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Treatment and Resource Recovery

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
Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.0c01495 • Publication Date (Web): 06 May 2020
Downloaded from pubs.acs.org on May 8, 2020

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1 Biodegradation of Polystyrene by Pseudomonas sp. Isolated from the Gut of Superworms

2 (Larvae of Zophobas atratus)
3
4
5 Hong Rae Kim1#, Hyun Min Lee1#, Hee Cheol Yu1, Eunbeen Jeon1, Sukkyoo Lee1,2, Jiaojie
6 Li3*, Dae-Hwan Kim1*

1
7 School of Undergraduate Studies, College of Transdisciplinary Studies, Daegu Gyeongbuk
8 Institute of Science and Technology, Daegu 42988, Republic of Korea

2
9 Department of Brain and Cognitive Sciences, Graduate School, Daegu Gyeongbuk Institute
10 of Science and Technology, Daegu 42988, Republic of Korea

3
11 Department of Chemistry, Gwangju Institute of Science and Technology, Gwangju 61005,
12 Republic of Korea

13

14 * Corresponding authors: Dae-Hwan Kim and Jiaojie Li

15 Address: School of Undergraduate Studies, Daegu Gyeongbuk Institute of Science and

16 Technology (DGIST), Daegu, Republic of Korea

17 Tel: (+82) 53-785-6692

18 Fax: (+82) 53-785-6639

19 E-mail: dhkim1@dgist.ac.kr

20

21 Address: Department of Chemistry, Gwangju Institute of Science and Technology (GIST)

22 Gwangju, Republic of Korea

23 Tel: (+82) 62-715-3655

24 Fax: (+82) 62-715-3609

25 E-mail: jjli@gist.ac.kr

26

27 # H.L. and H.K. contributed equally to this work.

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28 Abstract

29
30 Recently, various attempts have been made to solve plastic waste problems, such as
31 development of biodegradation without producing pollution. Polystyrene (PS) is the fifth most
32 used plastic in many industries; therefore, degrading PS becomes a critical global issue. Here,
33 we reported Pseudomonas aeruginosa strain DSM 50071, initially isolated from the gut of the
34 superworms, Zophobas atratus and the PS degradation by Pseudomonas sp. DSM 50071. We
35 examined PS degradation using electronic microscopy, and measured changes in atomic
36 composition and contact angles with water droplets on the PS surface that represent a chemical
37 change from hydrophobicity to hydrophilicity. We have further examined chemical structural
38 changes using X-ray photoelectron spectroscopy (XPS), Fourier-transform-infrared
39 spectroscopy (FT-IR) and nuclear magnetic resonance spectroscopy (NMR) to confirm the
40 formation of carbonyl group (C=O) in the oxidation pathway during PS biodegradation. In RT-
41 qPCR analysis, the gene expression level of serine hydrolase (SH) in Pseudomonas sp. DSM
42 50071 was highly increased during PS degradation and the enzyme-mediated biodegradation
43 of PS was further confirmed by SH inhibitor treatment test. Thus, the significance of these
44 findings goes beyond the discovery of a novel function of Pseudomonas sp. DSM 50071 in the
45 gut of superworms, highlighting a potential solution for PS biodegradation.
46

47

48 Key words: Polystyrene, Biodegradation, Pseudomonas sp., Superworm, Gut-Bacteria

49

50

51

52

53

54

55

56
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57 Introduction

58

59 The use of plastics across the globe has steeply increased, making it one of the most widely
60 used substances. However, natural plastic degradation is extremely slow, inevitably leading to
61 plastic waste accumulation, which represents a grave ecological issue. Due to the lack of
62 suitable degradation methods, plastic treatment involves 77 % reclamation, 13 % incineration,
63 and 10 % recycling. Among these, reclamation causes dangerous pollution in the soil and
64 groundwater, while incineration causes harmful substances to be released into the atmosphere,
65 rendering them both unsuitable as long-term solutions.1 Besides, plastic waste flowing into the
66 ocean is ingested by marine organisms in the form of microplastics, which causes serious health
67 problems to marine life, and subsequently could impact the health of humans.2-3 Thus, it is
68 essential to develop an efficient biodegradation method to degrade plastics.
69 Numerous researchers are making attempts to degrade plastics using microorganisms.
70 The majority searched bacteria for plastic-degrading found in landfill sites, soils, and marine
71 environments is a species belonging to the Pseudomonas genus.4 So far, Pseudomonas putida
72 and Pseudomonas fluorescens have been found to degrade Polyvinyl chloride (PVC),
73 Pseudomonas chlororaphis can degrade Polyurethane, Pseudomonas stutzri can degrade
74 polyethylene glycol, and Pseudomonas vesicularis PD can degrade polyvinyl alcohol.5
75 Furthermore, among the microorganisms isolated from soil or marine environments,
76 Pseudomonas sp. is known to mediate the most efficient degradation of polyethylene (PE).6-7
77 Specifically, the strain has been reported to reduce 20 % of the overall weight of PE during 120
78 days.8 In addition, Pseudomonas sp. is reported capable of degrading key aromatic compounds
79 in petroleum such as benzene and toluene.9
80 Polystyrene (PS) is a polymeric plastic used in packaging containers, disposable cups,
81 and insulating materials, which accounts for approximately 6.6 % of total plastic use.10 The
82 biodegradation of PS is known to progress very slowly in natural ecosystems and requires
83 several hundred years for complete degradation.5 Previous studies have reported that it can be
84 degraded by heat and photocatalysts.11-12 Nonetheless, such techniques themselves inflict
85 environmental pollution. There is no reported microorganism-mediated biodegradation method
86 currently in existence for PS, despite it being the most frequently used plastic globally.13-16
87 Recently, Pseudomonas sp. was reported to have been used in degrading High Impact PS
88 (HIPS), a polymer of PS and polybutadiene, while Pseudomonas aeruginosa was used in

