Biodegradation of Polystyrene by Dark (Tenebrio obscurus) and

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Biodegradation of Polystyrene by Dark (Tenebrio obscurus) and Yellow (Tenebrio molitor) Mealworms (Coleoptera: Tenebrionidae) Bo-Yu Peng, Yiming Su, Zhibin Chen, Jiabin Chen, Xuefei Zhou, Mark Eric Benbow, Craig Criddle, Wei-Min Wu, and Yalei Zhang Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b06963 • Publication Date (Web): 16 Apr 2019 Downloaded from http://pubs.acs.org on April 19, 2019

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Environmental Science & Technology

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Biodegradation of Polystyrene by Dark (Tenebrio obscurus)

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and Yellow (Tenebrio molitor) Mealworms (Coleoptera:

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Tenebrionidae)

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Bo-Yu Peng, † Yiming Su, †, ‖ Zhibin Chen, † Jiabin Chen, † Xuefei Zhou, † Mark Eric

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Benbow, § Craig S. Criddle, ‡ Wei-Min Wu, *, ‡ and Yalei Zhang *, †

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† State Key Laboratory of Pollution Control and Resource Reuse, College of

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Environmental Science and Engineering, Tongji University, Shanghai 200092, China

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‖ Department of Civil and Environmental Engineering, University of California, Los

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Angeles, California 90095, United States

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‡ Department of Civil and Environmental Engineering, William & Cloy Codiga Resource

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Recovery Center, Center for Sustainable Development & Global Competitiveness,

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Stanford University, Stanford, California 94305-4020, United States

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§ Department of Entomology, Department of Osteopathic Medical Specialties, Michigan

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State University, East Lansing, Michigan 48824, United States

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*

Corresponding Author

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Tel/Fax: +1650-7245310; E-mail: [email protected] (Weimin Wu).

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Tel/Fax: +86 21 65985811; E-mail: [email protected] (Yalei Zhang).

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ACS Paragon Plus Environment


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Abstract:

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Yellow mealworms (larvae of Tenebrio molitor, Coleoptera: Tenebrionidae) have been

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proven capable of biodegrading polystyrene (PS) products. Using four geographic sources,

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we found that dark mealworms (larvae of Tenebrio obscurus) ate PS as well. We

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subsequently tested T. obscurus from Shandong, China for PS degradation capability. Our

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results demonstrated the ability for PS degradation within the gut of T. obscurus at greater

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rates than T. molitor. With expanded PS foam as the sole diet, the specific PS consumption

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rate for T. obscurus and T. molitor at similar sizes (2.0 cm, 62-64 mg per larva) was 32.44

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± 0.51 and 24.30 ± 1.34 mg 100 larvae-1 d-1, respectively. After 31 days, the molecular

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weight (Mn) of residual PS in frass (excrement) of T. obscurus decreased by 26.03%,

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remarkably higher than that of T. molitor (11.67%). Fourier transform infrared

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spectroscopy (FTIR) indicated formation of functional groups of intermediates and

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chemical modification. Thermo gravimetric analysis (TGA) suggested that T. obscurus

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larvae degraded PS effectively based on the proportion of PS residue. Co-fed corn flour to

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T. obscurus and wheat bran to T. molitor increased total PS consumption by 11.6% and

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15.2%,

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depolymerization. High-throughput sequencing revealed significant shifts in the gut

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microbial community in both Tenebrio species that was associated with the PS diet and PS

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biodegradation, with changes in three predominate families (Enterobacteriaceae,

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Spiroplasmataceae and Enterococcaceae). The results indicate that PS biodegradability

respectively.

Antibiotic

gentamicin

almost

2


completely

inhibited

PS

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may be ubiquitous within the Tenebrio genus which could provide a bioresource for plastic

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waste biodegradation.

