Methane oxidation coupled to perchlorate reduction in a membrane biofilm batch reactor

Science of the Total Environment 667 (2019) 9–15

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Methane oxidation coupled to perchlorate reduction in a membrane biofilm batch reactor Pan-Long Lv a,b,c, Ling-Dong Shi a,b,c, Zhen Wang a,b,c, Bruce Rittmann d, He-Ping Zhao a,b,c,⁎ a

College of Environmental and Resource Science, Zhejiang University, Hangzhou, China Zhejiang Province Key Lab Water Pollut Control & Envi, Zhejiang University, Hangzhou, Zhejiang, China MOE Key Lab of Environmental Remediation and Ecosystem Health, College of Environmental and Resource Science, Zhejiang University, Hangzhou 310058, China d Biodesign Swette Center for Environmental Biotechnology, Arizona State University, P.O. Box 875701, Tempe, AZ 85287-5701, USA b c

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• Perchlorate was reduced using CH4 as the sole electron donor and carbon source. • The AOM relevant archaea, Methanosarcina was enriched in MBBR. • Archaea and bacteria synergistically complete the MO-PR.

a r t i c l e

i n f o

Article history: Received 3 December 2018 Received in revised form 21 February 2019 Accepted 21 February 2019 Available online 23 February 2019 Editor: Paola Verlicchi Keywords: Methane oxidation Perchlorate reduction Membrane-biofilm batch reactor Archaea Bacteria

a b s t r a c t A specially designed CH4-based membrane biofilm batch reactor (MBBR) was applied to investigate anaerobic methane oxidation coupled to perchlorate reduction (AnMO-PR). The 0.21 mM ClO4− added in the first stage of operation was completely reduced in 28 days, 0.40 mM ClO4− was reduced within 23 days in stage 2, and 0.56 mM of ClO4− was reduced within 30 days in stage 3. Although some chlorate (ClO3−) accumulated, the recovery of Cl− was over 92%. Illumina sequencing of the 16S rRNA gene documented that the bacterial community was mainly composed by perchlorate-reducing bacteria (PRB), methanotrophic bacteria, and archaea. Realtime quantitative PCR showed the archaeal 16S rRNA and mcrA genes increased as more ClO4− was reduced, and the predominant archaea belonged to Methanosarcina mazei, which is related to ANME-3, an archaeon able to perform reverse methanogenesis. Several pieces of evidence support that ClO4− reduction by the MBBR biofilm occurred via a synergism between Methanosarcina and PRB: Methanosarcina oxidized methane through reverse methanogesis and provided electron donor for PRB to reduce ClO4−. Because methanotrophs were present, we cannot rule out that they also were involved in AnMO-PR if they received O2 generated by disproportionation of ClO2− from the PRB. © 2019 Elsevier B.V. All rights reserved.

1. Introduction ⁎ Corresponding author at: College of Environmental and Resource Science, Zhejiang University, Hangzhou, China. E-mail address: [email protected] (H.-P. Zhao).

https://doi.org/10.1016/j.scitotenv.2019.02.330 0048-9697/© 2019 Elsevier B.V. All rights reserved.

Anaerobic methane oxidation (AnMO) has drawn interest because of its potential of controlling the potent greenhouse gas methane (Michaelis et al., 2002; Joye et al., 2004; Wang et al., 2017a) and its

