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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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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
‡
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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
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ABSTRACT: The retention of polyvinylchloride (PVC) microplastics in sewage sludge
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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
26
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
28
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
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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
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disposal has resulted in microplastics being ubiquitously found in aquatic water bodies,
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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
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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
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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
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present in seawater.13
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The most commonly found microplastics in sludge are polyethylene (PE), polyethylene
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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,
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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
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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.
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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
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methane potential tests. The impacts of PVC microplastics on both the hydrolysis rate
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and the methane production potential were then revealed through model-based analysis.
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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
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microplastics. Finally, the long-term effect of PVC microplastics on microbial
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community was explored.
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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
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(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
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WAS were identified and enumerated using the methods to be further described in the
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“Analytical Methods” section. Among the microplastics found in the raw WAS (i.e., PE,
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PET, PS and PP), PVC microplastic was found to be one of the most abundant
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microplastics with the content of 2 particles/g-TS. The PVC microplastics for 5
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experiments, in the form of white spherical powder, were purchased from Youngling Co.
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Ltd. (Shanghai, China). According to the manufacturer, the size of PVC microplastics
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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
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(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
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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,
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sealed by rubber stoppers with aluminum crimp-caps and stored in a constant-
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temperature (37 ± 1 °C) incubator. Raw WAS samples with 2 particles/g-TS PVC
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microplastics served as a control in this study. Based on the environmentally relevant
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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
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of inoculum to methane production, a blank was also created. The blank sample
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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
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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
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methane production was obtained. Methane production from WAS was calculated by
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subtracting its test value from that in the blank (pointwise) and recording the result as its
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volume per VS mass (L CH4/kg VS). After the BMP tests were operated for 45 d, the
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activities of those key enzymes (e.g., protease, cellulase, AK and F420) involved in
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anaerobic digestion steps were also analyzed. The detailed analytical procedures of these
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enzyme activities were presented in Supporting Information (SI).
131 132
Biochemical Methane Potential Tests Modeling. Methane production potential (B0)
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and hydrolysis coefficient (k), two primary parameters associated with methane
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production, were used to evaluate and compare the influence of PVC microplastics
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exposure on methane production potential and kinetics of WAS. Based on the cumulative
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methane curves, a one-substrate model was applied as described in Wang et al.,24 fitting
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B0 and k, with the apparent VS destruction (Y0) calculated from Y0=B0/380×RSS, where
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380 is the theoretical methane potential of sludge at standard temperature and pressure
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(STP, 25 °C, 1 atm) (L CH4/kg TCOD); RSS is measured as VS/TCOD of WAS (i.e.,
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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
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well known that methane production from WAS usually successively undergoes
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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
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organic macromolecular substances, such as soluble protein and soluble carbohydrate.
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Then, these were hydrolyzed to amino acid and monosaccharide constituents. Through
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acidification, volatile fatty acids (VFAs) were produced.27 The experiments regarding the
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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
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consume VFAs produced during acidification, it was necessary to deplete methanogens
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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
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polysaccharides and VFA before and after digestion were measured, respectively.
159 160
Determination of Chemicals Leaching from PVC Microplastics. According to
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manufacturers and Suhrhoff and Scholz-Böttcher,18 the bisphenol-A (BPA), diisononyl
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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
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chemicals leaching from PVC microplastics at four studied PVC microplastic levels
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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
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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
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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,
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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
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community. One, as control, was fed with raw WAS and the other experimental digester
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was fed with WAS containing 60 particles PVC microplastics/g TS. The two digesters
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were placed in a temperature-controlled shaker (37 ± 1 °C, 150 rmp). The pH in each
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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
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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
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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.
