Polyvinyl Chloride Microplastics Affect Methane Production from the

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Environmental Processes

Polyvinylchloride Microplastics Affect Methane Production from the Anaerobic Digestion of Waste Activated Sludge through Leaching Toxic Bisphenol-A Wei Wei, Qi-Su Huang, Jing Sun, Jun-Yue Wang, Shu-Lin Wu, and Bing-Jie Ni Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b07069 • Publication Date (Web): 13 Feb 2019 Downloaded from http://pubs.acs.org on February 14, 2019

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Environmental Science & Technology

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Polyvinylchloride Microplastics Affect Methane Production from the Anaerobic

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Digestion of Waste Activated Sludge through Leaching Toxic Bisphenol-A

3 Wei Wei,† Qi-Su Huang,† Jing Sun,†,‡ Jun-Yue Wang,† Shu-Lin Wu,† Bing-Jie Ni*,†,‡

4 5 6

†State

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

8

‡

9

China

Key Laboratory of Pollution Control and Resources Reuse, College of

Shanghai Institute of Pollution Control and Ecological Security, Shanghai200092, P.R.

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TOC Art

13

1

ACS Paragon Plus Environment


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ABSTRACT: The retention of polyvinylchloride (PVC) microplastics in sewage sludge

15

during wastewater treatment raises concerns recently. However, the effects of PVC

16

microplastics on methane production from anaerobic digestion of waste activated sludge

17

(WAS) have never been documented. In this work, the effects of PVC microplastics (1

18

mm, 10–60 particles/g TS) on anaerobic methane production from WAS were

19

investigated. The presence of 10 particles/g TS of PVC microplastics significantly (P =

20

0.041) increased methane production by 5.9 ± 0.1%, but higher levels of PVC

21

microplastics (i.e., 20, 40 and 60 particles/g TS) inhibited methane production to 90.6 ±

22

0.3%, 80.5 ± 0.1% and 75.8 ± 0.2% of the control, respectively. Model-based analysis

23

indicated that PVC microplastics at > 20 particles/g TS decreased both methane potential

24

(B0) and hydrolysis coefficient (k) of WAS. The mechanistic studies showed that

25

bisphenol-A (BPA) leaching from PVC microplastics was the primary reason for the

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decreased methane production, causing significant (P = 0.037, 0.01, 0.004) inhibitory

27

effects on the hydrolysis-acidification process. The long-term effects of PVC

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microplastics revealed that the microbial community was shifted in the direction against

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hydrolysis-acidification and methanation. In conclusion, PVC microplastic caused

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negative effects on WAS anaerobic digestion through leaching the toxic BPA.

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INTRODUCTION

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Microplastics, which are usually defined as plastic particles < 5 mm,1 have aroused

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increasing concerns as they pose threats to aquatic species as well as human beings.

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Microplastics with a size of 0.1-5 mm can be formed from break down of large plastic

36

debris through mechanical and chemical/biological actions.2,3 Microplastics can also be

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directly manufactured and are utilized in many personal care and cosmetic products

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(PCCPs).4,5 The massive usage of plastic products and poor management of waste

39

disposal has resulted in microplastics being ubiquitously found in aquatic water bodies,

40

including rivers, lakes, estuaries, coastlines and marine ecosystems.1,6-8

41 42

Recent studies have demonstrated that a mass of microplastics used in many PCCPs is

43

transported from the raw wastewater to wastewater treatment plants (WWTPs).9,10 Due to

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the strong hydrophobic nature of activated sludge in WWTPs,11 microplastics can be

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easily absorbed into activated sludge; thus, above 90% of microplastics in raw

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wastewater could be removed efficiently by WWTPs.12 The relatively high removal

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efficiency of microplastics by WWTPs indicated that most microplastics would be

48

retained in the sewage sludge. It is reported that every year in Europe and North America,

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the amount of microplastics transferred from WWTPs to biosolids is more than that

50

present in seawater.13

51 52

The most commonly found microplastics in sludge are polyethylene (PE), polyethylene

53

terephthalate (PET), polyvinyl chloride (PVC), polystyrene (PS) and polypropylene 3



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(PP).14 Among these microplastics, PVC ranked as the most hazardous microplastic with

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strong mutagenicity and carcinogenicity (category 1A and 1B).15 Several studies have

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examined the toxicity effect of PVC microplastics on microorganisms. For example,

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Zhang et al.16 reported that PVC microplastics (96 h exposure) inhibited growth of the

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microalgae Skeletonema costatum by up to 39.7% and clearly reduced photosynthetic

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efficiency. The additives incorporated into plastics to improve their properties could also

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represent toxicants to Amphibalanus amphitrite and Nitocra spinipes;8,17 potential

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toxicants are often easily leached out from microplastics.11 In particular, Suhrhoff and

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Scholz-Böttcher18 reported that bisphenol-A (BPA) is an extremely predominant additive

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which is highly toxic to microbes19 and can be leached from PVC microplastics,

64 65

Existing WWTPs are expanding to serve the demands of growing cities, which results in

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an increasing production of waste activated sludge (WAS).20 Anaerobic digestion, with

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20-30 % of VS destruction, is a highly effective method for WAS treatment that

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possesses many advantages, including high sludge reduction, effective pathogen killing

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and potential net recovery of energy through biogas.21,22 The retention of PVC

70

microplastics in WAS would inevitably lead to the presence of microplastics in the

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anaerobic digestion system, which might present a negative impact on anaerobic methane

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production from WAS. To date, however, the influence of PVC microplastics on methane

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production from WAS in anaerobic digestion has never been documented.

