus has been previously confirmed to
depolymerize PE isolated from soil, marine environment, and
the gut of Plodia interpunctella larvae.28−31 B. cereus and S.
marcescens were also significantly different in the PS-fed group
compared with the bran-fed only group, suggesting that these
two genera may also participate in PS degradation in the larval
gut (Figure 10c). Additionally, Pseudomonas, Geobacillus, and
Propionibacterium were also significantly related to the PS-fed
group when bran was the control diet. The OTU of
Enterococcus sp. had the highest 16S rRNA gene sequence
similarity (100%) to Enterococcus casselif lavus, Enterococcus
gallinarum, and Enterococcus saccharolyticus. A recent study
reported that E. casselif lavus with a resistance plasmid shows
resistance to erythromycin, kanamycin, and streptomycin.32
Thus, antibiotic dependence and independence of plastic
biodegradation relies on the antibiotic resistance genes in the
core gut microbiome.
PS is difficult to degrade, as only few bacteria and fungi can
colonize PS films or degrade PS at a very low rate.33 Bacillus
and Pseudomonas colonize and slowly degrade PS film.34−36
Bacillus, Serratia, and Pseudomonas isolated from soil grow on
PS functionalized with 14% maleic anhydride.37 S. marcescens
was also a core gut microbe in larvae fed PE in our study,
suggesting that this species can degrade PE. Although
Geobacillus has not been found to be capable of degrading
PS, it is in the same family Bacillaceae with Bacillus, and may
have similar characteristics as Bacillus that help depolymerize
the PS chain. However, we need pure culture evidence to
demonstrate direct degradation of PS and PE by Serratia,
Geobacillus, Enterococus, Bacillus, and Pseudomonas.
■ AUTHOR INFORMATION
Corresponding Author
Defeng Xing − State Key Laboratory of Urban Water Resource
and Environment, School of Environment, Harbin Institute of
Environmental Science & Technology pubs.acs.org/est Article
https://dx.doi.org/10.1021/acs.est.9b07044
Environ. Sci. Technol. 2020, 54, 2821−2831
2829
https://pubs.acs.org/action/doSearch?field1=Contrib&text1="Defeng+Xing"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdf
pubs.acs.org/est?ref=pdf
https://dx.doi.org/10.1021/acs.est.9b07044?ref=pdf
Technology, Harbin 150090, P. R. China; orcid.org/0000-
0002-4777-0390; Email: dxing@hit.edu.cn
Authors
Yu Lou − State Key Laboratory of Urban Water Resource and
Environment, School of Environment, Harbin Institute of
Technology, Harbin 150090, P. R. China
Pererva Ekaterina − State Key Laboratory of Urban Water
Resource and Environment, School of Environment, Harbin
Institute of Technology, Harbin 150090, P. R. China
Shan-Shan Yang − State Key Laboratory of Urban Water
Resource and Environment, School of Environment, Harbin
Institute of Technology, Harbin 150090, P. R. China
Baiyun Lu − State Key Laboratory of Urban Water Resource
and Environment, School of Environment, Harbin Institute of
Technology, Harbin 150090, P. R. China
Bingfeng Liu − State Key Laboratory of Urban Water Resource
and Environment, School of Environment, Harbin Institute of
Technology, Harbin 150090, P. R. China
Nanqi Ren − State Key Laboratory of Urban Water Resource
and Environment, School of Environment, Harbin Institute of
Technology, Harbin 150090, P. R. China
Philippe F.-X. Corvini − Institute for Ecopreneurship, School of
Life Sciences, University of Applied Sciences and Arts
Northwestern Switzerland, Muttenz CH-4132, Switzerland
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.est.9b07044
Notes
The authors declare no competing financial interest.
■ ACKNOWLEDGMENTS
This study was supported by the National Natural Science
Foundation of China (No. 31870114), the Science Fund for
Distinguished Young Scholars of Heilongjiang Province (Grant
No. JC201407), and the State Key Laboratory of Urban Water
Resource and Environment (Harbin Institute of Technology
(No. 2019DX02). The authors appreciate Dr. Wei-Min Wu,
Stanford University for his valuable suggestion and comments
during this research.