3



43

INTRODUCTION

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Plastic wastes have become a global environmental concern, with over 299 million tons

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of petroleum-based synthetic plastics industrially produced worldwide every year.1-7

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Recently, microplastic particles (i.e. particle diameter < 5 mm) have become an emerging

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environmental concern, as they have been found contaminating rivers, lakes, oceans, and

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wastewater treatment plants.8-13 Polystyrene (PS), [−CH(C6H5)CH2−]n, is one of the major

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sources of plastic products in the world, with an annual production rate exceeding 20

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million tons per year.14 Due to their high durability, plasticity and corrosion resistance, PS

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products are widely used in packing, building and food processing industries in forms of

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expanded PS (EPS), trade name Styrofoam, and as extruded PS (XPS). Due to its high

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prevalence in daily life, PS has become a major pollutant of soils, rivers, lakes, and oceans

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and is a source of microplastics.15-17

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Biodegradation of plastic wastes, including that of PS, has been studied since the

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1960s.18-23 Researchers have attempted to use mixed microbial cultures and isolated

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bacteria from various sources such as soil, garbage or sewage sludge to biodegrade PS into

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low molecular organics or mineralize to CO2.24-28 However, the efficiency of PS

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biodegradation by microorganisms or enzymes varied because of the persistent

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macromolecular structure of the plastics, and was still quite low even with precondition or

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pretreatment processes to the polymer.28-31

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Recently, PS has been shown to be susceptible to rapid biodegradation and 4


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mineralization in the gut of Tenebrio molitor.1,

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Linnaeus 1758, commonly referred to as yellow mealworms, are Coleoptera (beetles)

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within the cosmopolitan family Tenebrionidae (common name “darkling beetle”), which is

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comprised of more than 20,000 species. They have been found around the world (Fig.1a)

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and are capable of ingesting and biodegrading different PS foam products.1, 34, 35 A recent

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study indicated that T. molitor populations from 22 countries from Asia, North America,

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Europe, Africa, and Australia were able to consume PS foam and have an intrinsic capacity

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for biodegradation of PS through eating as well as chewing behaviors, and through gut

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microbe-dependent oxidative digestive machinery. Brandon et al. (2018) further

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investigated the biodegradation of mixed plastics [Polyethylene (PE) and PS] through next-

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generation sequencing and revealed that two OTUs (Citrobacter sp. and Kosakonia sp.)

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were strongly associated with both PE and PS biodegradation.35 In previous research, we

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hypothesized that the ability of insects to depolymerize diverse plastics is likely ubiquitous

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and not restricted to T. molitor.33 According to our observations and literature, dark

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mealworms, larvae of T. obscurus (Fabricius 1792), superworms (larvae of Zophobas

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morio; Coleoptera: Tenebrionidae) (Fig.S1a) and other beetles (Trogoderma variabile,

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Lasioderma serricorne, Rhyzopertha dominica, Tenebrioides mauretanicus, among others

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in Order Coleoptera) can chew, eat, and penetrate various plastic packing materials.36-38

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However, whether other Tenebrio species have the same or even greater biodegradability

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and plastic-degrading behavior, and if this ability is ubiquitous, remains unknown.

32-35

The larvae of Tenebrio molitor

5



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Currently, there are three extant Tenebrio species; two of them, Tenebrio molitor and

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Tenebrio obscurus, have been reported worldwide (Fig. 1), while T. opacus Duftschmid,

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1812 is only found in France.39-42 Different from the T. molitor, the larvae of T. obscurus

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darken as they mature.43 They are sold as a food source for pets in China and in the USA.

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Both species have been reported in North America, Asia, Europe and Australia (Fig. 1b).

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The adult beetle of T. obscurus appears similar to T. molitor but the larvae of T. obscurus

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have dark black rings on the abdomen (Fig.1d). Mature T. obscurus larvae are similar in

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size (1.5-2.5 cm) compared with T. molitor larvae (Fig.1d, Fig.1Sc-f) and much smaller

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than Z. morio (up to 5.0-6.0 cm) (Fig. S1a).44 T. obscurus larvae are more sensitive to light

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and have higher cysteine in their proteins (by 15.6 times) than T. molitor.45 The normal diet

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for commercial rearing of T. obscurus larvae is corn flour and oat while T. molitor are fed

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wheat bran.34 We find that T. obscurus larvae obtained from China and the USA also chew

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and eat PS foam (Fig. S1c-f).

