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org/est Article

Biodegradation of Polystyrene by Pseudomonas sp. Isolated from

the Gut of Superworms (Larvae of Zophobas atratus)
Hong Rae Kim,∥ Hyun Min Lee,∥ Hee Cheol Yu, Eunbeen Jeon, Sukkyoo Lee, Jiaojie Li,*
and Dae-Hwan Kim*
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ABSTRACT: Recently, various attempts have been made to solve

plastic waste problems, such as development of biodegradation
without producing pollution. Polystyrene (PS) is the fifth most
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used plastic in many industries; therefore, degrading PS becomes a

critical global issue. Here, we reported Pseudomonas aeruginosa
strain DSM 50071, initially isolated from the gut of the
superworms, Zophobas atratus, and the PS degradation by
Pseudomonas sp. DSM 50071. We examined PS degradation
using electronic microscopy and measured changes in atomic
composition and contact angles with water droplets on the PS
surface that represents a chemical change from hydrophobicity to
hydrophilicity. We have further examined chemical structural changes using X-ray photoelectron spectroscopy, Fourier-transform-
infrared spectroscopy, and nuclear magnetic resonance (NMR) to confirm the formation of carbonyl groups (CO) in the
oxidation pathway during PS biodegradation. In reverse transcription quantitative polymerase chain reaction analysis, the gene
expression level of serine hydrolase (SH) in Pseudomonas sp. DSM 50071 was highly increased during PS degradation, and the
enzyme-mediated biodegradation of PS was further confirmed by the SH inhibitor treatment test. Thus, the significance of these
findings goes beyond the discovery of a novel function of Pseudomonas sp. DSM 50071 in the gut of superworms, highlighting a
potential solution for PS biodegradation.

■ INTRODUCTION
The use of plastics across the globe has steeply increased,
can degrade polyethylene (PE) glycol, and Pseudomonas
vesicularis PD can degrade polyvinyl alcohol.5 Furthermore,
making it one of the most widely used substances. However, among the microorganisms isolated from soil or marine
natural plastic degradation is extremely slow, inevitably leading environments, Pseudomonas sp. is known to mediate the most
to plastic waste accumulation, which represents a grave efficient degradation of PE.6,7 Specifically, the strain has been
ecological issue. Because of the lack of suitable degradation reported to reduce 20% of the overall weight of PE during 120
methods, plastic treatment involves 77% reclamation, 13% days.8 In addition, Pseudomonas sp. is reported capable of
incineration, and 10% recycling. Among these, reclamation degrading key aromatic compounds in petroleum such as
causes dangerous pollution in the soil and groundwater, while benzene and toluene.9
Polystyrene (PS) is a polymeric plastic used in packaging
incineration causes harmful substances to be released into the
containers, disposable cups, and insulating materials, which
atmosphere, rendering them both unsuitable as long-term
accounts for approximately 6.6% of total plastic use.10 The
solutions.1 Besides, plastic waste flowing into the ocean is
biodegradation of PS is known to progress very slowly in
ingested by marine organisms in the form of microplastics,
natural ecosystems and requires several hundred years for
which causes serious health problems to marine life and
complete degradation.5 Previous studies have reported that it
subsequently could impact the health of humans.2,3 Thus, it is
can be degraded by heat and photocatalysts.11,12 Nonetheless,
essential to develop an efficient biodegradation method to
such techniques themselves inflict environmental pollution.
degrade plastics.
Numerous researchers are making attempts to degrade
plastics using microorganisms. The majority searched bacteria Received: March 10, 2020
for plastic-degrading found in landfill sites, soils, and marine Revised: May 5, 2020
environments are a species belonging to the Pseudomonas Accepted: May 6, 2020
genus.4 So far, Pseudomonas putida and Pseudomonas fluorescens Published: May 6, 2020
have been found to degrade polyvinyl chloride, Pseudomonas
chlororaphis can degrade Polyurethane, Pseudomonas stutzeri

