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Remediation and Control Technologies
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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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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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
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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.
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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
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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
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% 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 %,
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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.
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obscurus larvae showed slightly better survival than the T. molitor (Fig. 2b). The
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observation supported that addition of the nutrition-rich co-diet enhances PS consumption
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by both species.34,
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decrease in average weight of both species indicated that larvae received their energy
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source from digestion of PS but lacked a nutrition source for their growth, as observed
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previously.33
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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
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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
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were fed, respectively (Table 1). The rates are within the rate ranges of 12 T. molitor larvae
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collected from China, USA, and UK, i.e. 8.5-21 mg 100 worms-1 d-1 for T. molitor fed with
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PS only and 24-46 mg 100 worms-1 d-1 for the worms fed with PS plus co-diet.33 The results
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also confirmed that higher SPCR can be achieved in T. obscurus when the nutrition-rich
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co-diet is supplied, as reported with the tests with T. molitor.34, 35 The results of this study
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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
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larvae or for other, unknown reasons.
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Change of TER, i.e. THF extractable ratio (which represents the fraction of residual PS
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polymer in egested frass), can indirectly indicate the change of PS degradability in
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mealworm gut.34 The TER of frass from both species (Fig. 2d) fed with PS only decreased
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during the 31-day test from 57.8% ± 4.1% and 65.2% ± 2.3% on day 6 progressively, and
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finally stabilized at 27.3% ± 1.7% and 32.4% ± 0.7%, respectively, a value that was
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significantly different (p < 0.01). The decrease of TERs during the 31-day period indicated
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that PS degradation and then mineralization in the gut of larvae increased gradually, likely
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due to an increase in PS-degrading microbial activities. In addition, the final TER of the 15
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frass of T. obscurus was significantly lower than that of T. molitor (p < 0.01). Therefore,
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we concluded that T. obscurus was superior to T. molitor for PS biodegradability.
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Biodegradability and Evidence of Depolymerization
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GPC was conducted at the end of 31-day test to characterize the depolymerization and
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biodegradation of ingested PS using the established methods.1, 34 Frass samples from the T.
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obscurus fed with PS only contained polymer extracts with Mn value that was 26.0% lower
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than the feedstock and Mw value that was 59.2% lower than the feedstock (PS Feedstock
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with Mn of 107,000; Mw of 345,000). Frass samples from T. molitor had Mn values that
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were 11.7 % lower and Mw values that were 29.8% lower than the feedstock (Fig. 3a).
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These decreases in Mn and Mw were significant for all sources (t-test, p