74 75

Therefore, the aim of this study is to investigate the potential impacts of PVC

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microplastics (1 mm, 10–60 particles/g TS) on carbon transformation and methane 4


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production in the anaerobic digestion of WAS. The effects of different PVC microplastic

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levels on methane production from WAS were first investigated with biochemical

79

methane potential tests. The impacts of PVC microplastics on both the hydrolysis rate

80

and the methane production potential were then revealed through model-based analysis.

81

In addition, the mechanisms of PVC microplastics affecting methane production from

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WAS were explored from the aspects of the transformations of metabolic intermediates,

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the activities of key enzymes and the impacts of chemical additives leaching from PVC

84

microplastics. Finally, the long-term effect of PVC microplastics on microbial

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community was explored.

86 87

MATERIALS AND METHODS

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Sources of Sludge and PVC Microplastics. In this study, WAS and anaerobically

89

digested sludge (ADS) were used as the feedstock and inoculum, respectively. The

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feedstock was harvested from the secondary sedimentation tank of the Wastewater

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Treatment Plant, which is a biological nutrient removal plant in Shanghai, China. It was

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thickened by gravity and stored in a refrigerator (4 °C) for subsequent use. The inoculum

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was withdrawn from the sludge anaerobic digester with the loading rate of 2.1 gVS/L/d

94

(temperature: 37 ± 1 °C, SRT: 20d) in our laboratory. The main characteristics of the

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feedstock and inoculum are listed in SI Table S1. The various microplastics in the raw

96

WAS were identified and enumerated using the methods to be further described in the

97

“Analytical Methods” section. Among the microplastics found in the raw WAS (i.e., PE,

98

PET, PS and PP), PVC microplastic was found to be one of the most abundant

99

microplastics with the content of 2 particles/g-TS. The PVC microplastics for 5



100

experiments, in the form of white spherical powder, were purchased from Youngling Co.

101

Ltd. (Shanghai, China). According to the manufacturer, the size of PVC microplastics

102

was 1 mm. The microplastics’ sizes were confirmed using a microscope: in brief, 10

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particles of PVC microplastics were chosen from the samples used. The morphology of

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these PVC microplastics was observed and photographed using a Motic microscope

105

(BA410E, Motic Electric Group, Xiamen, China) and the sizes were measured using

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OPLENIC software.

107 108

Biochemical Methane Potential Tests at Different PVC Microplastic Levels. The

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influences of PVC microplastics exposure on anaerobic methane production from WAS

110

were investigated using a series of biochemical methane potential (BMP) tests. Briefly,

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each serum bottle (160 mL) was filled with 80 mL inoculum and 20 mL WAS with the

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VS ratios of 2.0 ± 0.1. Afterwards, all bottles were flushed with N2 (1 L/min) for 1 min,

113

sealed by rubber stoppers with aluminum crimp-caps and stored in a constant-

114

temperature (37 ± 1 °C) incubator. Raw WAS samples with 2 particles/g-TS PVC

115

microplastics served as a control in this study. Based on the environmentally relevant

116

concentration in the literature,23 four experimental dosages (10, 20, 40 and 60 particles/g-

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TS) of PVC microplastics were added in the raw WAS to assess the impact of PVC

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microplastics on methane production from WAS in BMP tests. To exclude contributions

119

of inoculum to methane production, a blank was also created. The blank sample

120

contained inoculum and an equivalent volume of Milli-Q water instead of WAS. All

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BMP tests were conducted in triplicate. When biogas production in each serum bottle

122

dropped to a negligible level, BMP tests were completed (i.e., 45 days in this study). The 6


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biogas (i.e., H2, N2, CH4 and CO2) volume and composition in each BMP test were

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measured every day in the first 10 d and every 3-6 d afterwards; based on these data, the

125

methane production was obtained. Methane production from WAS was calculated by

126

subtracting its test value from that in the blank (pointwise) and recording the result as its

127

volume per VS mass (L CH4/kg VS). After the BMP tests were operated for 45 d, the

128

activities of those key enzymes (e.g., protease, cellulase, AK and F420) involved in

129

anaerobic digestion steps were also analyzed. The detailed analytical procedures of these

130

enzyme activities were presented in Supporting Information (SI).

131 132

Biochemical Methane Potential Tests Modeling. Methane production potential (B0)

133

and hydrolysis coefficient (k), two primary parameters associated with methane

134

production, were used to evaluate and compare the influence of PVC microplastics

135

exposure on methane production potential and kinetics of WAS. Based on the cumulative

136

methane curves, a one-substrate model was applied as described in Wang et al.,24 fitting

137

B0 and k, with the apparent VS destruction (Y0) calculated from Y0=B0/380×RSS, where

138

380 is the theoretical methane potential of sludge at standard temperature and pressure

139

(STP, 25 °C, 1 atm) (L CH4/kg TCOD); RSS is measured as VS/TCOD of WAS (i.e.,

140

0.71 in this study).25 The parameter estimation procedure of Batstone et al.26 was used

141

with the sum of squared errors (Jopt) as objective function.

142 143

Impacts of PVC Microplastics on Solubilization, Hydrolysis and Acidification. It is

144

well known that methane production from WAS usually successively undergoes

145

solubilization, hydrolysis and acidification.24 During the solubilization process, 7



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particulate organic substrates and cells in WAS are destroyed and converted to soluble

147

organic macromolecular substances, such as soluble protein and soluble carbohydrate.