© 2020 American Chemical Society https://dx.doi.org/10.1021/acs.est.0c01495

6987 Environ. Sci. Technol. 2020, 54, 6987−6996
Environmental Science & Technology pubs.acs.org/est Article

There is no reported microorganism-mediated biodegradation purity, Myung-IL FOAMTEC, Daegu, South Korea) was
method currently in existence for PS, despite it being the most supplied as the sole carbon food source. To isolate the gut
frequently used plastic globally.13−16 Recently, Pseudomonas sp. bacteria from the larvae bred in an incubator at 25 °C for two
was reported to have been used in degrading high impact PS, a weeks, the larvae were anesthetized by immersing in 70%
polymer of PS and polybutadiene, while Pseudomonas ethanol and then washed with saline water (SW). The head
aeruginosa was used in degrading a polymer of polylactic acid and the tail were removed, and the gut was extracted using a
and PS.17,18 Although Rhodococcus ruber was found to have pair of sterilized forceps. The extracted gut was chopped and
mediated biofilm formation on the PS surface and a partial vortexed with 5 mL of SW for 5 min and then centrifuged for 5
reduction in the weight of PS, information on the physical and min at 800 rpm to remove the epithelial cells. The supernatant
chemical analyses of the plastic surface during degradation has layer was carefully transferred to a flask containing liquid
not been reported.19 Thus, the specific characterization of the carbon free basal medium (LCFBM) (pH: 6.51, 0.7 g of
degradation process has not so far been verified. KH2PO4, 0.7 g of K2HPO4, 0.7 g of MgSO4·7H2O, 1.0 g of
A recent study reported that various insect larvae were able NH4NO3, 0.005 g of NaCl, 0.002 g of FeSO4·7H2O, 0.002 g of
to ingest plastic and participate in its degradation. Most ZnSO4·7H2O, and 0.001 g of MnSO4·H2O in 1 L of deionized
notably, yellow and dark mealworms, the larvae of Tenebrio water) with sodium thioglycolate to maintain an anaerobic
molitor and Tenebrio obscurus, and superworms, the larvae of condition and then mixed thoroughly. After adding film-type
Zophobas atratus were reported capable of ingesting PS.20−23 PS (Mw = 316,900, Mn = 110,600, 99.9% purity, Goodfellow,
Although insects are known to be able to ingest plastic in the Huntingdon, UK), the mixture solution was cultured in a
past, their ability to degrade it remains controversial.24 Because shaking incubator at 25 °C with 180 rpm for 60 days under the
of a lack of overall research into this problem, there remains a anaerobic condition.
myriad of unknown facts. Nevertheless, several recent studies Identification of Gut Bacteria in Superworms. After 60
have reported that mealworms and superworms ingest PS and days of culture, the gut solution was diluted to 1/100 with
use it as an energy source. In particular, several studies on LCFBM solution, and then 2 mL of bacteria containing
mealworms and superworms that investigated degradation LCFBM solution was spread onto a solid nutrient medium
ability by feeding worms with antibiotics-containing bran (BD Difco, cat#: 90002-660, Franklin Lakes, NJ, USA; Beef
reported that the gut microbiota was critical for plastic Extract 3.0 g, Peptone 5.0 g, Agar 15.0 g in 1 L of deionized
degradation. The depolymerization of PS was significantly water, pH: 6.8). For bacterial culture, a completely oxygen-free
reduced in mealworms and superworms which ingested the environment was supplied. The culture was performed in an
antibiotic gentamicin-containing bran because of the suppres- anaerobic culture chamber (Anaerobic Jar, Mitsubishi gas
sion of their gut bacteria, suggesting that PS biodegradation is chemical company, Japan) containing a deoxygenation package
gut-microbiota-dependence.21−23 Yang’s group further isolated (Anaero Pack- Anaero, Mitsubishi gas chemical company,
the PS-degrading bacteria, Exiguobacterium sp. strain YT2, Japan) at 25 °C for 3−4 days.26,27 Each colony grown on the
from the gut of mealworms.25 solid nutrient medium was transferred to the liquid nutrient
The present study searched for the PS-degrading bacteria in medium and cultured for another 2 days at 25 °C under the
the gut of superworm, larvae of Z. atratus. Gut bacteria were anaerobic condition. To determine the species of bacteria
isolated from larvae that had ingested PS, and a PS-degrading grown in the liquid medium, genomic PCR was performed
strain of Pseudomonas was successfully isolated. A scanning after extracting the bacterial genomic DNA to obtain the 16S
electron microscope (SEM) allowed us to examine physical RNA sequence, and the primers used were as following:
changes during the process of PS degradation, and chemical forward primer 27-F (5′-AGAGTTTGATYMTGGCTCAG-
composition changes of PS were analyzed using X-ray 3′); reverse primer 1492-R (5′-GGTTACCTTGTTA-
photoelectron spectroscopy (XPS), attenuated total reflec- CGACTT-3′). 16S rRNA gene sequence analysis was carried
tance−fourier-transform infrared spectroscopy (ATR−FT-IR), out to identify the species. A T100 Thermal Cycler (Bio-Rad,
and nuclear magnetic resonance spectroscopy (NMR), in Singapore) was used for PCR, and amplified PCR DNA bands
order to verify Pseudomonas-mediated biodegradation of PS. were analyzed by electrophoresis. After sequencing the 16S
Moreover, we have also identified the PS depolymerization rRNA DNA, the bacterial species were determined using NCBI
enzyme, a serine hydrolase (SH), by reverse transcription BLAST analysis.
quantitative polymerase chain reaction (RT-qPCR) analysis PS Degradation by Pseudomonas sp. DSM 50071.
and the enzyme inhibitor test. Based on these findings, we Pseudomonas sp. was cultured with a PS film in a solid LCFBM
proposed the possible pathway during the degradation process. medium lacking an alternate carbon source to verify plastic
In summary, the study has shown that a Pseudomonas strain, degradation. After collecting 5 mL of bacteria solution cultured
previously known to degrade plastic in soils and marine in the liquid nutrient medium, it was centrifuged for 5 min at
environments, is also found in the gut of insects where the 4000 rpm. We removed the nutrient medium and then added 1
bacteria participate in the degradation of PS ingested by mL of LCFBM. Using a pipette, the bacteria were resuspended
superworms.