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potential importance in global nutrient cycles of carbon, sulfur, and nitrogen (Hinrichs et al., 1999; Anantharaman et al., 2018; Raghoebarsing et al., 2006). Devol and Ahmed (1981) first discovered AnMO, which was responsible for sulfate reduction in marine sediments. Subsequent research found AnMO coupled to denitrification (AnMO-D) (Raghoebarsing et al., 2006; Knittel and Boetius, 2009; Haroon et al., 2013), and the researchers proposed that AnMO-D was performed through a “reverse methanogenesis” pathway: Anaerobic methanotrophic archaea (ANME) generated electrons via reverse methanogenesis to reduce nitrate to nitrite. A contrasting explanation was offered by Ettwig et al. (2010), who reported that nitritedependent AnMO was performed by M. oxyfera through a new “intraaerobic” pathway: NO2− is reduced to NO by nitrite reductase (nirSJF), NO is disproportionated (by an unknown enzyme) to N2 and O2, and M. oxyfera oxidize methane intracellularly by a membrane-bound methane mono‑oxygenase (pmoCAB). Besides sulfate, nitrate, and nitrite, AnMO has been linked to the reduction of metal ions. Beal et al. (2009) reported that manganese (birnessite) and iron (ferrihydrite) could serve as electron accepters for AnMO by microorganisms from marine methane-seep sediment in the Eel River Basin in California. Ettwig et al. (2016) confirmed that archaea of the order Methanosarcinales, related to “Candidatus Methanoperedens nitroreducens,” coupled AnMO to the reduction of environmentally relevant forms of Fe3+ in a freshwater enrichment culture (AnMO-IR). Lu et al. (2016) found that Cr(VI) was reduced by an enriched AnMO culture with CH4 as the sole electron donor (AnMOCrR). Another electron acceptor is perchlorate (ClO4−), and our group achieved complete ClO4− reduction in a methane-based membrane biofilm reactor (MBfR) (Luo et al., 2015) in which AnMO coupled to perchlorate reduction (AnMO-PR) could have occurred through two pathways. In a one-microorganism intra-aerobic pathway, AnMO-PR is conducted by one bacterium that stepwise reduces ClO4− to ClO2−, and ClO2− is then intracellularly disproportionated to Cl− and O2, with O2 utilized to oxidize methane by mono‑oxygenation. In an alternate two-microorganism pathway (Miller et al., 2014), methane oxidation is coupled to perchlorate reduction based on exchange of O2 between PRB, which disproportionate ClO2− to produce O2 that was excreted, and methanotrophs, which use the trace O2 to activate methane oxidation via mono‑oxygenation. In the MBfR study, exogenous O2 entered the MBfR because of its continuous-flow operation (Luo et al., 2015). The entry of exogenous O2 made it impossible to conclude which of the two pathways was acting in the CH4-fed perchlorate-reducing MBfR. To eliminate this ambiguity, we designed a membrane biofilm batch reactor (MBBR) to preclude O2 entry. The MBBR had a bigger liquid volume and airtight connections, and batch operation precluded O2 entry in the feed medium. Once AnMO-PR was established, we increased the loading rate of ClO4− to enrich perchlorate-reducing biomass and to explore the reactor's perchlorate-removal capacity. We used quantitative realtime PCR (qPCR) to evaluate the abundance of key functional genes and high-throughput sequencing to define the structure and function of the biofilm. We placed special focus on the roles of archaea that can do methane oxidation due to its significance in AnMO (Wang et al., 2017b).

Fig. 1. Schematic of the bench-scale membrane biofilm batch reactor (MBBR).

other end was sealed (Ontiveros-Valencia et al., 2014). All fibers were ~14 cm long, and the total membrane surface was ~76 cm2. CH4 was supplied through Norprene tubing at a pressure of 15 psig (2.07 atm) for all experiments. To ensure air-tightness, all joints were sealed with an epoxy adhesive (EC 2216, 3 M Company, USA). The volume of MBBR was 1000 mL and the liquid phase was 700 mL. The MBBR was mixed with a magnetic stirrer bar (HJ-1, Xinbao, Ltd., China) to maintain homogeneous liquid contents and increase mass transfer. 2.2. Start up and operation

2. Materials and methods

We inoculated the MBBR with 50 mL of culture from an operating MBfR able to reduce perchlorate when fed with methane (Luo et al., 2015). We enriched the community by adding a mineral salt medium (described below) containing 0.21 mM ClO4−. The mineral salt medium formulation was (per L of demineralized water): CaCl2 1 mg, NH4Cl 20 mg, NaHCO3 0.3 g, MgSO4·7H2O 5 mg, KH2PO4 0.2 g, Na2HPO4·12H2O 0.4 g, 1 mL acid trace element solution (HCl 100 mM, 2.085 g of FeSO4·7H2O, 68 mg of ZnSO4·7H2O, 14 mg of H3BO3, 120 mg of CoCl2·6H2O, 500 mg of MnCl2·4H2O, 320 mg of CuSO4, 95 mg of NiCl2·6H2O per liter), and 1 mL alkaline trace element solution (NaOH 10 mM, 67 mg of SeO2, 50 mg of Na2WO4·2H2O, 242 mg of Na2MoO4·2H2O per liter). The medium was sparged with argon gas (Ar) for 30 min to establish an anaerobic condition. The MBBR was operated in batch mode at 35 ± 1 °C. After all perchlorate was removed (the end of stage 1), we re-fed the reactor with ClO4− at a concentration of 0.40 mM to start stage 2 and then 0.56 mM to start stage 3. Dissolved O2 in the medium was measured with a dissolved oxygen probe (Starter, model 300D, Ohaus Instruments Company, Germany); its concentration was ~30 μg/L at the beginning of the experiment.