148

Then, these were hydrolyzed to amino acid and monosaccharide constituents. Through

149

acidification, volatile fatty acids (VFAs) were produced.27 The experiments regarding the

150

influence of exposure to PVC microplastics (10, 20, 40 and 60 particles/g-TS) in these

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three stages were conducted under the same procedure as the BMP test but with 2-

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bromoethanesulfonic acid (BESA) addition. Since methanogens can easily and rapidly

153

consume VFAs produced during acidification, it was necessary to deplete methanogens

154

from the anaerobic digestion system. Hence, 50 mmol/L BESA was added in the mixture

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of WAS and inoculum to eliminate methanogen interference. All tests were conducted in

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triplicate and lasted for 3 d. After 3 d, methane production from each test was determined.

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For each test, the soluble chemical oxygen demand (SCOD), soluble proteins, soluble

158

polysaccharides and VFA before and after digestion were measured, respectively.

159 160

Determination of Chemicals Leaching from PVC Microplastics. According to

161

manufacturers and Suhrhoff and Scholz-Böttcher,18 the bisphenol-A (BPA), diisononyl

162

phthalate (DiNP), diisodecyl phthalate (DiDP) and diethylhexyl phthalate (DEHP) are the

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key additives in PVC microplastics. To measure the concentrations of those respective

164

chemicals leaching from PVC microplastics at four studied PVC microplastic levels

165

during anaerobic digestion, the corresponding amounts of PVC microplastics were dosed

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into 100 mL sludge mixtures. All tests were maintained in a constant-temperature (37 ±

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1 °C) incubator for 45 d. At different leaching times (0, 10, 20, 30, 45 d), samples were

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filtered through 0.22-μm cellulose acetate filter membrane, concentrated sludge was dried 8


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using a freeze-dryer (-20 °C, SCIENTZ-10N, China) and the concentrations of those

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chemicals in the filtrate and sludge solids were then measured using methods described in

171

the “Analytical Methods” section.

172 173

Impacts of Released Chemicals on Methane Production. The experiments on the

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effects of detected chemicals leaching from PVC microplastics on methane production

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from WAS were carried out with the same method as the BMP tests except that the

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detected chemical was used instead of PVC microplastics. On days 10, 20, 30 and 45, the

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chemicals with corresponding leaching concentration were gradually dosed in the serum

178

bottle using the syringe. In addition, the experiments regarding the effects of detected

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chemicals leaching from PVC microplastics on WAS solubilization were also conducted,

180

as detailed in SI.

181 182

Continuous Bench-Scale Anaerobic Digester Operation for Microbial Community

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Analysis. Two identical continuously operated anaerobic digesters (1 L working volume)

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were employed to investigate the impact of PVC microplastics on the microbial

185

community. One, as control, was fed with raw WAS and the other experimental digester

186

was fed with WAS containing 60 particles PVC microplastics/g TS. The two digesters

187

were placed in a temperature-controlled shaker (37 ± 1 °C, 150 rmp). The pH in each

188

digester was monitored but not controlled, varying between 7.0 and 7.2. The sludge

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retention time in each digester was controlled at 15 d by feeding 67 mL WAS on a daily

190

basis and simultaneously discharging 67 mL digested sludge. The two digesters were

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operated continuously for 75 d and then the microbial communities in both digesters were 9



192

analysed using methods described in the SI. The obtained sequences were deposited into

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National Center for Biotechnology Information (NCBI) with accession number

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MK471389-MK472051.

195 196

Analytical Methods. TS, VS and TCOD were determined according to Standard

197

Methods.28 For the analysis of SCOD, VFA, soluble protein and soluble polysaccharide,

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centrifugation (5000g, 15 min) was carried out first followed by filtration using a filter

199

with 0.45-μm pore size. VFA was analyzed using a gas chromatograph (Agilent, FID

200

with polar capillary column) and expressed as a sum of measured composition (i.e., acetic,

201

propionic, n-butyric, iso-butyric, n-valeric and iso-valeric acids in this study). Protein

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concentration was measured with the BCA method using bovine serum albumin as

203

standard.29 Polysaccharide concentration was determined according to the phenol-sulfuric

204

acid method using glucose as standard.30 The detailed measurement procedures of VFA,

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protein and polysaccharide were shown in SI. pH value was measured using the pH meter

206

(TPS miniCHEM). The equivalent COD conversion of protein, carbohydrate and VFA

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were performed according to Lu et al.:31 1.50 g-COD/g protein, 1.06 g-COD/g

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carbohydrate, 1.07 g-COD/g acetate, 1.51 g-COD/g propionate, 1.82 g-COD/g butyrate,

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and 2.04 g-COD/g valerate. Biogas volume was measured24 using the manometer at the

210

start of each sampling event. Cumulative volumetric biogas production was calculated

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from the pressure increase in the headspace volume (60 mL) and expressed under

212

standard conditions (25 °C, 1 atm). Then, the gas in the serum bottle was expelled out

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using a syringe needle until the pressure inside was equal to the atmosphere. Biogas

214

composition was analyzed using a gas chromatograph equipped with a thermal 10


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conductivity detector (GC-TCD, Lunan 6890), as described in our previous publication.32

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Methane production was calculated in terms of those products.

217 218

The detection of PVC microplastics in raw WAS contained three steps, i.e., sludge

219

collection, sludge pretreatment and quantification, according to Li et al..23 The detailed

220

procedures for PVC microplastics quantification were presented in SI. Chemicals (i.e.,

221

BPA, DiNP, DiDP and DEHP) leached from PVC microplastics were extracted and

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analyzed by gas chromatography combined with mass spectrometry (GC/MS, Saturn

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2000, Varian). The measured relative enzyme activities (%) were expressed as a biomass

224

specific enzyme activity dividing by the corresponding measured value of control with

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raw WAS. Further detailed information of the determination of four chemicals

226

concentrations and enzyme activities can be found in SI.