■ REFERENCES
(1) PlasticsEurope. Plasticsthe Facts 2017; https://www.
plasticseurope.org/application/files/1715/2111/1527/Plastics_the_
facts_2017_FINAL_for_website.pdf 2017.
(2) Adrados, A.; de Marco, I.; Caballero, B.M.; Lopez, A.; Laresgoiti,
M.F.; Torres, A. Pyrolysis of plastic packaging waste: A comparison of
plastic residuals from material recovery facilities with simulated plastic
waste. Waste Manage. 2012, 32 (5), 826−832.
(3) Thompson, R. C.; Moore, C. J.; vom Saal, F. S.; Swan, S. H.
Plastics, the environment and human health: Current consensus and
future trends. Philos. Trans. R. Soc., B 2009, 364 (1526), 2153−2166.
(4) Mor, R.; Sivan, A. Biofilm formation and partial biodegradation
of polystyrene by the actinomycete Rhodococcus ruber. Biodegradation
2008, 19 (6), 851−858.
(5) Koutny, M.; Lemaire, J.; Delort, A.-M. Biodegradation of
polyethylene films with prooxidant additives. Chemosphere 2006, 64
(8), 1243−1252.
(6) Yamada-Onodera, K.; Mukumoto, H.; Katsuyaya, Y.; Saiganji,
A.; Tani, Y. Degradation of polyethylene by a fungus, Penicillium
simplicissimum YK. Polym. Degrad. Stab. 2001, 72 (2), 323−327.
(7) Park, S. Y.; Kim, C. G. Biodegradation of micro-polyethylene
particles by bacterial colonization of a mixed microbial consortium
isolated from a landfill site. Chemosphere 2019, 222, 527−533.
(8) Syranidou, E.; Karkanorachaki, K.; Amorotti, F.; Avgeropoulos,
A.; Kolvenbach, B.; Zhou, N.-Y.; Fava, F.; Corvini, P. F.-X.;
Kalogerakis, N. Biodegradation of mixture of plastic films by tailored
marine consortia. J. Hazard. Mater. 2019, 375, 33−42.
(9) Delacuvellerie, A.; Cyriaque, V.; Gobert, S.; Benali, S.; Wattiez,
R. The plastisphere in marine ecosystem hosts potential specific
microbial degraders including Alcanivorax borkumensis as a key player
for the low-density polyethylene degradation. J. Hazard. Mater. 2019,
380, 120899.
(10) Muhonja, C. N.; Makonde, H.; Magoma, G.; Imbuga, M.
Biodegradability of polyethylene by bacteria and fungi from Dandora
dumpsite Nairobi-Kenya. PLoS One 2018, 13 (7), e0198446.
(11) Syranidou, E.; Karkanorachaki, K.; Amorotti, F.; Franchini, M.;
Repouskou, E.; Kaliva, M.; Vamvakaki, M.;Kolvenbach, B.; Fava, F.;
Corvini, P. F.-X.; Kalogerakis, N. Biodegradation of weathered
polystyrene films in seawater microcosms. Sci. Rep. 2017, 7 (1),
17991.
(12) Syranidou, E.; Karkanorachaki, K.; Amorotti, F.; Repouskou, E.;
Kroll, K.; Kolvenbach, B.; Corvini, P. F.-X.; Fava, F.; Kalogerakis, N.
Development of tailored indigenous marine consortia for the
degradation of naturally weathered polyethylene films. PLoS One
2017, 12 (8), e0183984.
(13) Roager, L.; Sonnenschein, E. C. Bacterial candidates for
colonization and degradation of marine plastic debris. Environ. Sci.
Technol. 2019, 53 (20), 11636−11643.
(14) Avio, C. G.; Gorbi, S.; Regoli, F. Plastics and microplastics in
the oceans: From emerging pollutants to emerged threat. Mar.