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89 degrading a polymer of polylactic acid (PLA) and PS.17-18 Although Rhodococcus ruber was
90 found to have mediated biofilm formation on the PS surface and a partial reduction in the
91 weight of PS, information on the physical and chemical analyses of the plastic surface during
92 degradation has not been reported.19 Thus, the specific characterization of the degradation
93 process have not so far been verified.
94 A recent study reported that various insect larvae were able to ingest plastic and
95 participate in its degradation. Most notably, yellow and dark mealworms, the larvae of Tenebrio
96 molitor and Tenebrio obscurus and superworms, the larvae of Zophobas atratus were reported
97 capable of ingesting PS.20-23 Although insects are known to be able to ingest plastic in the past,
98 their ability to degrade it remains controversial.24 Due to a lack of overall research into this
99 problem, there remains a myriad of unknown facts. Nevertheless, several recent studies have
100 reported that mealworms and superworms ingest PS and use it as an energy source. In particular,
101 several studies on mealworms and superworms that investigated degradation ability by feeding
102 worms with antibiotics-containing bran reported that the gut microbiota was critical for plastic
103 degradation. The depolymerization of PS was significantly reduced in mealworms and
104 superworms which ingested the antibiotic gentamicin-containing bran due to the suppression
105 of their gut bacteria, suggesting that PS biodegradation is gut-microbiota-dependence.21-23
106 Yang’s group further isolated the PS-degrading bacteria, Exiguobacterium sp. strain YT2, from
107 the gut of mealworms.25
108 The present study searched for the PS-degrading bacteria in the gut of superworm,
109 larvae of Zophobas atratus. Gut bacteria were isolated from larvae that had ingested PS, and a
110 PS-degrading strain of Pseudomonas was successfully isolated. Scanning electron microscope
111 (SEM) allowed us to examine physical changes during the process of PS degradation, and
112 chemical composition changes of PS were analyzed using X-ray photoelectron spectroscopy
113 (XPS), attenuated total reflectance-fourier-transform infrared spectroscopy (ATR-FTIR) and
114 nuclear magnetic resonance spectroscopy (NMR), in order to verify Pseudomonas-mediated
115 biodegradation of PS. Moreover, we have also identified the PS depolymerization enzyme, a
116 serine hydrolase (SH) by reverse transcription quantitative polymerase chain reaction (RT-
117 qPCR) analysis and the enzyme inhibitor test. Based on these findings, we proposed the
118 possible pathway during the degradation process. In summary, the study has shown that a
119 Pseudomonas strain, previously known to degrade plastic in soils and marine environments, is
120 also found in the gut of insects where the bacteria participate in the degradation of PS ingested
121 by superworms.
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122 Materials and Methods

123

124 Isolation of plastic-degrading bacteria from superworms

125 We purchased the 3-4 instar growth stage mealworms and superworms (NB mealworms,
126 Yangju, South Korea). Mealworms and superworms, the larva of Tenebrio molitor and
127 Zophobas atratus, were placed in a breeding chamber (n = 50 worms). To create a gut
128 environment enriched with plastic-degrading bacteria, Styrofoam PS (Mw = 302,300, Mn =
129 108,700, 99.5% purity, Myung-IL FOAMTEC, Daegu, South Korea) was supplied as the sole
130 carbon food source. To isolate the gut bacteria from the larvae bred in an incubator at 25 °C for
131 two weeks, the larvae were anesthetized by immersing in 70 % ethanol and then washed with
132 saline water (SW). The head and the tail were removed and the gut was extracted using a pair
133 of sterilized forceps. The extracted gut was chopped and vortexed with 5 mL SW for 5 mins
134 and then centrifuged for 5 mins at 800 rpm to remove the epithelial cells. The supernatant layer
135 was carefully transferred to a flask containing Liquid Carbon Free Basal Medium (LCFBM)
136 (pH: 6.51, 0.7 g of KH 2 PO 4 , 0.7 g of K 2 HPO 4 , 0.7 g of MgSO 4 ·7H 2 O, 1.0 g of NH 4 NO 3 ,
137 0.005 g of NaCl, 0.002 g of FeSO 4 ·7H 2 O, 0.002 g of ZnSO 4 ·7H 2 O, and 0.001 g of
138 MnSO 4 ·H 2 O in 1 L of deionized water) with sodium thioglycolate to maintain an anaerobic
139 condition, and then mixed thoroughly. After adding film-type PS (Mw = 316,900, Mn =
140 110,600, 99.9% purity, Goodfellow, Huntingdon, UK), the mixture solution was cultured in a
141 shaking incubator at 25 °C with 180 rpm for 60 days under the anaerobic condition.
142

143 Identification of gut bacteria in superworms

144 After 60 days of culture, the gut solution was diluted to 1/100 with LCFBM solution and then
145 2 mL of bacteria containing LCFBM solution was spread onto a solid nutrient medium (BD
146 Difco, cat#: 90002-660, Franklin Lakes, NJ, USA; Beef Extract 3.0 g, Peptone 5.0 g, Agar 15.0
147 g in 1 L of deionized water, pH: 6.8). For bacterial culture, a completely oxygen-free
148 environment was supplied. The culture was performed in an anaerobic culture chamber
149 (Anaerobic Jar, Mitsubishi gas chemical company, Japan) containing a deoxygenation package
150 (Anaero Pack- Anaero, Mitsubishi gas chemical company, Japan) at 25 °C for 3-4 days.26-27
151 Each colony grown on the solid nutrient medium was transferred to the liquid nutrient medium
152 and cultured for another two days at 25 °C under the anaerobic condition. To determine the
153 species of bacteria grown in the liquid medium, genomic PCR was performed after extracting
154 the bacterial genomic DNA to obtain the 16S RNA sequence and the primers used were as
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155 following: forward primer 27-F (5‘-AGAGTTTGATYMTGGCTCAG-3’); reverse primer