227 228

Statistical Analysis. The significance of the data was analyzed by variance (ANOVA)

229

analysis after checking normality (Shapiro-Wilk test) and homoscedasticity (Levene’s

230

test) using the SPSS 17.0 software (SPSS Inc., Chicago, USA). P < 0.05 was considered

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to be statistically significant.

232 233

RESULTS

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Impacts of PVC Microplastics on Methane Production from WAS. In this study, the

235

control represented raw WAS in sludge digestion experiments. To investigate the impact

236

of PVC microplastics on methane production from WAS, BMP testing at different levels

237

of PVC microplastics was performed and lasted for 45 d. The cumulative methane yield 11



238

vs. digestion time was recorded in Figure 1. After the 45-d period, complete anaerobic

239

digestion had been achieved with a stationary level of methane production in each case.

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For the control, the cumulative methane yield during the entire digestion period was 170

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± 5 L CH4/kg VS (mean ± 95% confidence interval). In comparison, the cumulative

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methane yield at a lower PVC microplastics dosage (10 particles/g TS) was 180 ± 3 L

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CH4/kg VS, representing a significant (P = 0.041) increase of 5.9 ± 0.1%. However, the

244

higher concentrations (i.e., 20, 40 and 60 particles/g TS) of PVC microplastics inhibited

245

the methane production from WAS throughout the entire digestion period. The

246

cumulative methane yield decreased gradually from 90.6 ± 0.3% (P = 0.018) to 80.5 ±

247

0.1% (P = 0.001) of the control with respect to the increasing PVC microplastics from 20

248

particles/g TS to 40 particles/g TS and then further significantly decreased (P = 0.001) to

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75.8 ± 0.2% of the control with increasing PVC microplastics to 60 particles/g TS. This

250

indicated that the influence of PVC microplastics on methane production during WAS

251

digestion was strongly related to the PVC level.

252 253

(Position for Figure 1)

254 255

Impacts of PVC Microplastics on Methane Production Kinetics. Methane potential

256

(B0) and hydrolysis coefficient (k), two primary parameters associated with methane

257

production, were determined using a one-substrate model. The simulated methane

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production profiles by the one-substrate model are shown in Figure S1, which indicate

259

that the fit of experimental data to the model was satisfactory (R2 > 0.95 in all cases).

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The determined B0 and k at different PVC microplastics concentrations were summarized

262

in Table 1. In general, B0 and k of WAS in the anaerobic digester decreased with

263

increased PVC microplastics level, except for the case of 10 particles/g TS. PVC

264

microplastics at a low level (i.e., 10 particles/g TS) did not affect k (P = 0.904) and

265

exerted a significant (P = 0.012) positive effect on B0 (also Figure 1) compared with the

266

control, which increased from 162 ± 2 to 171 ± 3 L CH4/kg VS. In contrast, PVC

267

microplastics at the higher levels (i.e., 20, 40 and 60 particles/g TS) decreased both B0

268

and k of WAS in comparison to the control. The lowest B0 and k were achieved at PVC

269

microplastics level of 60 particles/g TS and were determined as approximately 75.3 ± 0.1%

270

of the control (122 ± 1/162 ± 2 CH4/kg VS) and 81.9 ± 0.1% of the control (0.172 ±

271

0.007/0.210 ± 0.009 d−1), respectively. Correspondingly, apparent VS destruction (Y0)

272

also declined from 32.1 ± 0.6% to 22.9 ± 0.2% with increased PVC microplastics levels

273

(see Table 2). These results indicated that PVC microplastics at a low level (i.e., 10

274

particles/g TS) could enhance methane production from WAS, while the higher levels

275

(i.e., 20, 40 and 60 particles/g TS) of PVC microplastics inhibited methane production

276

and hydrolysis of WAS. The reason will be clarified in the following text.

277 278

(Position for Table 1)

279 280

Impacts of PVC Microplastics on Solubilization, Hydrolysis and Acidification.

281

Methane production from WAS during anaerobic digestion usually successively

282

undergoes solubilization, hydrolysis and acidification, as shown in Figure S2, with

283

several key enzymes (e.g., protease, cellulase, acetate kinase (AK) and coenzyme F420) 13



284

involved in each step. However, the impact of PVC microplastics on each step involved

285

in methane production has never been documented. In this study, it was investigated in

286

detail to explore the mechanisms of PVC microplastics affecting methane production

287

from WAS during anaerobic digestion.

288 289

The WAS with different levels of PVC microplastics (i.e., 10, 20, 40 and 60 particles/g

290

TS) were anaerobically digested under the condition of inhibiting methanogens. The

291

digestion lasted for 3 d. Methane was not detected in all tests during the 3-day test period.

292

Converting particulate organic matters of WAS to soluble substrates is the first step. The

293

effects of PVC microplastics exposure on solubilization of particulate organic substances

294

were expressed by the changes of SCOD in this study and the results were shown in

295

Figure 2. In general, a large amount of SCOD (i.e., 195-385 mg COD/L) was released

296

during anaerobic digestion in each case and increased PVC microplastics levels caused

297

increased SCOD release. The maximal SCOD of 3465 ± 25 mg COD/L was achieved

298

with PVC microplastics of 60 particles/g TS, while SCOD in the control situation was

299

only 3080 ± 15 mg COD/L, representing a relative increase (P = 0.000) of 12.5 ± 0.1%.

300

The unknown organics in the supernatant also significantly (P = 0.000) increased by a

301

factor of 2.8, which might be attributed to the release of lipids and nucleic acids in WAS.