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Analytical Methods. TS, VS and TCOD were determined according to Standard
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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
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with 0.45-μm pore size. VFA was analyzed using a gas chromatograph (Agilent, FID
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with polar capillary column) and expressed as a sum of measured composition (i.e., acetic,
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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
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standard.29 Polysaccharide concentration was determined according to the phenol-sulfuric
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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
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(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
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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
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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
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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
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collection, sludge pretreatment and quantification, according to Li et al..23 The detailed
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procedures for PVC microplastics quantification were presented in SI. Chemicals (i.e.,
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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
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concentrations and enzyme activities can be found in SI.
227 228
Statistical Analysis. The significance of the data was analyzed by variance (ANOVA)
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analysis after checking normality (Shapiro-Wilk test) and homoscedasticity (Levene’s
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test) using the SPSS 17.0 software (SPSS Inc., Chicago, USA). P < 0.05 was considered
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to be statistically significant.
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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
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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
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vs. digestion time was recorded in Figure 1. After the 45-d period, complete anaerobic
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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
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higher concentrations (i.e., 20, 40 and 60 particles/g TS) of PVC microplastics inhibited
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the methane production from WAS throughout the entire digestion period. The
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cumulative methane yield decreased gradually from 90.6 ± 0.3% (P = 0.018) to 80.5 ±
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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
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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
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(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
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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
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microplastics at a low level (i.e., 10 particles/g TS) did not affect k (P = 0.904) and
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exerted a significant (P = 0.012) positive effect on B0 (also Figure 1) compared with the
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control, which increased from 162 ± 2 to 171 ± 3 L CH4/kg VS. In contrast, PVC
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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
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microplastics level of 60 particles/g TS and were determined as approximately 75.3 ± 0.1%
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of the control (122 ± 1/162 ± 2 CH4/kg VS) and 81.9 ± 0.1% of the control (0.172 ±
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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
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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
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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
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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
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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.
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This revealed that the exposure of PVC microplastics at the studied levels enhanced the
303
solubilization of WAS in anaerobic digestion.
304 305
(Position for Figure 2)
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Soluble protein and soluble carbohydrate are usually the primary constituents of soluble
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substrates in WAS.33 As shown in Figure 2, the content of protein and soluble
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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
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Impacts of PVC microplastics on Key Enzyme Activities. As shown in Figure 3,
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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
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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
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(Position for Figure 3)
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
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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
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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
(Position for Figure 4)
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
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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
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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
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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
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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
REFERENCES
531
(1) Thompson, R.C.; Olsen, Y.; Mitchell, R.P.; Davis, A.; Rowland, S.J.; John, A.W.G.;
532
McGonigle, D.; Russell, A.E. Lost at sea: where is all the plastic? Science 2004, 304
533
(5672), 838-838.
534
(2) Barnes, D.K.; Galgani, F.; Thompson, R.C.; Barlaz, M. Accumulation and
535
fragmentation of plastic debris in global environments. Philos. Trans. R. Soc. B 2009,
536
364 (1526), 1985-1998.
537
(3) Nizzetto, L.; Futter, M.; Langaas, S. Are agricultural soils dumps for microplastics of 24
ACS Paragon Plus Environment
Page 24 of 37
Page 25 of 37
Environmental Science & Technology
538
urban origin? Environ. Sci. Technol. 2016, 50 (20), 10777-10779.
539
(4) Napper, I.E.; Bakir, A.; Rowland, S.J.; Thompson, R.C. Characterisation, quantity
540
and sorptive properties of microplastics extracted from cosmetics. Mar. Pollut. Bull. 2015,
541
99 (1), 178-185.
542
(5) Van Wezel, A.; Caris, I.; Kools, S.A.E. Release of primary microplastics from
543
consumer products to wastewater in the Netherlands. Environ. Toxicol. Chem. 2016, 35
544
(7), 1627-1631.
545
(6) Eerkes-Medrano, D.; Thompson, R.C.; Aldridge, D.C. Microplastics in freshwater
546
systems: a review of the emerging threats, identification of knowledge gaps and
547
prioritisation of research needs. Water Res. 2015, 75: 63-82.