Environ. Res. 2017, 128, 2−11.
(15) Kaplan, D. L.; Hartenstein, R.; Sutter, J. Biodegradation of
polystyrene, poly(metnyl methacrylate), and phenol formaldehyde.
Appl. Environ. Microbiol. 1979, 38 (3), 551−3.
(16) Riudavets, J.; Salas, I.; Pons, M. J. Damage characteristics
produced by insect pests in packaging film. J. Stored Prod. Res. 2007,
43 (4), 564−570.
(17) Kong, H.; Kim, H.; Chung, J.; Jun, J.; Lee, S.; Kim, H.; Jeon, S.;
Park, S.; Bhak, J.; Ryu, C. The Galleria mellonella hologenome
supports microbiota-independent metabolism of long-chain hydro-
carbon beeswax. Cell Rep. 2019, 26 (9), 2451−2465.
(18) Yang, S.-S.; Wu, W.-M.; Brandon, A. M.; Fan, H.-Q.; Receveur,
J. P.; Li, Y.; Wang, Z.-Y.; Fan, R.; McClellan, R. L.; Gao, S.-H.; Ning,
D.; Phillips, D. H.; Peng, B.-Y.; Wang, H.; Cai, S.-Y.; Li, P.; Cai, W.-
W.; Ding, L.-Y.; Yang, J.; Zheng, M.; Ren, J.; Zhang, Y.-L.; Gao, J.;
Xing, D.; Ren, N.-Q.; Waymouth, R. M.; Zhou, J.; Tao, H.-C.; Picard,
C. J.; Benbow, M. E.; Criddle, C. S. Ubiquity of polystyrene digestion
and biodegradation within yellow mealworms, larvae of Tenebrio
molitor Linnaeus (Coleoptera: Tenebrionidae). Chemosphere 2018,
212, 262−271.
(19) Yang, S.-S.; Brandon, A. M.; Flanagan, J. C. A.; Yang, J.; Ning,
D.; Cai, S.-Y.; Fan, H.-Q.; Wang, Z.-Y.; Ren, J.; Benbow, E.; Ren, N.-
Q.; Waymouth, R. M.; Zhou, J.; Criddle, C. S.; Wu, W.-M.
Biodegradation of polystyrene wastes in yellow mealworms (larvae
of Tenebrio molitor Linnaeus): Factors affecting biodegradation rates
and the ability of polystyrene-fed larvae to complete their life cycle.
Chemosphere 2018, 191, 979−989.
(20) Brandon, A. M.; Gao, S.-H.; Tian, R.; Ning, D.; Yang, S.-S.;
Zhou, J.; Wu, W.-M.; Criddle, C. S. Biodegradation of polyethylene
and plastic mixtures in mealworms (Larvae of Tenebrio molitor) and
effects on the gut microbiome. Environ. Sci. Technol. 2018, 52 (11),
6526−6533.
(21) Bombelli, P.; Howe, C. J.; Bertocchini, F. Polyethylene bio-
degradation by caterpillars of the wax moth Galleria mellonella. Curr.
Biol. 2017, 27 (8), R292−R293.
(22) Yang, Y.; Yang, J.; Wu, W.-M.; Zhao, J.; Song, Y.; Gao, L.; Yang,
R.; Jiang, L. Biodegradation and mineralization of polystyrene by
plastic-eating mealworms: Part 1. Chemical and physical character-
ization and isotopic tests. Environ. Sci. Technol. 2015, 49 (20),
12080−12086.