156 1492-R (5′-GGTTACCTTGTTA-CGACTT-3′). 16S rRNA gene sequence analysis was carried
157 out to identify the species. A T100 Thermal Cycler (Bio-Rad, Singapore) was used for PCR,
158 and amplified PCR DNA bands were analyzed by electrophoresis. After sequencing the 16S
159 rRNA DNA, the bacterial species were determined using NCBI BLAST analysis.
160

161 PS degradation by Pseudomonas sp. DSM 50071

162 Pseudomonas sp. was cultured with PS film in a solid LCFBM medium lacking an alternate
163 carbon source to verify plastic degradation. After collecting 5 mL of bacteria solution cultured
164 in the liquid nutrient medium, it was centrifuged for 5 mins at 4000 rpm. We removed the
165 nutrient medium and then added 1 mL of LCFBM. Using a pipette, the bacteria were
166 resuspended and centrifuged again to collect the bacterial sample. To completely remove the
167 remaining nutrient medium components, identical washing steps were carried out three times.
168 After the final washing step, each Pseudomonas sp. pellet was mixed with 0.1 mL LCFBM,
169 and then the suspended mixture was layered onto a solid LCFBM plate. After drying, a
170 sterilized PS film (Mw = 316,900, Mn = 110,600, 99.9% purity, Goodfellow, Huntingdon, UK)
171 was placed on the solid LCFBM plate. The plate was sealed with parafilm and placed in a
172 deoxygenating chamber (ThermoStable IR-250, Daihan Scientific Co., Seoul, South Korea).
173 After incubation at 25 °C for 60 days, bacterial colonies were examined.
174

175 Scanning Electron Microscopy (SEM) analysis

176 SEM (SU8230, Hitachi, Tokyo, Japan) was used to verify the proliferation of Pseudomonas sp.
177 DSM 50071 on the PS surface as well as PS degradation. For the observation of amplified
178 Pseudomonas sp. DSM 50071, the PS on which Pseudomonas sp. DSM 50071 had grown was
179 cut to a size of 1 cm × 1 cm, and the surface was investigated. To analyze changes on the PS
180 surface after degradation by Pseudomonas sp. DSM 50071, 2 % sodium dodecyl sulfate (SDS)
181 solution was used for 4 hours to remove bacteria attached to PS.28-29 Then, the degraded PS
182 surface was examined with SEM. First, PS was fixed on a copper tape and platinum coating
183 was added using an ion sputter for 15 seconds at 15 mA (E-1045, Hitachi High-Technologies
184 Corporation, Tokyo, Japan). Then, the degraded PS surface was imaged at 1.6 kV acceleration
185 voltage.
186

187 Analysis of the elemental composition on the PS surface

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188 To verify changes in the elemental composition on the PS surface during degradation by
189 Pseudomonas sp. DSM 50071, X-ray energy dispersive spectroscopy with a module attached
190 to the SEM was used to estimate the elemental composition between carbon and oxygen on the
191 PS surface in the areas exhibiting proliferated Pseudomonas sp. DSM 50071. We examined
192 changes in the elemental composition by analyzing the difference between the test group and
193 the control.
194

195 Examination of changes in hydrophilicity on the PS surface

196 During PS degradation by Pseudomonas sp. DSM 50071, contact angle analysis was used to
197 analyze changes in hydrophilicity on the PS surface. First, PS was fixed on a carbon tape to
198 measure the contact angle using the contact angle meter (Phoenix Multi, Seoul, Korea), and 35
199 μL of water was dropped onto the PS surface within a 20 mm2 area at 20 °C. Both left and right
200 contact angles were measured and the mean value was used for the comparative analysis.25, 29
201

202 Examination of PS degradation using X-ray photoelectron spectroscopy (XPS)

203 To verify the PS degradation by Pseudomonas sp. DSM 50071, changes in elemental
204 composition on the PS surface were examined by measuring the binding energy with an X-ray
205 photoelectron spectrometer (ESCALAB 250Xi, Thermo Scientific, Waltham, MA, USA). To
206 prepare the sample, prearranged PS (1 cm × 1 cm) fixed on the carbon tape was measured
207 within the energy range of 276–300 eV, C 1 s.
208

209 Examination of PS degradation using Fourier-Transform Nuclear Magnetic Resonance

210 spectroscopy (FT-NMR)
211 To further examine chemical structural changes during PS degradation, chemical structural
212 analysis was carried out using FT-NMR (AVANCE III 400, Bruker, Germany). The degraded

213 5 mg PS film was dissolved in 0.5 ml chloroform-d (≥ 99.96%, Sigma-Aldrich, St. Louis, MO,

214 USA), and the NMR analysis was carried out at 400 MHz and 25 °C.
215

216 Analysis of PS biodegradation with Attenuated Total Reflectance-Fourier-Transform-

217 Infrared spectroscopy (ATR-FT-IR)
218 FT-IR (Nicolet Continuum, Thermo Fisher Scientific) was used to verify PS biodegradation by
219 Pseudomonas sp. DSM 50071. To prepare samples, PS film was completely dried at 60 °C for

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220 24 h after removing bacteria. Then, FT-IR measurements were performed using smart single-
221 bounce attenuated total reflectance with a diamond tip in the 4400–400 cm-1 range.
222

