Tenebrio molitor

we found that dark mealworms (larvae of Tenebrio obscurus) ate PS as well. We ... mineralization in the gut of Tenebrio molitor.1, 32-35 The larvae of...
0 downloads 0 Views 1MB Size
Subscriber access provided by UNIV OF LOUISIANA

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 37

Environmental Science & Technology

1

Biodegradation of Polystyrene by Dark (Tenebrio obscurus)

2

and Yellow (Tenebrio molitor) Mealworms (Coleoptera:

3

Tenebrionidae)

4 5

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

6

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

7

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

8

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

9

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

10

Angeles, California 90095, United States

11

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

12

Recovery Center, Center for Sustainable Development & Global Competitiveness,

13

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

14

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

15

State University, East Lansing, Michigan 48824, United States

16

*

Corresponding Author

17

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

18

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

1

ACS Paragon Plus Environment

Environmental Science & Technology

20

Page 2 of 37

Abstract:

21

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

22

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

23

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

24

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

25

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

26

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

27

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

28

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

29

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

30

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

31

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

32

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

33

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

34

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

35

15.2%,

36

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

37

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

38

biodegradation, with changes in three predominate families (Enterobacteriaceae,

39

Spiroplasmataceae and Enterococcaceae). The results indicate that PS biodegradability

respectively.

Antibiotic

gentamicin

almost

2

ACS Paragon Plus Environment

completely

inhibited

PS

Page 3 of 37

Environmental Science & Technology

40

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

41

waste biodegradation.

3

ACS Paragon Plus Environment

Environmental Science & Technology

43

INTRODUCTION

44

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

45

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

46

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

47

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

48

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

49

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

50

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

51

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

52

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

53

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

54

and is a source of microplastics.15-17

55

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

56

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

57

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

58

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

59

biodegradation by microorganisms or enzymes varied because of the persistent

60

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

61

pretreatment processes to the polymer.28-31

62

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

ACS Paragon Plus Environment

Page 4 of 37

Page 5 of 37

Environmental Science & Technology

63

mineralization in the gut of Tenebrio molitor.1,

64

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

65

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

66

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

67

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

68

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

69

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

70

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

71

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

72

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

73

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

74

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

75

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

76

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

77

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

78

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

79

Lasioderma serricorne, Rhyzopertha dominica, Tenebrioides mauretanicus, among others

80

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

81

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

82

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

32-35

The larvae of Tenebrio molitor

5

ACS Paragon Plus Environment

Environmental Science & Technology

83

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

84

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

85

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

86

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

87

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

88

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

89

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

90

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

91

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

92

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

93

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

94

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

95

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

96

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

97

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

98

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

99

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

100

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

101

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

102

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

ACS Paragon Plus Environment

Page 6 of 37

Page 7 of 37

Environmental Science & Technology

103

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

104

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

105

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

106

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

107

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

108

abundance for T. obscurus than T. molitor larvae.

109 110

MATERIAL AND METHODS

111

Mealworm sources and feedstock

112

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

113

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

114

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

115

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

116

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

117

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

118

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

119

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

120

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

121

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

122

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

ACS Paragon Plus Environment

Environmental Science & Technology

123

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

124

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

125

as a source of carbon for mealworm growth.

126

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

127

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

128

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

129

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

130

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

131

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

132

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

133

1),

134

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

135

purchased from Aladdin, Shanghai, China.

136

Mealworm Survival Rates and Biodegradation of PS

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

137

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

138

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

139

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

140

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

141

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

142

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

ACS Paragon Plus Environment

Page 8 of 37

Page 9 of 37

Environmental Science & Technology

143

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

144

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

145

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

146

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

147

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

148

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

149

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

150

fed with respective feedstocks.

151

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

152

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

153

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

154

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

155

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

156

later period.33 All treatments were conducted in duplicate.

157

THF Extraction

158

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

159

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

160

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

161

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

162

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

ACS Paragon Plus Environment

Environmental Science & Technology

163

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

164

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

165

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

166

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

167

tests were conducted in triplicate.

168

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

169

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

170

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

171

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

172

Frass Collection and Analytical Methods

173

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

174

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

175

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

176

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

177

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

178

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

179

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

180

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

181

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

182

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

ACS Paragon Plus Environment

Page 10 of 37

Page 11 of 37

Environmental Science & Technology

183

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

184

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

185

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

186

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

187

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

188

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

189

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

190

homogenous KBr pellet for scanning.1, 32-34

191

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

192

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

193

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

194

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

195

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

196

heated again to 800 °C under air ambience.

197

Antibiotic Suppression Test

198

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

199

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

200

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

201

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

202

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

ACS Paragon Plus Environment

Environmental Science & Technology

203

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

204

analyze for the molecular weights using GPC.

205

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

206

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

207

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

208

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

209

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

210

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

211

as described elsewhere.33

212

Microbial Community Analysis

213

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

214

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

215

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

216

previously and detailed in supporting information (SI M3).

217

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

218

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

219

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

220

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

221

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

222

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

ACS Paragon Plus Environment

Page 12 of 37

Page 13 of 37

Environmental Science & Technology

223

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

224

microbial community composition, hierarchical cluster analysis, principal coordinate

225

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

226

Sanger Cloud Platform (Shanghai, China).

227

RESULTS AND DISCUSSION

228

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

229

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

230

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

231

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

232

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

233

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

234

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

235

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

236

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

237

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

238

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

239

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

240

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

241

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

242

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

ACS Paragon Plus Environment

Environmental Science & Technology

243

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

244

and as average numbers of survived mealworms.

245

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

246

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

247

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

248

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

249

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.

251

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

253

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

254

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

255

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

258

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

ACS Paragon Plus Environment

Page 14 of 37

Page 15 of 37

Environmental Science & Technology

263

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

265

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

266

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.

273

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

ACS Paragon Plus Environment

Environmental Science & Technology

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

286

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