■
and centrifuged again to collect the bacterial sample. To
completely remove the remaining nutrient medium compo-
MATERIALS AND METHODS nents, identical washing steps were carried out three times.
Isolation of Plastic-Degrading Bacteria from Super- After the final washing step, each Pseudomonas sp. pellet was
worms. We purchased the 3-4 instar growth stage mealworms mixed with 0.1 mL of LCFBM, and then, the suspended
and superworms (NB mealworms, Yangju, South Korea). mixture was layered onto a solid LCFBM plate. After drying, a
Mealworms and superworms, the larva of T. molitor and Z. sterilized PS film (Mw = 316,900, Mn = 110,600, 99.9% purity,
atratus, were placed in a breeding chamber (n = 50 worms). To Goodfellow, Huntingdon, UK) was placed on the solid
create a gut environment enriched with plastic-degrading LCFBM plate. The plate was sealed with parafilm and placed
bacteria, Styrofoam PS (Mw = 302,300, Mn = 108,700, 99.5% in a deoxygenating chamber (ThermoStable IR-250, Daihan
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Figure 1. Identification of Pseudomonas sp. DSM 50071 in the PS ingested superworms. (A) PS ingested and digested by the superworms. (B)
Measurements of PS ingestion between the same number (N: 50) of superworms and mealworms. The PS weight was measured every three days
during a twenty-one-day period to determine its reducing rate by worms. Superworms (blue line) consumed approximately 1.42 g from initially
supplied 2 g of PS, whereas mealworms ingested 0.22 g from 2 g of PS (red line). (C) Comparison of the PS degradation rates between mealworms
and superworms. Averagely, 68 mg of PS per day was consumed by the fifty superworms (blue bar), whereas 11 mg of PS per day was consumed by
the fifty mealworms (red bar) (left). Also, 2.9 mg of PS per day was consumed by 1 g of superworms (blue bar) and 4.8 mg of PS per day was
consumed by 1 g of mealworms (red bar) (right). (D) Measurement of biodegradation of PS using FT-IR and TGA. In TGA spectra, several
decomposition points were examined in the frass of mealworms and superworms unlike single decomposition point of PS control (upper). In
comparison to the control group, FT-IR spectra for frass of mealworms and superworms showed the biodegradation of PS (down). (E) Isolation of
bacteria inhabiting the guts of the superworms. The chopped guts of superworms mixed with PS-added LCFBM was cultured for 60 days, and the
surviving, amplified bacteria were isolated in a solid nutrient medium. Four different species of bacteria including Pseudomonas sp. DSM 50071 were
identified.