223 RT-qPCR
224 After the collection of Pseudomonas sp. DSM 50071 grown on LCFBM medium supplied with
225 PS and nutrient, total RNA was isolated using the Trizol method (Invitrogen, Carlsbad, CA, USA).
226 After quantification of total RNA, 1 µg of RNA was heated with 1 µL random primer at 65 °C
227 for 5 mins, and then 2 µL dNTPs and 4 µL 5X reaction buffer, 2 µL DTT, 1 µL Rnaseout and
228 0.5 µL SuperScript II (Invitrogen) were added to the cool-down mixture. cDNA synthesis was
229 performed at 42 °C for 50 mins and then terminated by inactivation at 75 °C for 5 mins. In
230 qRT-PCR, a CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA)
231 was used to measure the expression level of each gene with primer sets (Table S1).
232 Subsequently, 1 µL from 1/5 diluted cDNA was mixed with iQ SYBR Green Supermix (Bio-
233 Rad). The 16S rRNA gene was used as the endogenous control. The relative quantification of
234 each gene expression in the test group versus control was calculated by the Bio-Rad manager
235 3.0 software program and the expressed level of each gene was described in terms of its relative
236 value between the test group and the control.
237

238 Weight measurement of PS beads

239 To quantify the mass reduction of PS by Pseudomonas sp. DSM 50071-mediated degradation,
240 1 g of PS beads (diameter = 1500 μm, Mw = 371,100, Mn = 153,600, 99.5% purity, Myung-IL
241 FOAMTEC, Daegu, South Korea) were biodegraded by Pseudomonas sp. DSM 50071
242 (bacterial number = 1 × 108/mL) in liquid LCFBM for 15 days at 25 °C, with 180 rpm. The
243 biodegraded PS beads obtained through a 70 μm sieve and a 0.45 μm filter were processed with
244 2 % SDS, rinsed with DW, and then dried at 60 °C for 24 h and their masses were measured
245 independently in triplicate. We have further conducted the SH inhibitor test, different
246 concentrations of the purchased serine hydrolase inhibitor-7 (tert-Butyl 4-[{2-
247 fluorophenyl}carbamoyl]piperazine-1-carboxylate, SHI-7, CAS#: 931086-01-0, Sigma-
248 Aldrich) were added in liquid LCFBM including 1 g of PS beads and Pseudomonas sp. DSM
249 50071. For a control sample without Pseudomonas sp. DSM 50071, and SH inhibitor-added
250 samples, the weight changes of PS beads were measured using the same method.