2.1. MBBR setup

2.3. Chemical analyses

A batch MBBR system, illustrated in Fig. 1, consisting of two sets of 32 composite hollow fibers (280-μm outer diameter, 180-μm inner diameter, Model MHF-200TL, Mitsubishi, Ltd., Japan). One set received CH4 gas from both ends. All these fibers were glued together and sealed within Norprene tubing. The second set served as biofilm sampling coupons (Zhao et al., 2011); one end was glued in Norprene tubing, and the

We took liquid samples from the MBBR with 2-mL syringes and filtered them immediately through a 0.22-μm membrane filter (LC + PVDF membrane, Shanghai Xinya, China). ClO4− was measured using ion chromatography (DIONEX ICS-1000, USA) using an AS 16 column with a gradient concentration from 5 to 40 mM KOH and 1 mL/min flow rate, metabolites ClO3−, ClO2−, and Cl− were detected

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using an AS 19 column with the eluent concentration of 20 mM KOH and a 1 mL/min flow rate. According to the signal-to-noise ratio of the ion chromatography, the detection limits of ClO4−, ClO3−, ClO2− were 0.1 mg/L, and of Cl−, which has more sensitive responsiveness, the detection limit was 0.01 mg/L. O2 in the headspace of the MBBR was measured with gas chromatography (Agilent Technologies GC system, model 7890A, Agilent Technologies Inc., U.S.A), and the gas-phase concentration was used to calculate the dissolved O2 concentration in liquid phase according to Henry's Law (Campbell and Brand, 1998). pH values, measured by a pH meter (Seven Easy, Mettler Toledo, Switzerland), were between 7.0 and 7.7 for all stages. 2.4. Biofilm sampling, imaging, and DNA extraction Biofilm samples were collected at the end of each stage in a glove box to avoid any O2 permeation. We cut off one ~10-cm section from a coupon fiber and then sealed the remaining fiber by tying the end in a knot. DNA of biofilm samples was extracted using the Power Soil® DNA Isolation Kit (Qiagen, USA) as described by Zhao et al. (2013). At the end of experiment, a 10-cm section of a coupon fiber was cut into smaller pieces and fixed with 2.5% glutaraldehyde in phosphatebuffered saline (PBS) for 12 h. Samples were then dehydrated three times with gradient concentrations of ethanol for scanning electron microscope (SEM, Hitachi TM-1000, Japan). Other samples were immersed in absolute embedding agent overnight and then trimmed into 70–90nm-thick sections for transmission electron microscopy (TEM, Hitachi H-7650, Japan). 2.5. Archaeal 16S rRNA gene clone library and Illumina sequencing of bacterial 16S rRNA gene Primers ARCH 344F (5′-ACGGGGYGCAGCAGGCGCGA-3′) and 915R (5′-GTGCTCCCCCGCCAATTCCT-3′) were used to amplify the archaeal 16S rRNA gene. PCR amplification was performed using the following program: 94 °C for 5 min, followed by 30 cycles consisting of 94 °C for 30 s, 60 °C for 30 s, 72 °C for 40 s, and a final extension period of 72 °C for 10 min. The PCR products were then purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA). The purified amplicons were cloned in competent E. coli Trans-T1 by using the pEASY-T1 cloning vector (TransGen Biotech, Beijing, China). 24 clones were randomly picked from solid LB medium containing 100 μg/mL ampicillin and grown overnight in liquid LB medium for later sequencing. Phylogenetic analysis was based on aligned homologous nucleotides as reported by Suau et al. (1999). The sequences were aligned using ClustalX (Thompson et al., 1997) and guided tree files were utilized to create phylogenetic trees. The trees were constructed using the neighbor-joining method implemented in MEGA 6.0 (Tamura et al., 2007). Primers 341F (5′-CCTAYGGGRBGCASCAG-3′ and 806R (5′-GGAC TACNNGGGTATCTAAT-3′) were used to amplify a 466-bp fragment of the bacterial 16S rRNA gene flanking the V3 and V4 regions (Caporaso et al., 2010a). The purified amplicons were sent to Novogene company (Beijing, China) to process Illumina MiSeq sequencing with standard protocols and the data was processed using QIIME (version 1.7.0) pipeline (Caporaso et al., 2010b).