Scientific Co., Seoul, South Korea). After incubation at 25 °C We examined changes in the elemental composition by
for 60 days, bacterial colonies were examined. analyzing the difference between the test group and the
SEM Analysis. SEM (SU8230, Hitachi, Tokyo, Japan) was control.
used to verify the proliferation of Pseudomonas sp. DSM 50071 Examination of Changes in Hydrophilicity on the PS
on the PS surface and PS degradation. For the observation of Surface. During PS degradation by Pseudomonas sp. DSM
amplified Pseudomonas sp. DSM 50071, the PS on which 50071, contact angle analysis was used to analyze changes in
Pseudomonas sp. DSM 50071 had grown was cut to a size of 1 hydrophilicity on the PS surface. First, PS was fixed on a
cm × 1 cm, and the surface was investigated. To analyze carbon tape to measure the contact angle using the contact
changes on the PS surface after degradation by Pseudomonas angle meter (Phoenix Multi, Seoul, Korea), and 35 μL of water
sp. DSM 50071, 2% sodium dodecyl sulfate (SDS) solution was dropped onto the PS surface within a 20 mm2 area at 20
was used for 4 h to remove bacteria attached to PS.28,29 Then, °C. Both left and right contact angles were measured, and the
the degraded PS surface was examined with SEM. First, PS was mean value was used for the comparative analysis.25,29
fixed on a copper tape and platinum coating was added using Examination of PS Degradation Using XPS. To verify
an ion sputter for 15 s at 15 mA (E-1045, Hitachi High- the PS degradation by Pseudomonas sp. DSM 50071, changes
Technologies Corporation, Tokyo, Japan). Then, the degraded in elemental composition on the PS surface were examined by
PS surface was imaged at 1.6 kV acceleration voltage. measuring the binding energy with an X-ray photoelectron
Analysis of the Elemental Composition on the PS spectrometer (ESCALAB 250Xi, Thermo Scientific, Waltham,
Surface. To verify changes in the elemental composition on MA, USA). To prepare the sample, prearranged PS (1 cm × 1
the PS surface during degradation by Pseudomonas sp. DSM cm) fixed on the carbon tape was measured within an energy
50071, X-ray energy dispersive spectroscopy with a module range of 276−300 eV, C 1 s.
attached to the SEM was used to estimate the elemental Examination of PS Degradation Using Fourier-Trans-
composition between carbon and oxygen on the PS surface in form NMR Spectroscopy. To further examine chemical
the areas exhibiting proliferated Pseudomonas sp. DSM 50071. structural changes during PS degradation, chemical structural
6989 https://dx.doi.org/10.1021/acs.est.0c01495
Environ. Sci. Technol. 2020, 54, 6987−6996
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analysis was carried out using fourier-transform NMR several weeks, a much shorter time. In addition to the well-
spectroscopy (FT−NMR) (AVANCE III 400, Bruker, known mealworms,21 we characterized superworms (Z.
Germany). The degraded 5 mg PS film was dissolved in 0.5 atratus)an insect only recently observed to mediate PS
ml chloroform-d (≥99.96%, Sigma-Aldrich, St. Louis, MO, degradation after ingestion (Figures 1A and S1). To compare
USA), and the NMR analysis was carried out at 400 MHz and the changes in the ingested amount of PS supplied between
25 °C. superworms and mealworms, we measured changes in the
Analysis of PS Biodegradation with ATR−Fourier- weight of PS after twenty-one days (Figure 1B). The amounts
Transform-Infrared Spectroscopy. Fourier-transform-infra- of PS ingested by fifty superworms and fifty mealworms
red spectroscopy (FT-IR) (Nicolet Continuum, Thermo differed significantly. Superworms consumed approximately
Fisher Scientific) was used to verify PS biodegradation by 1.42 g from initially supplied 2 g of PS during the twenty-one-
Pseudomonas sp. DSM 50071. To prepare samples, PS film was day period, whereas fifty mealworms ingested 0.22 g from the
completely dried at 60 °C for 24 h after removing bacteria. same amount of PS supply (Figure 1B). Our weight and length
Then, FT-IR measurements were performed using smart measurements of each worm indicated that the average weight
single-bounce ATR with a diamond tip in the 4400−400 cm−1 and length of superworms were 0.4648 g (±0.0324 g) and 3.64
range. cm (±0.079 cm), respectively; whereas those of mealworms
RT-qPCR. After the collection of Pseudomonas sp. DSM were 0.0454 g (±0.0024 g) and 1.70 cm (±0.062 cm),
50071 grown on LCFBM medium supplied with PS and respectively. Because the amount of plastic ingested by worms
nutrient, total RNA was isolated using the Trizol method is related to the weights of worms, PS consumption rates per
(Invitrogen, Carlsbad, CA, USA). After quantification of total weight (g) of worms were measured (Figure 1C). The average
RNA, 1 μg of RNA was heated with 1 μL random primer at 65 rate of PS reduction by the fifty superworms was 68 mg per
°C for 5 min, and then, 2 μL of dNTPs and 4 μL of 5X day, which exceeded 11 mg per day measured in mealworms,
reaction buffer, 2 μL of DTT, 1 μL of RNaseOUT, and 0.5 μL but the average consuming rate per 1 g mealworms was around
of SuperScript II (Invitrogen) were added to the cool-down 1.7 times higher than that measured in superworms (Figure
mixture. cDNA synthesis was performed at 42 °C for 50 min 1C). Thus, mealworms showed better efficiency than super-
and then terminated by inactivation at 75 °C for 5 min. In worms for PS ingestion. Around 90% of both worms,
qRT-PCR, a CFX96 Touch Real-Time PCR Detection System mealworms and superworms, were survived after the 21-day
(Bio-Rad, Hercules, CA, USA) was used to measure the period.
expression level of each gene with primer sets (Table S1). Thermogravimetric analysis (TGA) and FT-IR analyses
Subsequently, 1 μL from 1/5 diluted cDNA was mixed with iQ were further conducted on the frass of PS-ingested mealworms
SYBR Green Supermix (Bio-Rad). The 16S rRNA gene was and superworms to confirm PS biodegradation. In the TGA
used as the endogenous control. The relative quantification of analysis, four maximum decomposition rates, 68, 251, 325, and
each gene expression in the test group versus control was 378 °C, were observed on the frass of PS-ingested mealworms,
calculated by the Bio-Rad manager 3.0 software program, and whereas three maximum decomposition rates, 88, 327, and 383
the expressed level of each gene was described in terms of its °C, were recorded on the frass of PS-ingested superworms
relative value between the test group and the control. (Figure 1D, upper). The frass of superworms showed 36%
Weight Measurement of PS Beads. To quantify the mass loss within 120−395 °C and 27% mass loss within 400−
mass reduction of PS by Pseudomonas sp. DSM 50071- 480 °C, whereas that of mealworms were 39% mass loss within
mediated degradation, 1 g of PS beads (diameter = 1500 μm, 121−380 °C and 33% mass loss within 400−480 °C (Figure
Mw = 371,100, Mn = 153,600, 99.5% purity, Myung-IL 1D, upper). The FT-IR analyses on the frass of both worms
FOAMTEC, Daegu, South Korea) were biodegraded by showed a broad O−H stretching absorption around 3300−
Pseudomonas sp. DSM 50071 (bacterial number = 1 × 108/ 3600 cm−1 and a sharp carbonyl (−CO) stretching
mL) in liquid LCFBM for 15 days at 25 °C, with 180 rpm. The absorption around 1700 cm−1, when compared with that of
biodegraded PS beads obtained through a 70 μm sieve and a PS in control (Figure 1D, down). These results confirmed that
0.45 μm filter were processed with 2% SDS, rinsed with DW, PS consumed by superworms and mealworms were biode-
and then dried at 60 °C for 24 h, and their masses were graded through their digestive system. In addition, the similar
measured independently in triplicate. We have further FT-IR patterns suggested that the biodegradation of PS could
conducted the SH inhibitor test, and different concentrations be progressed with the similar oxidation process by both
of the purchased SH inhibitor-7 (tert-butyl 4-[{2- worms.
fluorophenyl}carbamoyl]piperazine-1-carboxylate, SHI-7, As superworms readily ingest PS, we anticipated that they
CAS#: 931086-01-0, Sigma-Aldrich) were added in liquid might also contain active PS-degrading bacteria in the gut.
LCFBM including 1 g of PS beads and Pseudomonas sp. DSM Because a huge diversity of bacteria survives in the guts of the
50071. For a control sample without Pseudomonas sp. DSM superworms, it was deemed likely that some, but not all, would
50071 and SH inhibitor-added samples, the weight changes of be directly involved in PS degradation. Thus, isolation of each
PS beads were measured using the same method.