251

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252 Results and Discussion

253

254 Recently, several insects have been reported capable of degrading various plastics. For instance,
255 mealworms ingest and mediate rapid degradation of PS-consisting Styrofoam and other PS-
256 containing plastic products.30 PS biodegradation proceeds slowly in natural ecosystem,
257 requiring several hundred years for complete degradation but such insect-based systems
258 suggest that PS biodegradation can occur within several weeks, a much shorter time. In addition
259 to the well-known mealworms,21 we characterized superworms (Zophobas atratus)– an insect
260 only recently observed to mediate PS degradation after ingestion (Figure 1A and S1). To
261 compare the changes in the ingested amount of PS supplied between superworms and
262 mealworms, we measured changes in the weight of PS after twenty-one days (Figure 1B). The
263 amounts of PS ingested by fifty superworms and fifty mealworms differed significantly.
264 Superworms consumed approximately 1.42 g from initially supplied 2 g of PS during the
265 twenty-one-day period, whereas fifty mealworms ingested 0.22 g from the same amount PS
266 supply (Figure 1B). Our weight and length measurements of each worm indicated that the
267 average weight and length of superworms were 0.4648 g (±0.0324 g) and 3.64 cm (±0.079 cm),
268 respectively; whereas those of mealworms were 0.0454 g (±0.0024 g) and 1.70 cm (±0.062
269 cm), respectively. Since the amount of plastic ingested by worms is related to the weights of
270 worms, PS consumption rates per weight (g) of worms were measured (Figure 1C). The average
271 rate of PS reduction by the fifty superworms was 68 mg per day, which exceeded 11 mg per
272 day measured in mealworms, but the average consuming rate per 1 g mealworms was around
273 1.7 times higher than that measured in superworms (Figure 1C). Thus, mealworms showed
274 better efficiency than superworms for PS ingestion. Around 90 % of both worms, mealworms
275 and superworms were survived after the 21-day period.
276 TGA and FT-IR analyses were further conducted on the frass of PS-ingested mealworms
277 and superworms to confirm PS biodegradation. In the TGA analysis, four maximum
278 decomposition rates, 68, 251, 325, and 378 °C, were observed on the frass of PS-ingested
279 mealworms, whereas three maximum decomposition rates, 88, 327, and 383 °C, were recorded
280 on the frass of PS-ingested superworms (Figure 1D, upper). The frass of superworms showed
281 36 % mass loss within 120–395 °C and 27 % mass loss within 400–480 °C, whereas that of
282 mealworms were 39 % mass loss within 121–380 °C and 33 % mass loss within 400–480 °C
283 (Figure 1D, upper). The FT-IR analyses on the frass of both worms showed a broad O-H
284 stretching absorption around 3300-3600 cm-1 and a sharp carbonyl (-C=O) stretching
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285 absorption around 1700 cm-1, when compared with that of PS in control (Figure 1D, down).
286 These results confirmed that PS consumed by superworms and mealworms were biodegraded
287 through their digestive system. In addition, the similar FT-IR patterns suggested that the
288 biodegradation of PS could be progressed with the similar oxidation process by both worms.
289 As superworms readily ingest PS, we anticipated that they might also contain active PS-
290 degrading bacteria in the gut. Because a huge diversity of bacteria survives in the guts of the
291 superworms, it was deemed likely that some, but not all, would be directly involved in PS
292 degradation. Thus, isolation of each bacterium species to test PS biodegradation was required.
293 After identification of each single colony with 16S RNA sequencing, we found four different
294 strains of bacteria growing on the solid medium, each showing distinct morphology (Figure
295 1E). We surmised that these cultured bacteria could be candidates that utilize PS for their
296 viability and proliferation. Among the gut bacteria of superworms, Pseudomonas aeruginosa,
297 rod-shaped with 0.5 µm wide and 2 µm long, was identified as the bacterial strain exhibiting
298 the highest fraction, and comprised approximately 35 % of all sequenced bacteria. Few other
299 Pseudomonas strains participating in PS degradation have been identified, and most are derived
300 from soil and deep marine.5 Here, we identified for the first time a Pseudomonas aeruginosa
301 strain DSM 50071 (accession number in the GenBank: SUB6959368 16S MT045992), residing
302 in the guts of the superworms that might be engaged in PS degradation.
303 To confirm that isolated Pseudomonas sp. DSM 50071 could conduct the PS degradation,
304 we tested PS-degrading ability of Pseudomonas sp. DSM 50071 using a piece of PS in solid
305 LCFBM medium. Colonies of growing Pseudomonas sp. DSM 50071 were observed on the
306 piece of PS after culture for 30 days. In particular, large numbers of Pseudomonas sp. DSM
307 50071 were found around the edges of the PS (Figure 2A). SEM was used to examine the
308 shapes and characteristics of growing colonies, and the obtained images showed that
309 Pseudomonas sp. DSM 50071 firmly attached to the PS surface (Figure 2B). The viability and
310 proliferation of Pseudomonas sp. DSM 50071 on the PS surface indicated that PS degradation
311 could be utilized as the energy resource and cellular components in the absence of other
312 alternate carbon resources (Figure 2B). After 60 days of culture on PS-LCFBM solid medium,
313 Pseudomonas sp. DSM 50071 attached to the surface was removed with SDS containing buffer,
314 and the PS surface was re-examined by SEM. The edges of PS were changed to a smooth form
315 as a result of degradation by Pseudomonas sp. DSM 50071, whereas the edges remained rough
316 in the control (Figure 2C). In addition to edge smoothing, holes formed by degradation were
317 observed on the PS surface where colonies had been present, further confirming Pseudomonas
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318 sp. DSM 50071-mediated PS degradation. (Figure 2C). Despite SDS treatment, a proportion
319 of Pseudomonas sp. DSM 50071 in or near such holes was not entirely removed (Figure 2C,
320 middle row, right panel). Our findings provide the direct evidence that Pseudomonas sp. DSM
321 50071 isolated from the guts of superworms degrades PS.
322 To further confirm Pseudomonas sp. DSM 50071-mediated PS degradation, we have
323 analyzed the composition of carbon and oxygen atoms present on the PS surface in the
324 boundary region, where Pseudomonas sp. DSM 50071 had grown in large numbers. Results
325 showed that there was no significant difference in the number of carbon atoms between the
326 control and the areas in the PS exhibiting Pseudomonas sp. DSM 50071 growth (Figure 3A,
327 upper). On the contrary, a large number of oxygen atoms was detected in PS areas exhibiting
328 Pseudomonas sp. DSM 50071 growth, indicating that PS degradation-related enzymes secreted
329 by Pseudomonas sp. DSM 50071 had promoted oxidation as part of degradation (Figure 3A,
330 bottom). Such oxidation was absent in the control (Figure 3A). One of the most notable
331 characteristics of bacteria-mediated plastic degradation processes is the formation of a biofilm
332 on the plastic surface. The generated biofilm accelerates plastic degradation, by increasing the
333 contact area between the bacteria and the plastic (hydrophobic) surface, thereby speeding up
334 oxidation and overall degradation.28, 31 During the oxidation process, functional groups such as
335 hydroxyl or carbonyl groups could be formed via β-oxidation, which is known to be used in
336 the TCA cycle or energy metabolism in bacteria, thus increasing the hydrophilicity.32-33
337 Therefore, the increased number of oxygen atoms on the plastic surface in the areas exhibiting
338 Pseudomonas sp. DSM 50071 growth provides direct evidence of the PS degradation (Figure
339 3A).
340 Oxidation as the crucial step in Pseudomonas sp. DSM 50071-mediated PS degradation
341 converted the PS surface from hydrophobicity to hydrophilicity, which was confirmed by the
342 changes in the contact angles between water droplets and the PS surface.34 In comparison to
343 the control, the average value of the contact angles on the left and right sides of the water
344 droplets on a PS surface exhibiting Pseudomonas sp. DSM 50071 growth was highly reduced
345 (Figure 3B).25 As the left contact angle was 91.77° and the right contact angle was 91.35°, the
346 average contact angle was 91.56° in the case of the control (Table 1). Whereas on the PS surface
347 exhibiting Pseudomonas sp. DSM 50071 growth, this angle was 79.46° on the left and 79.29°
348 on the right, with an average contact angle of 79.39°. Thus, a 12.1° average contact angle
349 reduction was observed in comparison to the control (Table 1 and Figure 3B). The reduced
350 contact angle between the water droplets and the plastic surface represents the surface tension
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351 of water decreasing due to more abundant and stronger polar interactions resulted from oxygen
352 insertion on the PS surface in the oxidation process (Table 1). The oxidation step in
353 Pseudomonas sp. DSM 50071-mediated PS degradation converted hydrophobic regions to
354 hydrophilic ones, thereby changing the chemical properties of the PS surface (Figure 2C).35
355 XPS analysis has also confirmed that the chemical structural shift from hydrophobicity
356 to hydrophilicity is caused by oxidation during Pseudomonas sp. DSM 50071-mediated PS
357 degradation.25 Measurement of the binding energy at Pseudomonas sp. DSM 50071-degraded
358 areas showed a prominent increase in -C=O bonds and a decrease in C-C bonds. C-C bonds
359 were gradually replaced by C-O and -C=O bonds in areas where Pseudomonas sp. DSM 50071
360 had proliferated (Figure 3C, right). However, C-C bonds were mostly detected in areas of the
361 control (Figure 3C, left). This binding energy change confirmed oxidation occurred during
362 Pseudomonas sp. DSM 50071-mediated degradation, leading to the presence of conversion to
363 hydrophilicity at PS surface (Figure 3C).
364 In the ATR/FT-IR analysis, the multiple absorptions around 1000 cm-1 representing the
365 saturated C-C vibration in the PS chain and the multiple absorptions at 1450, 1550 and 1600
366 cm-1 indicating the presence of aromatic rings were observed in both Pseudomonas sp. DSM
367 50071 growing PS surface and the control (Figure 4A). A characteristic sharp absorption peak
368 at 1715 cm-1 was only detected on the PS surface inoculated with Pseudomonas sp. DSM 50071,
369 indicating the formation of carbonyl (-C=O) in the oxidation during PS degradation (Figure
370 4A, bottom)25, consistent with the XPS analysis. Interestingly, weak absorptions around 3600
371 cm-1 representing O-H stretching were also detected on Pseudomonas sp. DSM 50071 growing
372 PS surface (Figure 4A, middle panel), which suggested that alcohols (C-OH) seemed to be
373 generated as an intermediate stage on the oxidative pathway to carbonyl during PS
374 degradation.36 The formation of carbonyl groups is the well-accepted main indicative signal in
375 PS and other plastic degradation (Figure 4A).37
376 Furthermore, we have also conducted the NMR analysis on the degraded PS film.
377 Compared with the control, a new peak in the test group was detected at 2.15 ppm (Figure 4B,
378 right), which could be the benzylic proton signal right adjacent to the carbonyl group formed
379 in the oxidation. These chemical structural analyses, XPS, FT-IR and NMR, allow us to
380 understand the reaction pathway during PS degradation. We propose that PS degradation by
381 enzymes secreted from Pseudomonas sp. DSM 50071 is likely initiated by oxygen insertion in
382 the methylene C-H bonds to alcohols (C-OH), subsequently followed by further oxidation to
383 the carbonyl (-C=O), and finally fragmentation to small molecules via the enzyme-mediated
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384 hydrolysis. Therefore, further studies are required to identify plastic-degrading enzymes
385 secreted from Pseudomonas sp. DSM 50071 in PS degradation (Figure S2).
386 In general, plastic degradation by bacteria is mediated by various secreted enzymes to
387 form a biofilm. In the case of Pseudomonas sp. DSM 50071, several different enzymes
388 associated with plastic degradation have been reported. The most representative ones have the
389 characteristics of hydrolases, such as esterases, lipases, and cutinases.5 Such plastic-degrading
390 enzymes are secreted by bacteria and attached on the plastic surface to mediate fragment
391 degradation.38-39 For Pseudomonas sp. DSM 50071 to obtain energy and cellular components
392 from PS, the upregulation of enzymes involved in plastic degradation is necessary. To identify
393 plastic-degrading enzymes associated with Pseudomonas sp. DSM 50071-mediated PS
394 degradation, we selected six different hydrolases and esterases encoded in Pseudomonas sp.
395 DSM 50071 and examined their expression levels by RT-qPCR, in the PS-added nutrient-
396 limited medium (Figure 5A). Compared with Pseudomonas sp. DSM 50071 growing in regular
397 nutrient medium (control), expression levels of S-formylglutathione hydrolase (SGT) and
398 Serine hydrolase (SH) were increased in the Pseudomonas sp. DSM 50071 growing in the PS-
399 added nutrient-limited medium (Figure 5A). Specifically, a 7-fold upregulation was detected
400 for SH and a 2-fold increase was detected for SGT, as compared to the control (Figure 5A). On
401 the other hand, expression levels of the other four selected enzymes: alpha/beta fold hydrolase
402 (AB), arylesterase (AE), autotransport domain containing esterase (AT), and thioesterase (TE)
403 were reduced around 90, 30, 80 and 80 %, respectively (Figure 5A). Selective upregulation of
404 certain enzymes, SGT and SH, rather than many enzymes, could be essential for mediating PS
405 degradation to acquire more effectively the cellular components and energy required for
406 viability and amplification of Pseudomonas sp. DSM 50071 under nutrient-limited conditions.
18,40
407 The decreased levels of other hydrolases and esterases may be necessary to improve the
408 energy efficiency during PS degradation.
409 The essential role of SH in the PS biodegradation was further confirmed by blocking its
410 enzyme function with a specific SH inhibitor (Figure S3). As the plastic biodegradation rate
411 increases proportionally to its surface area, we used the bead-type PS with large surface areas
412 instead of the film-type PS in the SH inhibitor treatment test to obtain results in a short period
413 (Figure 5B).41 In the test group, the growth of Pseudomonas sp. DSM 50071 was not affected
414 in the nutrient broth treated with up to 50 µM of SH inhibitor and the growth curve was the
415 same as that obtained in the control without inhibitor (Figure 5B, upper and 5C, left). However,
416 the bacterial growth was affected and bacterial amplification via cell division was interfered in
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417 the presence of SH inhibitor concentration ≥ 1 µM in the PS-bead added LCFBM medium