548
(7) Van Cauwenberghe, L.; Vanreusel, A.; Mees, J.; Janssen, C.R. Microplastic pollution
549
in deep-sea sediments. Environ. Pollut. 2013, 182, 495-499.
550
(8) Li, H.X.; Getzinger, G.J.; Ferguson, P.L.; Orihuela, B.; Zhu, M.; Rittschof, D. Effects
551
of toxic leachate from commercial plastics on larval survival and settlement of the
552
barnacle Amphibalanus Amphitrite. Environ. Sci. Technol. 2015, 50 (2): 924-931.
553
(9) Browne, M.A.; Niven, S.J.; Galloway, T.S.; Rowland, S.J.; Thompson, R.C.
554
Microplastic moves pollutants and additives to worms, reducing functions linked to
555
health and biodiversity. Curr. Biol. 2013, 23, 2388-2392.
556
(10) Cheung, P.K.; Fok, L. Characterisation of plastic microbeads in facial scrubs and
557
their estimated emissions in Mainland China. Water Res. 2017, 122, 53-61.
558
(11) Jeong, C.B.; Won, E.J.; Kang, H.M, Lee, M.C.; Hwang, D.S.; Hwang, U.K.; Zhou,
559
B.S.; Souissi, S.; Lee, S.J.; Lee, J.S. Microplastic size-dependent toxicity, oxidative stress
560
induction, and p-JNK and p-p38 activation in the monogonont rotifer (Brachionus 25
ACS Paragon Plus Environment
Environmental Science & Technology
561
koreanus). Environ. Sci. Technol. 2016, 50 (16), 8849-8857.
562
(12) Murphy, F.; Ewins, C.; Carbonnier, F.; Quinn, B. Wastewater treatment works
563
(WwTW) as a source of microplastics in the aquatic environment. Environ. Sci. Technol.
564
2016, 50 (11), 5800-5808.
565
(13) Nizzetto, L.; Bussi, G.; Futter, M.N.; Butterfield, D.; Whitehead, P.G. A theoretical
566
assessment of microplastic transport in river catchments and their retention by soils and
567
river sediments. Environ. Sci. Proc. Impacts 2016, 18 (8), 1050-1059.
568
(14) Rochman, C.M.; Hoh, E.; Hentschel, B.T.; Kaye, S. Long-term field measurement of
569
sorption of organic contaminants to five types of plastic pellets: implications for plastic
570
marine debris. Environ. Sci. Technol. 2013, 47 (3), 1646-1654.
571
(15) Lithner, D.; Larsson, Å.; Dave, G. Environmental and health hazard ranking and
572
assessment of plastic polymers based on chemical composition. Sci. Total Environ. 2011,
573
409 (18), 3309-3324.
574
(16) Zhang, C.; Chen, X.; Wang, J, Tan, L. Toxic effects of microplastic on marine
575
microalgae Skeletonema costatum: interactions between microplastic and algae. Environ.
576
Pollut. 2017, 220, 1282-1288.
577
(17) Bejgarn, S.; MacLeod, M.; Bogdal, C.; Breitholtz, M. Toxicity of leachate from
578
weathering plastics: An exploratory screening study with Nitocra spinipes. Chemosphere
579
2015, 132, 114-119.
580
(18) Suhrhoff, T.J.; Scholz-Böttcher, B.M. Qualitative impact of salinity, UV radiation
581
and turbulence on leaching of organic plastic additives from four common plastics-A lab
582
experiment. Mar. Pollut. Bull. 2016, 102 (1), 84-94.
583
(19) Chen, M.; Ike, M.; Fujita, M. Acute toxicity, mutagenicity, and estrogenicity of 26
ACS Paragon Plus Environment
Page 26 of 37
Page 27 of 37
Environmental Science & Technology
584
bisphenol-A and other bisphenols. Environ. Toxicol. 2002, 17, 80-86.