■
bacterium species to test PS biodegradation was required. After
identification of each single colony with 16S RNA sequencing,
RESULTS AND DISCUSSION we found four different strains of bacteria growing on the solid
Recently, several insects have been reported capable of medium, each showing distinct morphology (Figure 1E). We
degrading various plastics. For instance, mealworms ingest surmized that these cultured bacteria could be candidates that
and mediate rapid degradation of PS-consisting Styrofoam and utilize PS for their viability and proliferation. Among the gut
other PS-containing plastic products.30 PS biodegradation bacteria of superworms, P. aeruginosa, rod-shaped with 0.5 μm
proceeds slowly in natural ecosystem, requiring several wide and 2 μm long, was identified as the bacterial strain
hundred years for complete degradation, but such insect- exhibiting the highest fraction and comprised approximately
based systems suggest that PS biodegradation can occur within 35% of all sequenced bacteria. Few other Pseudomonas strains
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participating in PS degradation have been identified, and most had been present, further confirming Pseudomonas sp. DSM
are derived from soil and deep marine.5 Here, we identified for 50071-mediated PS degradation (Figure 2C). Despite SDS
the first time a P. aeruginosa strain DSM 50071 (accession treatment, a proportion of Pseudomonas sp. DSM 50071 in or
number in the GenBank: SUB6959368 16S MT045992), near such holes was not entirely removed (Figure 2C, middle
residing in the guts of the superworms that might be engaged row, right panel). Our findings provide the direct evidence that
in PS degradation. Pseudomonas sp. DSM 50071 isolated from the guts of
To confirm that isolated Pseudomonas sp. DSM 50071 could superworms degrades PS.
conduct the PS degradation, we tested PS-degrading ability of To further confirm Pseudomonas sp. DSM 50071-mediated
Pseudomonas sp. DSM 50071 using a piece of PS in solid PS degradation, we have analyzed the composition of carbon
LCFBM medium. Colonies of growing Pseudomonas sp. DSM and oxygen atoms present on the PS surface in the boundary
50071 were observed on the piece of PS after culture for 30 region, where Pseudomonas sp. DSM 50071 had grown in large
days. In particular, large numbers of Pseudomonas sp. DSM numbers. Results showed that there was no significant
50071 were found around the edges of the PS (Figure 2A). difference in the number of carbon atoms between the control
and the areas in the PS exhibiting Pseudomonas sp. DSM 50071
growth (Figure 3A, upper). On the contrary, a large number of
oxygen atoms were detected in PS areas exhibiting
Pseudomonas sp. DSM 50071 growth, indicating that PS
degradation-related enzymes secreted by Pseudomonas sp.
DSM 50071 had promoted oxidation as part of degradation
(Figure 3A, bottom). Such oxidation was absent in the control
(Figure 3A). One of the most notable characteristics of
bacteria-mediated plastic degradation processes is the for-
mation of a biofilm on the plastic surface. The generated
biofilm accelerates plastic degradation by increasing the
contact area between the bacteria and the plastic (hydro-
phobic) surface, thereby speeding up oxidation and overall
degradation.28,31 During the oxidation process, functional
groups such as hydroxyl or carbonyl groups could be formed
via β-oxidation, which is known to be used in the TCA cycle or
energy metabolism in bacteria, thus increasing the hydro-
philicity.32,33 Therefore, the increased number of oxygen atoms
on the plastic surface in the areas exhibiting Pseudomonas sp.
Figure 2. PS degradation by Pseudomonas sp. DSM 50071. (A)
DSM 50071 growth provides direct evidence of the PS
Pseudomonas sp. DSM 50071 normally grows on PS. Pseudomonas sp. degradation (Figure 3A).
DSM 50071 were viable and proliferated on the PS, especially around Oxidation as the crucial step in Pseudomonas sp. DSM
its edges. Several colonies of Pseudomonas sp. DSM 50071 were 50071-mediated PS degradation converted the PS surface from
observed at the center of the PS surface. (B) SEM detection of the hydrophobicity to hydrophilicity, which was confirmed by the
inhabited Pseudomonas sp. DSM 50071 attached to PS. Most changes in the contact angles between water droplets and the
Pseudomonas sp. DSM 50071 attached to the PS surface were PS surface.34 In comparison to the control, the average value of
observed under SEM in proliferated colonies. (C) PS digestion by the contact angles on the left and right sides of the water
Pseudomonas sp. DSM 50071. Unlike the control, the PS inhabiting droplets on a PS surface exhibiting Pseudomonas sp. DSM
Pseudomonas sp. DSM 50071 exhibited degradation, which was
50071 growth was highly reduced (Figure 3B).25 As the left
investigated in the areas around the edges of the PS (upper panel) and
several large newly generated holes in the PS surface (lower panel). contact angle was 91.77° and the right contact angle was
Although SDS buffer was used to remove Pseudomonas sp. DSM 91.35°, the average contact angle was 91.56° in the case of the
50071, there still remained a small number of attached Pseudomonas control (Table 1), whereas on the PS surface exhibiting
sp. DSM 50071, which were investigated near PS degraded areas Pseudomonas sp. DSM 50071 growth, this angle was 79.46° on
(upper right). the left and 79.29° on the right, with an average contact angle
of 79.39°. Thus, a 12.1° average contact angle reduction was
SEM was used to examine the shapes and characteristics of observed in comparison to the control (Table 1 and Figure
growing colonies, and the obtained images showed that 3B). The reduced contact angle between the water droplets
Pseudomonas sp. DSM 50071 firmly attached to the PS surface and the plastic surface represents the surface tension of water
(Figure 2B). The viability and proliferation of Pseudomonas sp. decreasing because of more abundant and stronger polar
DSM 50071 on the PS surface indicated that PS degradation interactions resulted from oxygen insertion on the PS surface
could be utilized as the energy resource and cellular in the oxidation process (Table 1). The oxidation step in
components in the absence of other alternate carbon resources Pseudomonas sp. DSM 50071-mediated PS degradation
(Figure 2B). After 60 days of culture on PS-LCFBM solid converted hydrophobic regions to hydrophilic ones, thereby
medium, Pseudomonas sp. DSM 50071 attached to the surface changing the chemical properties of the PS surface (Figure
was removed with SDS containing buffer, and the PS surface 2C).35
was re-examined by SEM. The edges of PS were changed to a XPS analysis has also confirmed that the chemical structural
smooth form as a result of degradation by Pseudomonas sp. shift from hydrophobicity to hydrophilicity is caused by
DSM 50071, whereas the edges remained rough in the control oxidation during Pseudomonas sp. DSM 50071-mediated PS
(Figure 2C). In addition to edge smoothing, holes formed by degradation. 25 Measurement of the binding energy at
degradation were observed on the PS surface where colonies Pseudomonas sp. DSM 50071-degraded areas showed that a
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Environ. Sci. Technol. 2020, 54, 6987−6996
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Figure 3. Chemical structural and component changes during PS degradation by Pseudomonas sp. DSM 50071. (A) Changes in atomic composition
due to PS degradation by Pseudomonas sp. DSM 50071. In the areas of the PS surface displaying proliferation of Pseudomonas sp. DSM 50071,
change in the quantity of carbon atoms was not observed (upper), but a prominent increase in oxygen atoms was observed (lower). Thus, during
PS degradation, the increased oxygen atoms on PS surface led to the conversion of a hydrophobic to a hydrophilic surface. (B) Changes in the
contact angle between water droplets and the PS surface. In the Pseudomonas sp. DSM 50071 growing PS surface, the contact angle formed
between water droplets and PS surface was reduced compared to the control. This result suggested that hydrophobicity was converted to
hydrophilicity during PS degradation by Pseudomonas sp. DSM 50071. (C) Surface binding energy changes during PS degradation by Pseudomonas
sp. DSM 50071. The binding energy on the PS surface increased in the test group but not in the control. This result indicated the conversion of C−
C bonds to CO bonds on the PS surface during degradation by Pseudomonas sp. DSM 50071.