302

This revealed that the exposure of PVC microplastics at the studied levels enhanced the

303

solubilization of WAS in anaerobic digestion.

304 305


306 14


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Soluble protein and soluble carbohydrate are usually the primary constituents of soluble

308

substrates in WAS.33 As shown in Figure 2, the content of protein and soluble

309

carbohydrate in the control were comparable (P = 0.986, 0.885) to those in the test with

310

10 particles/g TS of PVC microplastics. Nevertheless, PVC microplastics at higher

311

concentrations (i.e., 20, 40 and 60 particles/g TS) resulted in the accumulation of soluble

312

protein. The content of soluble protein in the presence of 60 particles PVC microplastics

313

/g TS was 935 ± 30 mg COD/L, significantly (P = 0.01) higher than that in the control

314

(815 ± 35 mg COD/L). The reason has been revealed by model analysis above; that is,

315

PVC microplastics at a low level (i.e., 10 particles/g TS) had no effect on hydrolysis of

316

WAS, while the higher levels (i.e., 20, 40 and 60 particles/g TS) of PVC microplastics

317

inhibited hydrolysis of WAS.

318 319

VFA including acetate, propionate, butyrate and valerate were the products of the

320

acidification. From Figure 2, PVC microplastics at 10 particles/g TS could enhance VFA

321

production by 4.9 ± 0.1% compared to control, which may be due to the increased SCOD

322

release. This further resulted in the increased methane production from WAS. In contrast,

323

with the increase of PVC microplastics from 20 to 60 particles/g TS, the VFA decreased

324

from 6.9 ± 0.1 to 16.8 ± 0.2% in comparison to the control, although increased PVC

325

microplastics levels caused increased SCOD release, which indicated that PVC

326

microplastics at higher levels (i.e., 20, 40 and 60 particles/g TS) could restrain the

327

acidification process of WAS. This finding might represent one reason for the decreased

328

methane production when exposed to higher concentrations of PVC microplastics.

329 15



330

Impacts of PVC microplastics on Key Enzyme Activities. As shown in Figure 3,

331

protease and cellulase are responsible for the decomposition of proteins and

332

polysaccharide, respectively. Acetyl-CoA is converted to acetate by AK. F420 is the key

333

enzyme with respect to methanation (see Figure S2). In this study, these key enzymes

334

responsible for sludge anaerobic digestion were also analyzed. In the presence of PVC

335

microplastics with varying levels, the relative enzyme activities of control were listed in

336

Table 2. PVC microplastics at 10 particles/g TS did not affect the activities of these four

337

key enzymes. However, at higher levels of 20, 40 and 60 particles/g TS, the impacts of

338

PVC microplastics on protease and AK were significantly dosage dependent. The relative

339

enzyme activities of the control declined as PVC microplastics level increased. The

340

protease activity decreased gradually from 96.4 ± 0.02 % (P = 0.000) to 90.2 ± 0.04 % (P

341

= 0.000) of the control with respect to increasing PVC microplastics from 20 particles/g

342

TS to 40 particles/g TS and then further significantly decreased (P = 0.000) to 87.1 ±

343

0.02 % of the control with increasing PVC microplastics to 60 particles/g TS. The lowest

344

AK activity was achieved at the PVC microplastics concentration of 60 particles/g TS,

345

representing 87.2 ± 0.02 % (P = 0.000) of the control. The cellulase activity did not

346

change significantly with PVC microplastics levels (P = 0.791, 0.608, 0.175, 0.081). For

347

the F420, 60 particles/g TS of PVC microplastics remarkably (P = 0.000) reduced its

348

activity to 79.3 ± 0.02% of the control. All of these results were in accordance with the

349

above observed influences of PVC microplastics on each step involved in methane

350

production, i.e., solubilization, hydrolysis and acidification (Figure 2).

351 352

(Position for Table 2) 16


Page 16 of 37

Page 17 of 37


353 354

Impacts of Chemicals Released from PVC Microplastics on Methane Production.

355

During the whole leaching experiments, BPA was found to be the only dominant

356

chemical leached from PVC microplastics, while additional chemicals including DiNP,

357

DiDP and DEHP were not detected. This result was in accordance with the research of

358

Suhrhoff and Scholz-Böttcher,18 which demonstrated that BPA was the dominant additive

359

leached from PVC microplastics under the experimental condition that played the key

360

role in the system. The corresponding cumulative concentrations of BPA leaching from

361

PVC microplastics at studied levels (i.e., 10, 20, 40 and 60 particles/g TS) vs. time were

362

recorded in Figure S3. In each case, the cumulative BPA concentration followed a

363

saturation curve. The leaching rate of BPA from PVC microplastics was high at the

364

beginning and gradually decreased with time. In addition, an enhanced leaching amount

365

of BPA with increasing PVC microplastics level was observed. At PVC microplastics

366

levels of 10, 20, 40 and 60 particles/g TS, the corresponding maximum leaching

367

concentrations of BPA over 40 d were 0.5, 1.8, 3.0 and 3.6 μg/L, respectively. To

368

investigate the impacts of BPA leaching from PVC microplastics on methane production

369

during WAS anaerobic digestion, BPA with corresponding concentration was dosed at

370

the corresponding leaching times instead of PVC microplastics. The results in Figure 3(A)

371

demonstrated that BPA leaching from 10 particles/g TS of PVC microplastics enhanced

372

methane production from WAS, while methane production was inhibited by higher

373

concentrations of BPA leaching from PVC microplastics at higher levels (i.e., 20, 40 and

374

60 particles/g TS).