585
(20) Wang, Q.; Wei, W.; Gong, Y.; Yu, Q.; Li, Q.; Sun, J.; Yuan, Z. Technologies for
586
reducing sludge production in wastewater treatment plants: state of the art. Sci. Total
587
Environ. 2017, 587, 510-521.
588
(21) Dai, X.; Hu, C.; Zhang, D.; Dai, L.; Duan, N. Impact of a high ammonia-
589
ammonium-pH system on methane-producing archaea and sulfate-reducing bacteria in
590
mesophilic anaerobic digestion. Bioresource Technol. 2017, 245, 598-605.
591
(22) Wei, W.; Zhou, X.; Wang, D.; Sun, J.; Wang, Q. Free ammonia pre-treatment of
592
secondary sludge significantly increases anaerobic methane production. Water Res. 2017,
593
118, 12-19.
594
(23) Li X, Chen L, Mei Q, Dong, B.; Dai, X.; Ding, G.; Zeng, E.Y. Microplastics in
595
sewage sludge from the wastewater treatment plants in China. Water Res. 2018, 142, 75-
596
85.
597
(24) Wang, Q.; Ye, L.; Jiang, G.; Jensen, P.; Batstone, D.; Yuan, Z. Free nitrous acid
598
(FNA)-Based pre-treatment enhances methane production from waste activated sludge.
599
Environ. Sci. Technol. 2013, 47 (20), 11897-11904.
600
(25) Metcalf; Eddy. Wastewater Engineering: Treatment and Reuse. McGraw- Hill Inc.
601
2003.
602
(26) Batstone, D.J.; Tait, S.; Starrenburg, D. Estimation of hydrolysis parameters in full-
603
scale anaerobic digesters. Biotechnol. Bioeng. 2009, 102 (5), 1513-1520.
604
(27) Mu, H.; Chen, Y. Long-term effect of ZnO nanoparticles on waste activated sludge
605
anaerobic digestion. Water Res. 2011, 45 (17), 5612-5620.
606
(28) APHA. Standard Methods for Water and Wastewater Examination. American Public 27
ACS Paragon Plus Environment
Environmental Science & Technology
607
Health Association, Washington, DC. 2005.
608
(29) Smith, P.K.; Krohn, R.I.; Hermanson, G.T.; Mallia, A.K.; Gartner, F.H.; Provenzano,
609
M.D.; Fujimoto, E.K.; Goeke, N.M.; Olson, B.J.; Klenk, D.C. Measurement of protein
610
using bicinchoninic acid. Anal. Biochem. 1985, 150 (1), 76-85.
611
(30) Raunkjaer, K.; Hvitved-Jacobsen, T.; Nielsen, P.H. Measurement of pools of protein,
612
carbohydrate and lipid in domestic wastewater. Water Res. 1994, 28 (2), 251-262.
613
(31) Lu, L.; Xing, D.; Liu, B.; Ren, N. Enhanced hydrogen production from waste
614
activated sludge by cascade utilization of organic matter in microbial electrolysis cells.
615
Water Res. 2012, 46 (4), 1015-1026.
616
(32) Wei, W.; Wang, Q.; Zhang, L.; Laloo, A.; Duan, H.; Batstone, D.J.; Yuan, Z. Free
617
nitrous acid pre-treatment of waste activated sludge enhances volatile solids destruction
618
and improves sludge dewaterability in continuous anaerobic digestion. Water Res. 2018,
619
130, 13-19.
620
(33) Jimenez, J.; Vedrenne, F.; Denis, C.; Mottet, A.; De´ le´ ris, S.; Steyer, J.-P.; Cacho
621
Rivero, J.A. A statistical comparison of protein and carbohydrate characterisation
622
methodology applied on sewage sludge samples. Water Res. 2012, 47 (5), 1751-1762.
623
(34) Ariesyady, H.D.; Ito, T.; Okabe, S. Functional bacterial and archaeal community
624
structures of major trophic groups in a full-scale anaerobic sludge digester. Water Res.