Table 1. Measurements of the Contact Angles between Furthermore, we have also conducted the NMR analysis on
Water Droplets and PS Surface the degraded PS film. Compared with the control, a new peak
in the test group was detected at 2.15 ppm (Figure 4B, right),
left angle right angle contact angle (average)
sample (°) (°) (°) which could be the benzylic proton signal right adjacent to the
carbonyl group formed in the oxidation. These chemical
control 91.77 91.35 91.56
Pseudomonas sp. 79.46 79.29 79.38
structural analyses, XPS, FT-IR, and NMR, allowed us to
understand the reaction pathway during PS degradation. We
propose that PS degradation by enzymes secreted from
prominent increase in −CO bonds and a decrease in C−C Pseudomonas sp. DSM 50071 is likely initiated by oxygen
bonds. C−C bonds were gradually replaced by C−O and insertion in the methylene C−H bonds to alcohols (C−OH),
−CO bonds in areas, where Pseudomonas sp. DSM 50071 subsequently followed by further oxidation to the carbonyl
had proliferated (Figure 3C, right). However, C−C bonds (−CO) and finally fragmentation to small molecules via the
were mostly detected in areas of the control (Figure 3C, left). enzyme-mediated hydrolysis. Therefore, further studies are
This binding energy change confirmed oxidation occurred required to identify plastic-degrading enzymes secreted from
during Pseudomonas sp. DSM 50071-mediated degradation, Pseudomonas sp. DSM 50071 in PS degradation (Figure S2).
leading to the presence of conversion to hydrophilicity at the In general, plastic degradation by bacteria is mediated by
PS surface (Figure 3C). various secreted enzymes to form a biofilm. In the case of
In the ATR/FT-IR analysis, the multiple absorptions around Pseudomonas sp. DSM 50071, several different enzymes
1000 cm−1 representing the saturated C−C vibration in the PS associated with plastic degradation have been reported. The
chain and the multiple absorptions at 1450, 1550 and 1600 most representative ones have the characteristics of hydrolases,
cm−1 indicating the presence of aromatic rings were observed such as esterases, lipases, and cutinases.5 Such plastic-
in both Pseudomonas sp. DSM 50071 growing PS surface and degrading enzymes are secreted by bacteria and attached on
the control (Figure 4A). A characteristic sharp absorption peak the plastic surface to mediate fragment degradation.38,39 For
at 1715 cm−1 was only detected on the PS surface inoculated Pseudomonas sp. DSM 50071 to obtain energy and cellular
with Pseudomonas sp. DSM 50071, indicating the formation of components from PS, the upregulation of enzymes involved in
carbonyl (−CO) in the oxidation during PS degradation plastic degradation is necessary. To identify plastic-degrading
(Figure 4A, bottom),25 consistent with the XPS analysis. enzymes associated with Pseudomonas sp. DSM 50071-
Interestingly, weak absorptions around 3600 cm−1 representing mediated PS degradation, we selected six different hydrolases
O−H stretching were also detected on Pseudomonas sp. DSM and esterases encoded in Pseudomonas sp. DSM 50071 and
50071 growing PS surface (Figure 4A, middle panel), which examined their expression levels by RT-qPCR in the PS-added
suggested that alcohols (C−OH) seemed to be generated as an nutrient-limited medium (Figure 5A). Compared with
intermediate stage on the oxidative pathway to carbonyl during Pseudomonas sp. DSM 50071 growing in regular nutrient
PS degradation.36 The formation of carbonyl groups is the medium (control), expression levels of S-formylglutathione
well-accepted main indicative signal in PS and other plastic hydrolase (SGT) and SH were increased in the Pseudomonas
degradation (Figure 4A).37 sp. DSM 50071 growing in the PS-added nutrient-limited
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Environmental Science & Technology pubs.acs.org/est Article