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In this study, we tested the hypothesis that PS degradation in members of the Tenebrio

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genus is ubiquitous by investigating the biodegradation of T. obscurus larvae obtained from

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an insect farm in Shandong Province, China, compared to that of T. molitor. We tested PS

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degradation using previously established protocols including (1) PS mass balance to

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determine the specific rates of PS degradation; (2) Gel permeation chromatography (GPC)

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to assess changes in molecular weight; and (3) Fourier transform infrared (FTIR)

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spectroscopy of frass residues (excrement of larvae) to identify chemical modifications 6


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resulting from PS digestion.1, 34 We also examined larval microbial communities using

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high-throughput sequencing before and after PS feeding. We found that T. obscurus larvae

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had the capacity to biodegrade PS at higher levels of depolymerization than same sized T.

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molitor larvae. PS degradation was also dependent on gut microbial community

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composition for both species, and Enterobacteriaceae showed a faster shift in relative

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abundance for T. obscurus than T. molitor larvae.

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MATERIAL AND METHODS

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Mealworm sources and feedstock

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In this study, Tenebrio obscurus larvae (approximately 2 cm in length) purchased from

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Zaozhuang Insects Breeding Plant, Shandong, and Tenebrio molitor larvae (approximately

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2 cm in length) purchased from Binzhou Insects Breeding Plant, Shandong, China were

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utilized for PS biodegradation tests. Additional T. obscurus larvae were purchased to

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evaluate their ability to eat and chew PS foam from one USA source, Rocky Mountain

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Mealworms, Colorado, as well as from two sources in China: Guangyuan Insects Breeding

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Plant, Sichuan, and Luoyang Insects Breeding Plant, Henan (Fig.S1).

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The larvae for both species were identified based on morphology and coloration (Fig. 1)

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as described by Robinson (2005) and Calmont and Soldati (2008).41, 42 Larvae of both

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species were not fed with any antibiotics according to suppliers. Prior to the tests, T. molitor

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larvae were fed with a normal diet of wheat bran while T. obscurus was fed with a normal 7



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diet of corn flour. Elementary ratios of C:H:O:N:S (w/w) were 35.7:6.5:41.5:2.7:0.5 for

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bran and 38.5:7.4:48.0:1.2:0.3 for corn flour. Both diets provide sufficient nutrients as well

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as a source of carbon for mealworm growth.

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Expanded PS foam (or Styrofoam as commercial name) was used as feedstock for the

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larvae of both species and was purchased from Lan Tian Plastic Company, Guangdong,

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China. The PS feedstock contained polystyrene purity over 96.3%. Number-average

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molecular weight (Mn) of the PS was 107,000 and weight-average molecular weight (Mw)

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was 345,000 with a density of 0.0197 ± 0.0009 g/cm3 and a water angle of 104.2 ± 2.1°.

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No extra additives or catalysts were added, according to the manufacturer. According to

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our analysis, the bromide content in the PS foam was under the detection limit (< 1 mg g-

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1),

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GC grade purity ≥ 99.9%), gentamicin sulfate, and trypticase soy agar (TSA) were

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purchased from Aladdin, Shanghai, China.

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Mealworm Survival Rates and Biodegradation of PS

indicating that there is no brominated flame retardants in it. The tetrahydrofuran (THF,

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To compare the PS consumption and biodegradation between the larvae of the two

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species, six treatments were prepared based on feeding conditions: starvation of T. molitor,

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PS-fed T. molitor, PS plus bran-fed T. molitor, starvation T. obscurus, PS-fed T. obscurus,

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and PS plus corn flour-fed T. obscurus. Wheat bran or corn flour can provide sufficient

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nutrients for their growth. For each treatment, initial larvae (410) were randomly selected

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and placed in a food grade polypropylene container (volume of 3300 mL) under controlled 8


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conditions (25 ± 1 °C, 70 ± 5% humidity, and dark environment). To assess PS consuming

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capacity initially, PS blocks (7.2 g) were added. Co-diet treatments were PS plus bran (1.2

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g) for T. molitor larvae, and PS plus corn flour (1.2 g) for T. obscurus larvae. An additional

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1.2 g of the co-diet was supplemented every 5 days to reach a final ratio of PS to co-diet of

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1.0:1.0 at the end of test. This ratio was the same to that previously used to test the effect

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of the co-diet on PE and PS degradation in T. molitor larvae.35 Prior to the test, larvae were

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fed their regular food for at least 3 days, then they were fasted for one day, and finally were

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fed with respective feedstocks.