(23) Yang, Y.; Yang, J.; Wu, W.-M.; Zhao, J.; Song, Y.; Gao, L.; Yang,
R.; Jiang, L. Biodegradation and mineralization of polystyrene by
Environmental Science & Technology pubs.acs.org/est Article
https://dx.doi.org/10.1021/acs.est.9b07044
Environ. Sci. Technol. 2020, 54, 2821−2831
2830
http://orcid.org/0000-0002-4777-0390
http://orcid.org/0000-0002-4777-0390
mailto:dxing@hit.edu.cn
https://pubs.acs.org/action/doSearch?field1=Contrib&text1="Yu+Lou"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdf
https://pubs.acs.org/action/doSearch?field1=Contrib&text1="Pererva+Ekaterina"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdf
https://pubs.acs.org/action/doSearch?field1=Contrib&text1="Shan-Shan+Yang"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdf
https://pubs.acs.org/action/doSearch?field1=Contrib&text1="Baiyun+Lu"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdf
https://pubs.acs.org/action/doSearch?field1=Contrib&text1="Bingfeng+Liu"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdf
https://pubs.acs.org/action/doSearch?field1=Contrib&text1="Nanqi+Ren"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdf
https://pubs.acs.org/action/doSearch?field1=Contrib&text1="Philippe+F.-X.+Corvini"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdf
https://pubs.acs.org/doi/10.1021/acs.est.9b07044?ref=pdf
https://www.plasticseurope.org/application/files/1715/2111/1527/Plastics_the_facts_2017_FINAL_for_website.pdf
https://www.plasticseurope.org/application/files/1715/2111/1527/Plastics_the_facts_2017_FINAL_for_website.pdf
https://www.plasticseurope.org/application/files/1715/2111/1527/Plastics_the_facts_2017_FINAL_for_website.pdf
https://dx.doi.org/10.1016/j.wasman.2011.06.016
https://dx.doi.org/10.1016/j.wasman.2011.06.016
https://dx.doi.org/10.1016/j.wasman.2011.06.016
https://dx.doi.org/10.1098/rstb.2009.0053
https://dx.doi.org/10.1098/rstb.2009.0053
https://dx.doi.org/10.1007/s10532-008-9188-0
https://dx.doi.org/10.1007/s10532-008-9188-0
https://dx.doi.org/10.1016/j.chemosphere.2005.12.060
https://dx.doi.org/10.1016/j.chemosphere.2005.12.060
https://dx.doi.org/10.1016/S0141-3910(01)00027-1
https://dx.doi.org/10.1016/S0141-3910(01)00027-1
https://dx.doi.org/10.1016/j.chemosphere.2019.01.159
https://dx.doi.org/10.1016/j.chemosphere.2019.01.159
https://dx.doi.org/10.1016/j.chemosphere.2019.01.159
https://dx.doi.org/10.1016/j.jhazmat.2019.04.078
https://dx.doi.org/10.1016/j.jhazmat.2019.04.078
https://dx.doi.org/10.1016/j.jhazmat.2019.120899
https://dx.doi.org/10.1016/j.jhazmat.2019.120899
https://dx.doi.org/10.1016/j.jhazmat.2019.120899
https://dx.doi.org/10.1371/journal.pone.0198446
https://dx.doi.org/10.1371/journal.pone.0198446
https://dx.doi.org/10.1038/s41598-017-18366-y
https://dx.doi.org/10.1038/s41598-017-18366-y
https://dx.doi.org/10.1371/journal.pone.0183984
https://dx.doi.org/10.1371/journal.pone.0183984
https://dx.doi.org/10.1021/acs.est.9b02212
https://dx.doi.org/10.1021/acs.est.9b02212
https://dx.doi.org/10.1016/j.marenvres.2016.05.012
https://dx.doi.org/10.1016/j.marenvres.2016.05.012
https://dx.doi.org/10.1128/AEM.38.3.551-553.1979
https://dx.doi.org/10.1128/AEM.38.3.551-553.1979
https://dx.doi.org/10.1016/j.jspr.2007.03.006
https://dx.doi.org/10.1016/j.jspr.2007.03.006
https://dx.doi.org/10.1016/j.celrep.2019.02.018
https://dx.doi.org/10.1016/j.celrep.2019.02.018
https://dx.doi.org/10.1016/j.celrep.2019.02.018
https://dx.doi.org/10.1016/j.chemosphere.2018.08.078
https://dx.doi.org/10.1016/j.chemosphere.2018.08.078
https://dx.doi.org/10.1016/j.chemosphere.2018.08.078
https://dx.doi.org/10.1016/j.chemosphere.2017.10.117
https://dx.doi.org/10.1016/j.chemosphere.2017.10.117
https://dx.doi.org/10.1016/j.chemosphere.2017.10.117
https://dx.doi.org/10.1021/acs.est.8b02301
https://dx.doi.org/10.1021/acs.est.8b02301
https://dx.doi.org/10.1021/acs.est.8b02301
https://dx.doi.org/10.1016/j.cub.2017.02.060
https://dx.doi.org/10.1016/j.cub.2017.02.060
https://dx.doi.org/10.1021/acs.est.5b02661
https://dx.doi.org/10.1021/acs.est.5b02661
https://dx.doi.org/10.1021/acs.est.5b02661
https://dx.doi.org/10.1021/acs.est.5b02663
pubs.acs.org/est?ref=pdf
https://dx.doi.org/10.1021/acs.est.9b07044?ref=pdf
plastic-eating mealworms: Part 2. Role of gut microorganisms.