375 17



376


377 378

The effects of PVC microplastics at different levels and the corresponding leached BPA

379

on methane production during WAS digestion were compared and the relative methane

380

production of the control was calculated. Compared to the control, 10 particles/g TS of

381

PVC microplastics increased methane production by 6.1 ± 0.2% (Figure 3(B)), which

382

was comparable (P = 0.455) with the enhanced methane production by the corresponding

383

BPA leaching from 10 particles/g TS of PVC microplastics. At higher levels of PVC

384

microplastics (i.e., 20, 40 and 60 particles/g TS), the cumulative methane production

385

values after 45 d were 90.6 ± 1.3, 80.5 ± 1.5 and 75.8 ± 2.1 of the control, respectively.

386

The cumulative methane production results with corresponding BPA leaching over the

387

same period were 93.5 ± 0.3, 84.4 ± 0.8 and 78.5 ± 1.1 of the control, respectively. The

388

above results revealed that PVC microplastics affected methane production during WAS

389

digestion mainly due to the leached BPA, which appeared to be the main reason for the

390

inhibition of methane production at higher levels of PVC microplastics.

391 392

Microbial Community. To further understand the mechanisms of the PVC microplastics

393

effects on methane production from WAS anaerobic digestion, the microbial community

394

compositions in the continuously operated control and experimental anaerobic digesters

395

were analyzed and compared. The control digester was fed with raw WAS, whereas the

396

experimental digester was fed with WAS containing 60 particles PVC microplastics/g TS.

397

The number of operational taxonomic units (OTUs) in the control digester was 705 and

398

the quantity of OTUs in the experimental digester was 711, in which 701 OTUs were 18


Page 18 of 37

Page 19 of 37


399

shared with the control (see Figure S4). The rarefaction curves of the OUT numbers in

400

the control and experimental group were shown in Figure S5. The curves for each group

401

were near saturation, suggesting that the sequencing depth was enough to cover the whole

402

microbial diversity for each sample and the sequencing data had a high quality. The

403

Alpha diversity results showed that the presence of PVC microplastics did not

404

significantly change the Chao index (718 ± 33 vs 710 ± 31; P = 0.49) and the Shannon

405

index (4.98 ± 0.01 vs 5.00 ± 0.01; P = 0.07). Moreover, the mean of PD whole-tree index

406

in each group was 55. All these results revealed that the presence of PVC microplastics

407

did not significantly affect the microbial structure and the microbial diversity.

408 409

Phylum level distributions of microbial populations in the two digesters were presented in

410

Figure 4(A). The most abundant bacterial populations in the control and experimental

411

digesters were Proteobacteria, Bacteroidetes, Chloroflexi and Firmicutes, which together

412

accounted for 79.6 ± 0.5% and 73.6 ± 0.2% of the total bacterial community respectively.

413

Many microbes in these four phyla were reported to possess abilities to convert organic

414

compounds (e.g., proteins and carbohydrates) into VFA.34,35 Compared to the control, the

415

abundances of Proteobacteria, Chloroflexi and Firmicutes in the experimental digester

416

decreased by 10.8 ± 0.1%, 4.3 ± 0.1% and 10.2 ± 0.1% of the total sequences respectively

417

and no variation was found in the abundance of Bacteroidetes. In addition to bacterial

418

sequences, archaeal sequences belonging to the phylum Euryarchaeota were detected in

419

the two digesters. Methanogens have been documented to be a part of Euryarchaeota.

420

The presence of 60 particles PVC microplastics/g TS significantly (P = 0.004) reduced

421

the abundance of Euryarchaeota from 10.2 ± 0.4% to 8.7 ± 0.2% of the total sequences.

422

Further exploration at the genus level (see Figure 4(B)) found that the microbial 19



423

communities in the two digesters consisted of various anaerobes relevant to hydrolysis

424

(e.g., Rhodobacter sp., Bacteroides sp.), acidogenesis (e.g., Proteiniborus sp., Garciella

425

sp., Collinsella sp. and Caldilinea sp.) and methanogenesis (e.g., Methanosaeta sp.).36,37

426

Some of these microbes abundances were affected by PVC microplastics. For example,

427

the abundance of Rhodobacter sp. in the control digester accounted for 9.9 ± 0.3% of the

428

total bacterial sequences, significantly (P = 0.002) higher than that (8.6 ± 0.1%) in the

429

experimental digester. The presence of PVC microplastics significantly decreased the

430

abundances of Proteiniborus sp. (P = 0.010) and Garciella sp. (P = 0.031) from 2.4 ± 0.1%

431

and 1.9 ± 0.3% to 1.8 ± 0.2% and 1.1 ± 0.3%, respectively. Methanosaeta sp. occupied

432

9.7 ± 0.2% of the total bacterial sequences in the control, while only 8.1 ± 0.3% was

433

detected in the experimental digester, representing a significant (P = 0.002) reduction of

434

16.5 ± 0.1%. These results suggested that the microbial community in the presence of 60

435

particles PVC microplastics/g TS was shifted in the direction against hydrolysis-

436

acidification and methanation, which was consistent with the lower methane production

437

observed in BMP testing with 60 particles PVC microplastics/g TS.