performed qPCR as previously described by Zhao et al. (2011). Negative controls included water instead of template DNA in the PCR reaction mixture. Triplicate PCR reactions were performed for all samples and negative controls. The slopes of the plasmid standard curves and efficiency values for quantification by qPCR are shown in Table S1, too. 3. Results and discussion 3.1. Perchlorate reduction in MBBR fed with CH4 as the sole electron donor Fig. 2 shows ClO4− reduction in the MBBR fed with CH4 as the sole electron donor and carbon source throughout the whole experiments. CH4 was not rate-limiting, since the actual CH4 fluxes were far higher than the maximum theoretical CH4 demand in each stage (Table S2). 0.21 mM of ClO4− was completely reduced in 28 days in stage 1, while ~0.40 and ~0.56 mM of ClO4− were fully reduced within 23 and 31 days in stage 2 and 3, respectively. The reduction rates of ClO4− were 0.94, 2.30, and 2.34 mM/m2-d in stage1, 2, and 3. The larger reduction rates after stage 1 may be explained by a combination of microbial adaptation in first stage and enrichment of perchlorate reducing bacteria in the following two stages. ClO3− accumulated up to ~0.23 mM in stage 1, but was fully reduced about 7 days after ClO4− was completely consumed. When higher concentrations of ClO4− were introduced in stages 2 and 3, the accumulation of ClO3− was greater (up to 0.27 mM at day 66), but it was completely removed by day 108. Dudley et al. (2008) also found significant amounts of chlorate accumulation by Dechlorosoma sp. HCAP-C during high loading of perchlorate reduction, and they ascribed it to competitive inhibition of the (per)chlorate reductase. The final reduction product, Cl−, constantly increased in parallel to ClO4− and ClO3− reductions, and Cl− reached 0.88 mM at the end of experiment, which was 92% of the theoretical Cl− yield from full reductive dechlorination of all input ClO4− (Fig. S1). No ClO2− was detected throughout the experiment in the MBBR. The total theoretical yield of disproportionated O2 was ~1.17 mM, but O2 was not detected (the detection limit of O2 was 8 μg/L) from day 2 to the end of the experiment in the MBBR. Two sinks could reduce disproportionated O2: first, most O2 could be utilized by perchloratereducing bacteria inside the cell as a means to avoid O2 toxicity (Dudley et al., 2008); second, a small amount of O2 released from cells could have been consumed quickly by methanotrophic or other heterotrophic bacteria in the MBBR. 3.2. The enrichment of functional genes Fig. 3 shows gene copies for pcrA, mcrA, and the 16S rRNA gene of archaea in all stages. Because the (per)chlorate-reductase gene is general for many perchlorate-reducing bacteria (PRB), the pcrA gene can be used to represent PRB. From stage 1 to stage 3, the copy number of the pcrA gene increased from 1.5 × 1010 to 2.2 × 1011 copies/m2 fiber

2.6. Quantification of pcrA, mcrA, and archaeal 16S rRNA genes The plasmids containing target fragments were constructed and used to generate standard curves based on serial dilutions containing series of target gene copies. For plasmid construction, we used primers that target the perchlorate reductase gene (pcrA), methyl coenzyme M reductase gene (mcrA), and the 16S rRNA gene for archaea. The primer names, primer sequences, and PCR conditions for each target gene are in Table S1. We used SYBR Premix Ex Taq Kit (Takara Bio Inc., Japan) and

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Fig. 2. ClO− 4 reduction in the CH4-MBBR through all stages.

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from 1.0 × 109 copies/m2 in stage 1 to 8.0 × 109 copies/m2 in stage 3, with the mcrA gene accounted for 14% and 33% of archaeal 16S rRNA gene abundance in stages 1 and 3, respectively. Xie et al. (2018) also reported that mcrA gene copies were correlated with the perchlorate flux in a methane-based membrane biofilm reactor. Zehnder and Brock (1980) first noted that some archaea, specifically anaerobic methanotrophic archaea (ANME), could do reverse methanogenesis, in which methane was activated by mcrA in the first step. Thus, the increase of the mcrA gene in the MBBR system is consistent with an increase in reverse methanogenesis, which could generate electron donors for the PRB to reduce ClO4−.

3.3. Phylogenetic analysis of archaea Fig. 3. Abundances of the functional genes pcrA and mcrA and the archaeal 16S rRNA gene through all stages. The corresponding values for the inoculum are 2.72 × 104, 3.80 × 104, and 6.72 × 103 copies/μL, respectively.