Environ. Sci. Technol. 2015, 49 (20), 12087−12093.
(24) van Zyl, C.; Malan, A. P. Cost-effective culturing of Galleria
mellonella and Tenebrio molitor and entomopathogenic nematode
production in various hosts. Afr. Entomol. 2015, 23 (2), 361−375.
(25) Zhou, H.; Liu, B.; Wang, Q.; Sun, J.; Xie, G.; Ren, N.; Ren, Z.
J.; Xing, D. Pulse electromagnetic fields enhance extracellular electron
transfer in magnetic bioelectrochemical systems. Biotechnol. Biofuels
2017, 10, 238.
(26) Liu, Q.; Ren, Z. J.; Huang, C.; Liu, B.; Ren, N.; Xing, D.
Multiplesyntrophic interactions drive biohythane production from
waste sludge in microbial electrolysis cells. Biotechnol. Biofuels 2016, 9,
162.
(27) Liu, Q.; Liu, B.; Li, W.; Zhao, X.; Zuo, W.; Xing, D. Impact of
ferrous iron on microbial community of the biofilm in microbial fuel
cells. Front. Microbiol. 2017, 8, 920.
(28) Yang, J.; Yang, Y.; Wu, W.-M.; Zhao, J.; Jiang, L. Evidence of
polyethylene biodegradation by bacterial strains from the guts of
plastic-eating waxworms. Environ. Sci. Technol. 2014, 48 (23), 13776−
13784.
(29) Sudhakar, M.; Doble, M.; Murthy, P. S.; Venkatesan, R. Marine
microbe-mediated biodegradation of low- and high-density poly-
ethylenes. Int. Biodeterior. Biodegrad. 2008, 61 (3), 203−213.
(30) Roy, P. K.; Titus, S.; Surekha, P.; Tulsi, E.; Deshmukh, C.;
Rajagopal, C. Degradation of abiotically aged LDPE films containing
pro-oxidant by bacterial consortium. Polym. Degrad. Stab. 2008, 93
(10), 1917−1922.
(31) Yang, Y.; Chen, J.; Wu, W.-M.; Zhao, J.; Yang, J. Complete
genome sequence of Bacillus sp. YP1, a polyethylene-degrading
bacterium from waxworm’s gut. J. Biotechnol. 2015, 200, 77−78.
(32) Yin, M.; Jiang, Y.; Qian, C.; Wu, F.; Ying, Y.; Wu, C.; Li, P.;
Ying, J.; Li, K.; Xu, T.; Bao, Q.; Sun, C. Molecular characteristics and
comparative genomics analysis of a clinical Enterococcus casselif lavus
with a resistance plasmid. Infect. Drug Resist. 2018, 11, 2159−2167.
(33) Ho, B. T.; Roberts, T. K.; Lucas, S. An overview on
biodegradation of polystyrene and modified polystyrene: the micro-
bial approach. Crit. Rev. Biotechnol. 2018, 38 (2), 308−320.