Figure 4. ATR−FT-IR and NMR analyses on the chemical structure of the degraded PS. (A) ATR−FT-IR analysis of degraded PS by Pseudomonas
sp. DSM 50071. A carbonyl (−CO) absorption at 1715 cm−1 (black arrow, green square) and O−H stretching absorption around 3600 cm−1
(gray arrow, blue square) were detected on the ATR−FT-IR spectra of the degraded PS surface. Each square was enlarged and represented
separately. (B) 1H NMR analysis on the chemical structure of the degraded PS by Pseudomonas sp. DSM 50071. A new singlet signal at 2.15 ppm
(red arrow) corresponding to the benzylic C−H proton right adjacent to carbonyl formed in the oxidation was detected in the test group compared
to the control.

medium (Figure 5A). Specifically, a 7-fold upregulation was ditions.18,40 The decreased levels of other hydrolases and
detected for SH and a 2-fold increase was detected for SGT, as esterases may be necessary to improve the energy efficiency
compared to the control (Figure 5A). On the other hand, during PS degradation.
expression levels of the other four selected enzymes alpha/beta The essential role of SH in the PS biodegradation was
fold hydrolase (AB), arylesterase (AE), autotransport domain further confirmed by blocking its enzyme function with a
containing esterase (AT), and thioesterase were reduced specific SH inhibitor (Figure S3). As the plastic biodegradation
around 90, 30, 80, and 80%, respectively (Figure 5A). Selective rate increases proportionally to its surface area, we used the
upregulation of certain enzymes, SGT and SH, rather than bead-type PS with large surface areas instead of the film-type
many enzymes, could be essential for mediating PS PS in the SH inhibitor treatment test to obtain results in a
degradation to acquire more effectively the cellular compo- short period (Figure 5B).41 In the test group, the growth of
nents and energy required for viability and amplification of Pseudomonas sp. DSM 50071 was not affected in the nutrient
Pseudomonas sp. DSM 50071 under nutrient-limited con- broth treated with up to 50 μM of SH inhibitor, and the
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Figure 5. Identification of PS-degrading related enzymes from Pseudomonas sp. DSM 50071. (A) Examination of the expression levels of six
selected hydrolase genes in Pseudomonas sp. DSM 50071. Expression levels of six different enzymes were measured in RT-qPCR. The expression
levels of two enzymes, SGT and SH, related to plastic degradation, were increased in Pseudomonas sp. DSM 50071 growing in the PS-added
nutrient-limited medium, compared with Pseudomonas sp. DSM 50071 growing in a regular nutrient medium (control). (B) Survival test of
Pseudomonas sp. DSM 50071 in the presence of the SH inhibitor. Pseudomonas sp. DSM 50071 was cultured in nutrient broth (upper) and liquid
LCFBM media mixed with PS beads (down) with different concentrations of the SH inhibitor, 1, 10, and 50 μM. (C) Growth rates of Pseudomonas
sp. DSM 50071 in the presence of the SH inhibitor. Growth curves of Pseudomonas sp. DSM 50071 were measured in NB (left) and liquid LCFBM
mixed with PS beads (right) by optical density measurement at 600 nm. (D) Inhibition of Pseudomonas sp. DSM 50071-mediated PS
biodegradation by the SH inhibitor. The SH inhibitor (chemical compound in the graph) blocked its enzyme function and thus inhibited the
Pseudomonas sp. DSM 50071-mediated biodegradation in the PS-added nutrient-limited medium. (E) FT-IR analysis of biodegraded PS beads by
Pseudomonas sp. DSM 50071. A carbonyl (−CO) absorption at 1715 cm−1 (purple arrow) and O−H stretching absorption near 3600 cm−1
(green arrow) were examined on the FT-IR spectra of biodegraded PS beads (red line), but both peaks were not detected in treatment of 50 μM
SH inhibitor (blue line) like control PS beads (black line).