418 (Figures 5B, down and 5C, right). During a 15-day period, 2.6 % weight reduction of PS beads
419 by the degradation was observed in the test without SH inhibitor (Figure 5D). Whereas around
420 1.3 % weight reductions were observed in the presence of SH inhibitor, 1 and 10 µM, and the
421 PS biodegradation was completely blocked at 50 µM, similar with what was observed without
422 bacteria (negative control) (Figure 5D). SEM images also confirmed that the dimeters of PS
423 beads at 50 µM SH inhibitor were same as control (no bacteria), different from significantly
424 reduced PS beads cultured with Pseudomonas sp. DSM 50071 without the inhibitor (Figure
425 S4A). However, reduced number of Pseudomonas sp. DSM 50071 still adhered to the surface
426 of PS beads in the presence of 1 µM SH inhibitor (Figure S4B). Inhibiting SH function limited
427 the use of essential PS source for survival and amplification of Pseudomonas sp. DSM 50071
428 under nutrient-limited condition, and thus the decreased bacterial cell number further reduced
429 PS biodegradation (Figure 5C and D). In the FT-IR analysis, the representative carbonyl (-C=O)
430 absorption at 1715 cm-1 and the hydroxyl (-O-H) stretching absorption around 3300-3600 cm-
1
431 were both detected on PS beads cultured with Pseudomonas sp. DSM 50071. However, these
432 absorptions were not detected on the sample with 50 µM SH inhibitor treatment, which showed
433 the same IR pattern as the control without bacteria (Figure 5E). Thus, the SH inhibitor blocked
434 the Pseudomonas sp. DSM 50071-mediated PS biodegradation by inactivation of SH enzyme
435 instead of detaching bacteria from PS surface, which suggested that SH acted as one of the
436 pivotal enzymes required in PS biodegradation. Considering SH as a well-known hydrolase,
437 we believe it plays an important role in mediating the hydrolysis step to depolymerize PS and/or
438 intermediates formed from depolymerized PS to small molecules in the degradation (Figure
439 S2).
440 In summary, polystyrene (PS) is widely used across all industries, but previous reports
441 related to its biodegradation are limited, unlike for other plastics. Herein, we show PS
442 biodegradation, analyze chemical structural changes and identify the enzymes responsible for
443 PS degradation in Pseudomonas sp. DSM 50071 isolated from the guts of superworms which
444 ingest PS. We found that the isolated Pseudomonas sp. DSM 50071 efficiently biodegraded PS
445 in a manner similar to other plastic-degrading bacteria. We discovered that a conversion from
446 hydrophobicity to hydrophilicity on the PS surface via biofilm formation is crucial for PS
447 degradation. Using a combination of TGA, XPS, FT-IR and NMR, we confirmed that carbonyl
448 groups were produced in the oxidation during PS degradation by PS digestive enzymes secreted