(34) Mohan, A. J.; Sekhar, V. C.; Bhaskar, T.; Nampoothiri, K. M.
Microbial assisted high impact polystyrene (HIPS) degradation.
Bioresour. Technol. 2016, 213, 204−207.
(35) Hadad, D.; Geresh, S.; Sivan, A. Biodegradation of polyethylene
by the thermophilic bacterium Brevibacillus borstelensis. J. Appl.
Microbiol. 2005, 98 (5), 1093−1100.
(36) Novotny, C.; Malachova, K.; Adamus, G.; Kwiecien, M.ł; Lotti,
N.; Soccio, M.; Verney, V.; Fava, F. Deterioration of irradiation/high-
temperature pretreated, linear low-density polyethylene (LLDPE) by
Bacillus amyloliquefaciens. Int. Biodeterior. Biodegrad. 2018, 132, 259−
267.
(37) Galgali, P.; Varma, A. J.; Puntambekar, U. S.; Gokhale, D. V.
Towards biodegradable polyolefins: Strategy of anchoring minute
quantities of monosaccharides and disaccharides onto functionalized
polystyrene, and their effect on facilitating polymer biodegradation.
Chem. Commun. 2002, No. 23, 2884−2885.
Environmental Science & Technology pubs.acs.org/est Article
https://dx.doi.org/10.1021/acs.est.9b07044
Environ. Sci. Technol. 2020, 54, 2821−2831
2831
https://dx.doi.org/10.1021/acs.est.5b02663
https://dx.doi.org/10.4001/003.023.0232
https://dx.doi.org/10.4001/003.023.0232
https://dx.doi.org/10.4001/003.023.0232
https://dx.doi.org/10.1186/s13068-017-0929-3
https://dx.doi.org/10.1186/s13068-017-0929-3
https://dx.doi.org/10.1186/s13068-016-0579-x
https://dx.doi.org/10.1186/s13068-016-0579-x
https://dx.doi.org/10.3389/fmicb.2017.00920
https://dx.doi.org/10.3389/fmicb.2017.00920
https://dx.doi.org/10.3389/fmicb.2017.00920
https://dx.doi.org/10.1021/es504038a
https://dx.doi.org/10.1021/es504038a
https://dx.doi.org/10.1021/es504038a
https://dx.doi.org/10.1016/j.ibiod.2007.07.011
https://dx.doi.org/10.1016/j.ibiod.2007.07.011
https://dx.doi.org/10.1016/j.ibiod.2007.07.011
https://dx.doi.org/10.1016/j.polymdegradstab.2008.07.016
https://dx.doi.org/10.1016/j.polymdegradstab.2008.07.016
https://dx.doi.org/10.1016/j.jbiotec.2015.02.034
https://dx.doi.org/10.1016/j.jbiotec.2015.02.034
https://dx.doi.org/10.1016/j.jbiotec.2015.02.034
https://dx.doi.org/10.2147/IDR.S180254
https://dx.doi.org/10.2147/IDR.S180254
https://dx.doi.org/10.2147/IDR.S180254
https://dx.doi.org/10.1080/07388551.2017.1355293
https://dx.doi.org/10.1080/07388551.2017.1355293
https://dx.doi.org/10.1080/07388551.2017.1355293
https://dx.doi.org/10.1016/j.biortech.2016.03.021
https://dx.doi.org/10.1111/j.1365-2672.2005.02553.x
https://dx.doi.org/10.1111/j.1365-2672.2005.02553.x
https://dx.doi.org/10.1016/j.ibiod.2018.04.014
https://dx.doi.org/10.1016/j.ibiod.2018.04.014
https://dx.doi.org/10.1016/j.ibiod.2018.04.014
https://dx.doi.org/10.1039/b209254a
https://dx.doi.org/10.1039/b209254a
https://dx.doi.org/10.1039/b209254a
pubs.acs.org/est?ref=pdf
https://dx.doi.org/10.1021/acs.est.9b07044?ref=pdf