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The measurement of survival rates and plastic mass loss was carried out every 5 days

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and ended on day 31. We selected a 31-day period to avoid pupation and a high survival

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rate of T. molitor larvae could be maintained within 4-5 weeks with PS as only diet.34

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During the measurement time, dead larvae and molted exoskeletons were removed from

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containers to prevent being eaten by the remaining larvae since cannibalism existed in the

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later period.33 All treatments were conducted in duplicate.

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THF Extraction

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THF extraction was used to determine the mass of PS residue in frass during the test

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period as described previously.34, 35 The egested frass contained an extractable part and a

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non-extractable part; the extractable part consisted of the undigested PS polymer and

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modified PS polymers, while the non-extractable part consisted of other residues, such as

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undigested exoskeletons.34, 35 After being dried at ambient temperature, frass samples (0.5 9



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g) were extracted with 10 mL of THF for 3 hours with a magnetic stirrer. Then, the

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extracted THF solution was filtered with a 0.22 μm polyethersulfone (PES) membrane

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filter, and collected in a pre-weighed glass vial. After evaporation, polymer residue in the

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vial was weighed to calculate the mass of THF-extracted polymer.34, 35 All THF extraction

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tests were conducted in triplicate.

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THF extractable ratio (TER) was used as an indicator of PS biodegradation or digestion

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in larval guts, and was calculated on the basis of the weight of extracted polymer divided

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by the weight of the sample for THF extraction34 PS feedstock showed 100% while TER

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of frass depended on the content of residual or not-degraded PS.

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Frass Collection and Analytical Methods

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To obtain enough frass for characterization, additional larvae (approximately 2000 for

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each group) were raised as described above. Likewise, mass of PS and co-feeds were

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increased proportionately. At the end of 31-day test, larvae were cleansed by streamed air

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and transferred to a clean iron container to collect frass within 24 hours. The collected frass

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was immediately stored at -20 °C for final characterization. The morphology of frass of

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PS-fed T. molitor and T. obscurus larvae was observed by SEM-EDX (SI M2, Fig. S3 S4).

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Gel permeation chromatography (GPC) (Agilent 1260, Agilent Technologies Inc., USA)

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was applied to analyze changes of molecular weight of the polymer. Preparation of samples

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was similar to THF extraction test described previously. PS was extracted from PS

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feedstock (1.0 g) and frass samples (1.0 g) from PS-fed larvae by THF. After filtration, 10


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THF solution was mixed on a magnetic stirrer with gentle heating (60 °C). After the

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extracted solution was concentrated to 5 mL in volume, the extract (20 μL in volume) was

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injected into the GPC analyzer, with a flow rate of 0.8 mL/min.1, 34, 35

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Fourier transform infrared spectroscopy (FTIR) (Nicolet iS05 FTIR Spectrometer,

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Thermo Fisher Scientific, USA) was applied to characterize major functional groups of PS

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feedstock sample and frass in the range of 4000-500 cm-1. Prior to the analyses, samples

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were dried in a freeze drier for at least 36 hours, and then grinded with KBr to prepare a

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homogenous KBr pellet for scanning.1, 32-34

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Thermal gravimetric analysis (TGA) (TA-Q500, TA Instruments, USA) was applied to

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characterize thermal changes from PS to frass. The heating program included two different

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atmospheres so as to study pyrolysis of the sample under nitrogen and air ambience,

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respectively. Samples (5 mg) were heated from 40 °C to 800 °C at a rate of 20 °C/min

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under high-purity nitrogen ambience (99.999%), then cooled down to 500°C, and finally

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heated again to 800 °C under air ambience.