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449 from Pseudomonas sp. DSM 50071. Using molecular biological experiments, we found that S-
450 formylglutathione hydrolase (SGT) and Serine hydrolase (SH) were overexpressed during PS
451 degradation. And the specific SH inhibitor treatment test further confirmed that it is a PS
452 degradation-related enzyme. Therefore, our research elucidates the possible mechanism for
453 Pseudomonas sp. DSM 50071-mediated PS degradation in the gut of superworms, and
454 highlights the potential utility of SGT and SH to maximize the efficiency of PS degradation
455 following further studies.
456

457 Supporting Information

458 Detailed information about the sequence of primers used for RT-qPCR performance, pictures
459 for PS ingesting superworms and mealworms, the proposed PS degradation pathway by
460 Pseudomonas sp. DSM 50071, and SEM pictures of PS beads for demonstrating the effect of
461 SH inhibitor.
462

463 Conflicts of interest

464 There are no conflicts to declare.
465

466 Acknowledgements

467 This research was supported by the grants from the CJ Blossom idea lab in CJ company and
468 Under-Graduate Research Program (UGRP 2019) in Daegu Gyeongbuk Institute of Science
469 and Technology (DGIST). Also, this work was also supported by the INGE funds of Gwangju
470 Institute of Science and Technology (GIST) (GK11750).
471

472

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595

596 Table 1. Measurements of the contact angles between water droplets and PS surface
Contact angle
Sample Left angle (°) Right angle (°)
(Average)(°)
Control 91.77 91.35 91.56