625
2007, 41 (7), 1554-1568.
626
(35) Zheng, X.; Su, Y.L.; Li, X.; Xiao, N.D.; Wang, D.B.; Chen, Y.G. Pyrosequencing
627
reveals the key microorganisms iInvolved in sludge alkaline fermentation for efficient
628
short-chain fatty acids production. Environ. Sci. Technol. 2013, 47 (9), 4262-4268.
28
ACS Paragon Plus Environment
Page 28 of 37
Page 29 of 37
Environmental Science & Technology
629
(36) Wang, Y.; Zhao, J.; Wang, D.; Liu, Y.; Wang, Q.; Ni, B.J.; Chen, F.; Yang, Q.; Li,
630
X.; Zeng, G.; Yuan, Z. Free nitrous acid promotes hydrogen production from dark
631
fermentation of waste activated sludge. Water Res. 2018, 145, 113-124.
632
(37) Luo, J.; Chen, Y.; Feng, L. Polycyclic aromatic hydrocarbon affects acetic acid
633
production during anaerobic fermentation of waste activated sludge by altering activity
634
and viability of acetogen. Environ. Sci. Technol. 2016, 50 (13), 6921-6929.
635
(38) Cydzik-Kwiatkowska, A.; Bernat K, Zielińska M, Bułkowska, K.; Wojnowska-
636
Baryła, I. Aerobic granular sludge for bisphenol A (BPA) removal from wastewater. Int.
637
Biodeter. Biodegr. 2017, 122, 1-11.
638
(39) Zhang, C.; Qin, Y.; Xu, Q.; Liu, X.; Liu, Y.; Ni, B.J.; Yang, Q.; Wang, D.; Li, X.;
639
Wang, Q. Free ammonia-based pretreatment promotes short-chain fatty acid production
640
from waste activated sludge. ACS Sustain. Chem. Eng. 2018, 6 (7), 9120-9129.
641
(40) e Silva, P.P.G.; Nobre, C.R.; Resaffe, P.; Pereira, C.D.S.; Gusmao, F. Leachate from
642
microplastics impairs larval development in brown mussels. Water Res. 2016, 106, 364-
643
370.
644
(41) Li, K.; Wei, D.; Zhang, G.; Shi, L.; Wang, Y.; Wang, B.; Wang, X.; Du, B.; Wei, Q.
645
Toxicity of bisphenol A to aerobic granular sludge in sequencing batch reactors. J. Mol.
646
Liq. 2015, 209, 284-288.
647
(42) Yoshida, S.; Hiraga, K.; Takehana, T.; Taniguchi, I.; Yamaji, H.; Maeda, Y.;
648
Toyohara, K.; Miyamoto, K.; Kimura, Y.; Oda, K. A bacterium that degrades and
649
assimilates poly(ethylene terephthalate). Science 2016, 351(6278), 1196-1199.
650
(43) Yang, Y.; Yang, J.; Wu, W.M.; Zhao, J.; Song, Y.; Gao, L.; Yang, R.; Jiang, L.
651
Biodegradation and mineralization of polystyrene by plastic-eating mealworms: Part 2. 29
ACS Paragon Plus Environment
Environmental Science & Technology
652
Role of gut microorganisms. Environ. Sci. Technol. 2015, 49(20), 12087-12093.
653
(44) Mahon, A.M.; O’connell, B.; Healy, M.G.; O’connor, I.; Officer, R.; Nash, R.;
654
Morrison, L. Microplastics in sewage sludge: effects of treatment. Environ. Sci. Technol.
655
2016, 51(2), 810-818.
30
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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.
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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.
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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
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Y0 (%)
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Environmental Science & Technology
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
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F420
Environmental Science & Technology
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.
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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.
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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
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Environmental Science & Technology
(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.
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ACS Paragon Plus Environment
Euryarchaeota