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Antibiotic Suppression Test

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The effect of antibiotic suppression on PS depolymerization and degradation was tested

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using T. obscurus larvae with gentamicin as an inhibitor. Gentamicin was selected based

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on previous results.32, 33 The gentamicin suppressive group (410 larvae) was fed antibiotic

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feedstock (AF) containing corn flour versus gentamicin sulfate at 100:3 (w/w) for 3 days,

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and then fed with PS feedstock. AF was continuously fed with PS feedstock and resupplied 11



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every 3 days. On day 15, frass samples from the antibiotics treatment were collected to

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analyze for the molecular weights using GPC.

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On day 0 (prior to feeding PS), 7, and 15, 15 T. obscurus larvae were randomly chosen

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from each source of larval populations to prepare a gut suspension for counting of gut

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microorganisms. The larvae were decontaminated by 80 % ethanol, rinsed by Milli-Q water,

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and anatomized to obtain the guts. Subsequently, gut contents were extracted and

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suspended in 5 ml sterile saline water, serial diluted (10-1 to 10-7) and cultivated on non-

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selective TSA plates at 37 °C for 24 hours. Finally, the number of colonies were counted

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as described elsewhere.33

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Microbial Community Analysis

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Larvae used for microbial community analysis were collected from PS-fed T. obscurus

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and PS-fed T. molitor treatments at day 0, day 6, and day 11 for microbial community

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analysis. The DNA extraction and PCR amplification methods were similar to that reported

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previously and detailed in supporting information (SI M3).

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Phasing amplicon sequencing was applied to sequence the V3-V4 region of 16S rRNA

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gene. Purified amplicons were paired-end sequenced on an Illumina MiSeq platform.

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Sequencing data was demultiplexed, quality-filtered on Trimmomatic, and merged

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according to criteria.46 Operational Taxonomic Units (OTUs) were clustered with 0.97

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identity threshold using UPARSE.47 Chimeric sequences were identified and removed

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using UCHIME. Taxonomy of each 16S rRNA gene sequence was analyzed by RDP 12


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Classifier against the Silva 16S rRNA database with a confidence threshold of 70%. Finally,

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microbial community composition, hierarchical cluster analysis, principal coordinate

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analysis (PCoA), and ternary analysis were run on the free online platform of Majorbio I-

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Sanger Cloud Platform (Shanghai, China).

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RESULTS AND DISCUSSION

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PS Consumption of T. obscurus - versus T. molitor larvae

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All T. obscurus larvae purchased from Shandong, Sichuan and Henan Provinces, China

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and Colorado, USA chewed and ate PS foam (Fig. 1 and Fig. S1). We surmise that that

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chewing and ingestion of PS foam is likely an adaptive behavior intrinsic to T. obscurus.

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They behaved similarly with each other but differently from T. molitor larvae. They were

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all sensitive to light and mostly hid below PS foam in clusters (Fig. 1d, Fig S1 c-f). The

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larvae of T. molitor were less sensitive to light and spread on the foam surface or penetrated

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the inside matrix (Fig. 1a, Fig.1c). The T. obscurus larvae purchased from Shandong, China

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were selected for further study because of stable supply. The PS consumption by both

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species increased progressively (Fig. 2a). The T. obscurus were capable of rapid PS

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consumption at rates which were even greater than those of T. molitor. During the 31-day

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test with PS as the only diet, the PS mass consumption by the T. obscurus larvae was 55.4%

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± 1.5% while that by T. molitor was 41.5% ± 3.0% (Fig. 2a). The PS consumption increased

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when co-diets were added, i.e. the T. obscurus consumed 67.1% ± 1.8% of PS and T.

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molitor consumed 56.8% ± 1.9%. The specific rate of PS consumption was calculated as 13



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the amount of ingested PS per day for 100 mealworms based on the mass loss of PS foam,

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and as average numbers of survived mealworms.

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At the end of the 31-day test at 25 °C, the survival rates (SRs) of both species fed EPS

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alone were 91.5 ± 1.5% and 89.3 ± 2.7% , respectively, significantly greater than those of

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unfed controls (67.6 ± 2.2 % and 62.0 ± 2.9%) (Table 1), and not significantly less than

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corn flour-fed and bran-fed larvae (95.0 ± 1.7% and 93.2 ± 1.0%) (Table 1 and Fig. 2b).