Pseudomonas sp. 79.46 79.29 79.38

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625 Figure legends

626
627
628 Figure 1. Identification of Pseudomonas sp. DSM 50071 in the PS ingested superworms.
629 (A) PS ingested and digested by the superworms. (B) Measurements of PS ingestion between
630 the same number (N: 50) of superworms and mealworms. The PS weight was measured every
631 three days during a twenty-one-day period to determine its reducing rate by worms.
632 Superworms (blue line) consumed approximately 1.42 g from initially supplied 2 g of PS,
633 whereas mealworms ingested 0.22 g from 2 g of PS (red line). (C) Comparison of the PS
634 degradation rates between mealworms and superworms. Averagely, 68 mg of PS per day was
635 consumed by the fifty superworms (blue bar), whereas 11 mg of PS per day was consumed by
636 the fifty mealworms (red bar) (left). Also, 2.9 mg of PS per day was consumed by 1 g of
637 superworms (blue bar) and 4.8 mg of PS per day was consumed by 1 g of mealworms (red bar)
638 (right). (D) Measurement of biodegradation of PS using FT-IR and TGA. In TGA spectra,
639 several decomposition points were examined in the frass of mealworms and superworms unlike
640 single decomposition point of PS control (upper). In comparison to the control group, FT-IR
641 spectra for frass of mealworms and superworms showed the biodegradation of PS (down). (E)
642 Isolation of bacteria inhabiting the guts of the superworms. The chopped guts of superworms
643 mixed with PS-added LCFBM was cultured for 60 days and the surviving, amplified bacteria
644 were isolated in a solid nutrient medium. Four different species of bacteria including
645 Pseudomonas sp. DSM 50071, were identified.
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666 Figure 2. PS degradation by Pseudomonas sp. DSM 50071. (A) Pseudomonas sp. DSM
667 50071 normally grows on PS. Pseudomonas sp. DSM 50071 were viable and proliferated on
668 the PS, especially around its edges. Several colonies of Pseudomonas sp. DSM 50071 were
669 observed at the center of PS surface. (B) SEM detection of the inhabited Pseudomonas sp.
670 DSM 50071 attached to PS. Most Pseudomonas sp. DSM 50071 attached to PS surface were
671 observed under SEM in proliferated colonies. (C) PS digestion by Pseudomonas sp. DSM
672 50071. Unlike the control, the PS inhabiting Pseudomonas sp. DSM 50071 exhibited
673 degradation, which was investigated in the areas around the edges of the PS (upper panel) and
674 several large newly generated holes in the PS surface (lower panel). Although SDS buffer was
675 used to remove Pseudomonas sp. DSM 50071, there still remained a small number of attached
676 Pseudomonas sp. DSM 50071, which were investigated near PS degraded areas (upper right).
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699 Figure 3. Chemical structural and component changes during PS degradation by

700 Pseudomonas sp. DSM 50071. (A) Changes in atomic composition due to PS degradation by
701 Pseudomonas sp. DSM 50071. In the areas of the PS surface displaying proliferation of
702 Pseudomonas sp. DSM 50071, change in the quantity of carbon atoms was not observed (upper)
703 but a prominent increase in oxygen atoms was observed (lower). Thus, during PS degradation,
704 the increased oxygen atoms on PS surface led to the conversion of a hydrophobic to a
705 hydrophilic surface. (B) Changes in the contact angle between water droplets and the PS
706 surface. In the Pseudomonas sp. DSM 50071 growing PS surface, the contact angle formed
707 between water droplets and PS surface was reduced, compared to the control. This result
708 suggested that hydrophobicity was converted to hydrophilicity during PS degradation by
709 Pseudomonas sp. DSM 50071. (C) Surface binding energy changes during PS degradation by
710 Pseudomonas sp. DSM 50071. The binding energy on the PS surface increased in the test group
711 but not in the control. This result indicated the conversion of C-C bonds to C=O bonds on the
712 PS surface during degradation by Pseudomonas sp. DSM 50071.
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740 Figure 4. ATR-FTIR and NMR analyses on the chemical structure of the degraded PS.

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741 (A) ATR-FTIR analysis of degraded PS by Pseudomonas sp. DSM 50071. A carbonyl (-C=O)
742 absorption at 1715 cm-1 (black arrow, green square) and O-H stretching absorption around 3600
743 cm-1 (gray arrow, blue square) were detected on the ATR-FTIR spectra of the degraded PS
744 surface. Each square was enlarged and represented separately. (B) 1H NMR analysis on the
745 chemical structure of the degraded PS by Pseudomonas sp. DSM 50071. A new singlet signal
746 at 2.15 ppm (red arrow) corresponding to the benzylic C-H proton right adjacent to carbonyl
747 formed in the oxidation was detected in the test group, compared to the control.
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773 Figure 5. Identification of PS-degrading related enzymes from Pseudomonas sp. DSM
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774 50071. (A) Examination of the expression levels of six selected hydrolase genes in
775 Pseudomonas sp. DSM 50071. Expression levels of six different enzymes were measured in
776 RT-qPCR. The expression levels of two enzymes, S-formylglutathione hydrolase (SGT) and
777 Serine hydrolase (SH), related to plastic degradation, were increased in Pseudomonas sp. DSM
778 50071 growing in the PS-added nutrient-limited medium, compared with Pseudomonas sp.
779 DSM 50071 growing in a regular nutrient medium (control). (B) Survival test of Pseudomonas
780 sp. DSM 50071 in the presence of the SH inhibitor. Pseudomonas sp. DSM 50071 was cultured
781 in nutrient broth (upper) and liquid LCFBM media mixed with PS beads (down) with different
782 concentrations of the SH inhibitor, 1, 10, and 50 µM. (C) Growth rates of Pseudomonas sp.
783 DSM 50071 in the presence of the SH inhibitor. Growth curves of Pseudomonas sp. DSM
784 50071 were measured in NB (left) and liquid LCFBM mixed with PS beads (right) by optical
785 density (OD) measurement at 600 nm. (D) Inhibition of Pseudomonas sp. DSM 50071-
786 mediated PS biodegradation by the SH inhibitor. The SH inhibitor (chemical compound in the
787 graph) blocked its enzyme function, and thus inhibited the Pseudomonas sp. DSM 50071-
788 mediated biodegradation in the PS-added nutrient-limited medium. (E) FT-IR analysis of
789 biodegraded PS beads by Pseudomonas sp. DSM 50071. A carbonyl (-C=O) absorption at 1715
790 cm-1 (purple arrow) and O-H stretching absorption near 3600 cm-1 (green arrow) were
791 examined on the FTIR spectra of biodegraded PS beads (red line) but both peaks were not
792 detected in treatment of 50 µM SH inhibitor (blue line) like control PS beads (black line).
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