438 439


440 441

DISCUSSION

442

This study revealed for the first time the manner in which PVC microplastics exposure

443

affected the methane production in WAS anaerobic digestion. BMP tests clearly showed

444

that PVC microplastics at 10 particles/g TS can increase (P = 0.041) anaerobic methane

445

production by 5.9 ± 0.1%, which was due to the enhanced sludge solubilization. 20


Page 20 of 37

Page 21 of 37


446

Moreover, PVC microplastics at this low level had no effect on the hydrolysis-

447

acidification process (Table 1 and Figure 2). The increased methane production was

448

primarily induced by BPA leaching from PVC microplastics, which is reflected in the

449

comparable effects of PVC microplastics and released BPA on methane production. The

450

effects of PVC microplastics at different levels and the corresponding leached BPA on

451

sludge solubilization were compared (Figure S6). In all cases, the SCODs of the PVC

452

microplastics dosed were comparable (P = 0.076, 0.055, 0.069, 0.071) with those of the

453

corresponding leached BPA dosed. This finding revealed that PVC microplastics affected

454

sludge solubilization during digestion mainly due to the BPA leached. Therefore, with the

455

release of BPA, microbial cell walls and extracellular polymeric substances in WAS

456

increasingly ruptured and formed more soluble substances from particulate substances.38

457

On the contrary, hydrolysis-acidification was a microbially related process. Since the

458

released BPA from 10 particles/g TS of PVC microplastics occurred at a relatively low

459

level,37 it had no effect on the hydrolysis-acidification process, as reflected in the similar

460

(P = 0.904) hydrolysis coefficient (k) to the control. This was also supported by the

461

results of relevant enzyme activity changes. These combined facts resulted in the

462

increased (P = 0.041) anaerobic methane production at 10 particles/g TS PVC of

463

microplastics.

464 465

In contrast, the higher levels of PVC microplastics (i.e., 20, 40 and 60 particles/g TS)

466

inhibited the methane production from WAS during anaerobic digestion. Although PVC

467

microplastics at > 20 particles/g TS improved sludge solubilization, they strongly

468

inhibited the subsequent sludge hydrolysis-acidification process (Figure 2), thereby 21



469

resulting in the overall decreasing trends of methane production.39 This was also

470

confirmed by the results of relevant enzyme activities (Table 2). BPA leaching from PVC

471

microplastics was proven to be the main reason for the inhibition of methane production

472

at higher levels of PVC microplastics (Figure 3(B)). Note that some microparticles had a

473

much smaller size than those that we used (1 mm). However, a similar amount (mass) of

474

these smaller microplastics might cause a different impact on anaerobic digestion

475

performance from this study, because the size of the microplastics could affect the rate of

476

chemical leaching through equilibrium partitioning. Model analysis results showed that

477

PVC microplastics at > 20 particles/g TS decreased the hydrolysis coefficient (k), which

478

indicated that a larger anaerobic digester or a longer hydraulic retention time was

479

required in order to achieve a similar methane production to the control. In addition, the

480

apparent VS destruction (Y0) also declined from 30.4% to 28.0-22.9% in the presence of

481

20 to 60 particles of PVC microplastics /g TS, which indicates a greater requirement for

482

sludge transport and disposal, thus increasing the overall costs for subsequent sludge

483

transport and disposal.

484 485

The long-term effect experiments found that PVC microplastics with 60 particles/g TS

486

decreased the abundance of various anaerobes relevant to hydrolysis (e.g., Rhodobacter

487

sp.), acidogenesis (e.g., Proteiniborus sp. and Garciella sp.) and methanogenesis (e.g.,

488

Methanosaeta sp.). Several studies demonstrated that the toxicity effects of microplastics

489

on different aquatic species were primarily due to their leachate. Li et al.8 reported that

490

leachates from seven categories of microplastics (i.e., PET, HDPE, PVC, LDPE, PP, PS

491

and PC) increased the mortality of Amphibalanus amphitrite. Leachates also caused acute 22


Page 22 of 37

Page 23 of 37


492

toxicity in other freshwater species, such as Daphnia magna and Perna perna.17,40

493

Specifically, the BPA was found to be the most toxic,8 which could also inhibit the

494

activities of crustaceans, heterotrophic bacteria and nitrifying bacteria.19,41 BPA leaching

495

from PVC microplastics was demonstrated to be the main reason for the inhibition of

496

methane production at higher levels of PVC microplastics. Therefore, the high toxicity of

497

BPA leaching from PVC microplastics might be responsible for the changes of microbial

498

community.

499 500

Recent studies showed that plastics can be biologically degraded under aerobic

501

conditions. For example, Ideonella sakaiensis 201-F6 was able to degrade the PET film

502

surface at a rate of 0.13 mg/cm2/d at 30°C.42 Over a period of 60 d, 7.4 ± 0.4% of the PS

503

pieces (2500 mg/L) were degraded by Exiguobacterium sp. strain YT2.43 Mahon et al.44

504

found that the anaerobic digestion progress significantly reduced the abundances of

505

microplastics in sewage sludge, but there is no evidence to prove the breakdown of

506

microplastics in an anaerobic digester. Therefore, microplastics possibly follow digested

507

sludge, entering the soil and water systems and causing risks through their leachate. New

508

and less toxic chemical additives with low migration potential are required. Two such

509

alternatives might be tris(2-ethylhexyl) trimellitate (TOTM) and acetyltri-n-butyl citrate

510

(ATBC).15

511 512

ASSOCIATED CONTENT

513

Supporting Information. Detailed description of the experimental methods, and the

514

additional figures (Figures S1−S6) and table (Tables S1) about sludge characteristics, 23



515

model evaluation, metabolic pathways, BPA leaching, venn analysis, rarefaction curves

516

and SCOD concentrations.

517 518

AUTHOR INFORMATION

519

Corresponding Author

520

*Phone: +86 21 65986849; fax: +86 21 65983602; e-mail: [email protected]

521 522

Notes

523

The authors declare no competing financial interest.