growth curve was the same as that obtained in the control DSM 50071 without the inhibitor (Figure S4A). However, the
without inhibitor (Figure 5B, upper and 5C, left). However, reduced number of Pseudomonas sp. DSM 50071 still adhered
the bacterial growth was affected and bacterial amplification via to the surface of PS beads in the presence of 1 μM SH
cell division was interfered in the presence of SH inhibitor inhibitor (Figure S4B). Inhibiting SH function limited the use
concentration ≥1 μM in the PS-bead added LCFBM medium of essential PS source for survival and amplification of
(Figures 5B, down and 5C, right). During a 15-day period, Pseudomonas sp. DSM 50071 under nutrient-limited condition,
2.6% weight reduction of PS beads by the degradation was and thus, the decreased bacterial cell number further reduced
observed in the test without SH inhibitor (Figure 5D), whereas PS biodegradation (Figure 5C,D). In the FT-IR analysis, the
around 1.3% weight reductions were observed in the presence representative carbonyl (−CO) absorption at 1715 cm−1
of SH inhibitor, 1 and 10 μM, and the PS biodegradation was and the hydroxyl (−O−H) stretching absorption around
completely blocked at 50 μM, similar with what was observed 3300−3600 cm−1 were both detected on PS beads cultured
without bacteria (negative control) (Figure 5D). SEM images with Pseudomonas sp. DSM 50071. However, these absorptions
also confirmed that the diameters of PS beads at 50 μM SH were not detected on the sample with 50 μM SH inhibitor
inhibitor were the same as control (no bacteria), different from treatment, which showed the same IR pattern as the control
significantly reduced PS beads cultured with Pseudomonas sp. without bacteria (Figure 5E). Thus, the SH inhibitor blocked
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the Pseudomonas sp. DSM 50071-mediated PS biodegradation Hee Cheol Yu − School of Undergraduate Studies, College of
by inactivation of SH enzyme instead of detaching bacteria Transdisciplinary Studies, Daegu Gyeongbuk Institute of Science
from the PS surface, which suggested that SH acted as one of and Technology, Daegu 42988, Republic of Korea
the pivotal enzymes required in PS biodegradation. Consid- Eunbeen Jeon − School of Undergraduate Studies, College of
ering SH as a well-known hydrolase, we believe it plays an Transdisciplinary Studies, Daegu Gyeongbuk Institute of Science
important role in mediating the hydrolysis step to depoly- and Technology, Daegu 42988, Republic of Korea
merize PS and/or intermediates formed from depolymerized Sukkyoo Lee − School of Undergraduate Studies, College of
PS to small molecules in the degradation (Figure S2). Transdisciplinary Studies and Department of Brain and
In summary, PS is widely used across all industries, but Cognitive Sciences, Graduate School, Daegu Gyeongbuk Institute
previous reports related to its biodegradation are limited, of Science and Technology, Daegu 42988, Republic of Korea
unlike for other plastics. Herein, we show PS biodegradation, Complete contact information is available at:
analyze chemical structural changes, and identify the enzymes https://pubs.acs.org/10.1021/acs.est.0c01495
responsible for PS degradation in Pseudomonas sp. DSM 50071
isolated from the guts of superworms which ingest PS. We Author Contributions
found that the isolated Pseudomonas sp. DSM 50071 efficiently ∥
H.L. and H.K. contributed equally to this work.
biodegraded PS in a manner similar to other plastic-degrading
Notes
bacteria. We discovered that a conversion from hydrophobicity
The authors declare no competing financial interest.

■
to hydrophilicity on the PS surface via biofilm formation is
crucial for PS degradation. Using a combination of TGA, XPS, ACKNOWLEDGMENTS
FT-IR, and NMR, we confirmed that carbonyl groups were
produced in the oxidation during PS degradation by PS This research was supported by the grants from the CJ
digestive enzymes secreted from Pseudomonas sp. DSM 50071. Blossom idea lab in CJ company and Under-Graduate
Using molecular biological experiments, we found that SGT Research Program (UGRP 2019) in Daegu Gyeongbuk
and SH were overexpressed during PS degradation, and the Institute of Science and Technology (DGIST). Also, this
specific SH inhibitor treatment test further confirmed that it is work was also supported by the INGE funds of Gwangju
Institute of Science and Technology (GIST) (GK11750).

■
a PS degradation-related enzyme. Therefore, our research
elucidates the possible mechanism for Pseudomonas sp. DSM
50071-mediated PS degradation in the gut of superworms and
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