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Over the 31-day test period, starved larvae of both species lost 13.2 ± 2.7 % and 18.2 ± 6.0

250

% of their average weight, respectively. As expected, the larvae of T. obscurus and T.

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molitor fed with PS alone lost their average weight slightly by 8.1 ± 3.7 % and 8.6 ±1.2 %,

252

respectively, while those fed with corn flour and bran experienced a 15.9 ± 4.1 % and 14.6

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± 1.8% weight gain, respectively (Table 1). As expected, the survival rates of both

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starvation species were much lower than those fed with PS only and PS plus co-diet (corn

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flour or bran) at the end of 31 day period, as observed previously with T. molitor.33 The T.

256

obscurus larvae showed slightly better survival than the T. molitor (Fig. 2b). The

257

observation supported that addition of the nutrition-rich co-diet enhances PS consumption

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by both species.34,

259

decrease in average weight of both species indicated that larvae received their energy

260

source from digestion of PS but lacked a nutrition source for their growth, as observed

261

previously.33

262

35

When PS was applied as the sole diet, the higher SRs and slight

The specific PS consumption rates (SPCRs) were calculated for day 0 to day 10, day 11 14


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to day 20, day 21 to day 31 (Fig. 2c), and for the overall 31-day period (Fig. 2c insert

264

figure). Overall, T. obscurus larvae exhibited comparatively greater PS consuming ability

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than T. molitor, i.e. 32.44 ± 0.51 versus 24.30 ± 1.34 mg 100 worms-1 d-1 fed with PS only

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and 39.24 ± 1.73 versus 33.23 ± 0.80 mg 100 worms-1 d-1 when co-diet corn flour and bran

267

were fed, respectively (Table 1). The rates are within the rate ranges of 12 T. molitor larvae

268

collected from China, USA, and UK, i.e. 8.5-21 mg 100 worms-1 d-1 for T. molitor fed with

269

PS only and 24-46 mg 100 worms-1 d-1 for the worms fed with PS plus co-diet.33 The results

270

also confirmed that higher SPCR can be achieved in T. obscurus when the nutrition-rich

271

co-diet is supplied, as reported with the tests with T. molitor.34, 35 The results of this study

272

also showed that SPCRs were increased during the initial 21 days and then declined (Fig.

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2c). Further research is needed to understand whether this was due to maturation of the

274

larvae or for other, unknown reasons.

275

Change of TER, i.e. THF extractable ratio (which represents the fraction of residual PS

276

polymer in egested frass), can indirectly indicate the change of PS degradability in

277

mealworm gut.34 The TER of frass from both species (Fig. 2d) fed with PS only decreased

278

during the 31-day test from 57.8% ± 4.1% and 65.2% ± 2.3% on day 6 progressively, and

279

finally stabilized at 27.3% ± 1.7% and 32.4% ± 0.7%, respectively, a value that was

280

significantly different (p < 0.01). The decrease of TERs during the 31-day period indicated

281

that PS degradation and then mineralization in the gut of larvae increased gradually, likely

282

due to an increase in PS-degrading microbial activities. In addition, the final TER of the 15



283

frass of T. obscurus was significantly lower than that of T. molitor (p < 0.01). Therefore,

284

we concluded that T. obscurus was superior to T. molitor for PS biodegradability.

285

Biodegradability and Evidence of Depolymerization

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GPC was conducted at the end of 31-day test to characterize the depolymerization and

287

biodegradation of ingested PS using the established methods.1, 34 Frass samples from the T.

288

obscurus fed with PS only contained polymer extracts with Mn value that was 26.0% lower

289

than the feedstock and Mw value that was 59.2% lower than the feedstock (PS Feedstock

290

with Mn of 107,000; Mw of 345,000). Frass samples from T. molitor had Mn values that

291

were 11.7 % lower and Mw values that were 29.8% lower than the feedstock (Fig. 3a).

292

These decreases in Mn and Mw were significant for all sources (t-test, p

Biodegradation of Polystyrene by Dark (Tenebrio obscurus) and

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