524 525

ACKNOWLEDGMENTS

526

This work was supported by the Recruitment Program of Global Experts, the National

527

Natural Science Foundation of China (51578391 and 51608374), and the State Key

528

Laboratory of Pollution Control and Resource Reuse Foundation (No. PCRRK18007).

529 530

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30


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Page 31 of 37

656


List of Figures and Tables

657 658

Table 1. Estimated hydrolysis coefficient (k), methane production potential (B0) and

659

apparent VS destruction (Y0) at different PVC microplastics levels using a one-substrate

660

model (with 95% confidence intervals).

661

Table 2. Relative activities of key enzymes of control in the presence of PVC

662

microplastics with different levels (with 95% confidence intervals).

663 664

Figure 1. Cumulative methane yield from waste activated sludge at different PVC

665

microplastics levels. Error bars represent 95% confidence intervals.

666

Figure 2. Effects of PVC microplastics at different levels on the components of

667

supernatant fermentation liquor after 3 d. Error bars represent 95% confidence intervals.

668

Figure 3. Effects of BPA leaching from PVC microplastics at different levels (A) and

669

PVC microplastics at different levels (B) on methane production during WAS digestion.

670

Error bars represent 95% confidence intervals.

671

Figure 4. Distributions of microbial populations at the phylum level (A) and at the genus

672

level (B) in the control and experimental anaerobic digesters.

31



Page 32 of 37

673

Table 1. Estimated hydrolysis coefficient (k), methane production potential (B0) and

674

apparent VS destruction (Y0) at different PVC microplastics levels using a one-substrate

675

model (with 95% confidence intervals). Parameters

PVC (particles/g-TS)

k (d-1)

control

0.210 ± 0.009

162 ± 2

30.4 ± 0.4

10

0.209 ± 0.010

171 ± 3

32.1 ± 0.6

20

0.193 ± 0.005

149 ± 1

28.0 ± 0.3

40

0.178 ± 0.008

129 ± 2

24.2 ± 0.4

60

0.172 ± 0.007

122 ± 1

22.9 ± 0.2

B0 (L CH4/kg VS)

676

32


Y0 (%)

Page 33 of 37


677

Table 2. Relative activities of key enzymes of control in the presence of PVC

678

microplastics with different levels (with 95% confidence intervals). PVC

Relative activities of control (%)

(particles/gProtease

Cellulase

AK

10

101.1 ± 0.05

100.2 ± 0.03

101.4 ± 0.02

99.8 ± 0.02

20

96.4 ± 0.02

99.9 ± 0.01

95.3 ± 0.01

94.4 ± 0.01

40

90.2 ± 0.04

98.7 ± 0.02

91.3 ± 0.03

85.7 ± 0.03

60

87.1 ± 0.02

97.6 ± 0.01

87.2 ± 0.02

79.3 ± 0.02

TS)

679

33


F420


Page 34 of 37

680

200 160 (L CH4/ kg VS added)

Cumulative methane yield

180 140 120 100

control 10 particles/g-TS 20 particles/g-TS 40 particles/g-TS 60 particles/g-TS

80 60 40 20 0

0

5

10

15

20

25

30

35

40

45

50

Digestion time (d) 681 682

Figure 1. Cumulative methane yield from waste activated sludge at different PVC

683

microplastics levels. Error bars represent 95% confidence intervals.

34



Soluble organic substrates (mg COD/L)

Page 35 of 37

684

5000 4000

Unknown Polysaccharide Protein VFA

3000 2000 1000 0

initial initial

a control

b 10

c 20

d 40

e 60

PVC concentrations (particles/g-TS)

685

Figure 2. Effects of PVC microplastics at different levels on the components of

686

supernatant fermentation liquor after 3 d. Error bars represent 95% confidence intervals.

35



200 160

(L CH4/ kg VS added)

Cumulative methane yield

(A)

Page 36 of 37

120 80 40 0

(B)

BPA from control/g-TS BPA from10 particles/g-TS BPA from 20 particles/g-TS BPA from 40 particles/g-TS BPA from 60 particles/g-TS

0

10

20

30

40

50

Digestion time (d)

687 688

Figure 3. Effects of BPA leaching from PVC microplastics at different levels (A) and

689

PVC microplastics at different levels (B) on methane production during WAS digestion.

690

Error bars represent 95% confidence intervals. 36


Page 37 of 37


(A)

10.2%

8.7%

Proteobacteria

3.6%

9.0%

Bacteroidetes Proteobacteria

2.1% 2.3% 2.4% 3.2%

28.7%

Chloroflexi

Firmicutes

Firmicutes

Actinobacteria Actinobacteria

Spirochaetae Spirochaetae

4.7%

Proteobacteria

Bacteroidetes Chloroflexi

Acidobacteria Acidobacteria Aminicenantes Aminicenantes

25.6%

2.3%

Bacteroidetes

25.6%

2.5% 2.7%

Actinobacteria Spirochaetae

3.6%

Acidobacteria

4.0%

Aminicenantes Thermotogea

Thermotogea

8.8%

Thermotogea Others

Others Euryarchaeota 14.1%

Others

7.9%

Euryarchaeota

19.9%

Chloroflexi Firmicutes

Control

20.2% 13.5%

Experimental

(B) Methanogens

Hydrolytic microbes

Acidogens

Control Experimental

691 692

Figure 4. Distributions of microbial populations at the phylum level (A) and at the genus

693

level (B) in the control and experimental anaerobic digesters.

37


Euryarchaeota

Polyvinyl Chloride Microplastics Affect Methane Production from the

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