(14-fold increase), a trend that correlates well with the increase of the ClO4− loading (Table S3). De Long et al. (2012) also found that the copy number of pcrA genes was strongly correlated with perchlorate removal. The copy number of the archaeal 16S rRNA gene, which was 7.2 × 109 copies/m2 in stage 1, increased about 3 times higher, to 2.4 × 1010 copies/m2 in stage 3. The increase of the archaeal 16S rRNA gene demonstrates that enrichment of archaea mirrored the increase in AnMO-PR. Previous studies reported that archaea were enriched during nitrate and nitrite reduction in AnMO and likely were responsible for AnMO-D (Raghoebarsing et al., 2006; Welte et al., 2016). Lu et al. (2016) also found the copy number of the archaeal 16S rRNA gene increased during AnMO coupled to chromate (Cr(VI)) reduction after 210 days of incubation. The abundance of the mcrA gene, also used to represent archaea (by reverse methanogesis) (Hallam et al., 2003), increased

After constructing archaeal 16S rRNA gene clone libraries, we randomly picked clones for sequencing. 24 clones were successfully sequenced and analyzed using phylogenetic tree (GenBank accession: MH122735-MH122758). Fig. 4 shows that 17 of clones (71%) had sequences close to uncultured archaea (relatively far from ANME in phylogenetic tree), while the other 7 clones (29%) belonged to Methanosarcina, which is a versatile genus of methanogens able to produce methane using as substrates H2 and CO2, acetate, methylamines, and methanol. Methanosarcina is closely related to ANME-3, an archaean known for performing AnMO. ANME-3, clustered within the order Methanosarcinales, are reported to play essential roles in sulfateand manganese-dependent AnMO (Niemann et al., 2006; Losekann et al., 2007; Beal et al., 2009). Methanosarcina barkeri was reported to mediate methane oxidation and produce acetate as an oxidation product (Zehnder and Brock, 1979). Soo et al. (2016) authenticated that Methanosarcina acetivorans could grow anaerobically on methane in a pure culture, and it could convert methane into acetate instead of CO2 and H2. Recently, Luo et al. (2017) and Lai et al. (2018) reported that Methanosarcina was the dominant microorganism responsible for AnMO coupled to bromate and antimonate reductions.

Fig. 4. Phylogenetic relationships among the dominant archaeal 16S rRNA genes in the MBBR biofilm. The evolutionary history was inferred using the neighbor-joining method. The clones are highlighted in bold.

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Considering that O2 was not detected after day 2 in the MBBR system, we propose that AnMO was carried out by Methanosarcina following the “reverse methanogenesis” pathway, which generated electron donors (such as acetate and H2) for PRB to reduce ClO4−. 3.4. Evaluation of bacterial microbial community Fig. 5A shows the bacterial abundances at the class level of the biofilm for all stages, while Fig. 5B shows the relative abundances of the key bacterial genera. As the inoculum was from a micro-aerobic methane-fed perchlorate-reducing system, the bacterial community in the biofilm was mainly composed of two groups: PRB and methanotrophs. Denitratisoma (in the β-proteobacteria) accounted for about 25% of the total bacteria in stage 1, and they increased to ~ 30% in stages 2 and 3. Known to be denitrifiers, Denitratisoma are phylogenetically close to the PRB Azospira and Dechloromonas (Fig. S2), and both belong to the order of Rhodocyclales, which is important in communities exposed to nitrate and perchlorate (Hesselsoe et al., 2009). Since most denitrifiers are able to reduce ClO4− using either nitrate or (per)chlorate reductase (Coates and Achenbach, 2004; Nerenberg et al., 2006), Denitratisoma probably utilized ClO4− as its electron acceptor (Oren, 2014). Azospira (in the β-proteobacteria) also is a typical PRB that was present throughout the experiment. Methanotrophs Methylococcus, Methylomonas, and Methylocystis also were detected in all stages: Their abundance was ~ 27% in stage 1, but decreased to 22% and 20% in stage 2 and 3, respectively. It was obviously different from the case in MBfR system where methanotrophs were enriched in the early stages of the experiment (Chen et al., 2016; Zhong et al., 2017). Numerous researchers have shown that these genera are responsible for aerobic activation of CH4 (Eisentraeger et al., 2001; Kits et al., 2015; Lai et al., 2016). The decrease of their abundance may be due to the long-term incubation without exogenous oxygen in the MBBR. Though Zahn and Dispirito (1996) reported that Methylococcus could oxidize CH4 with trace O2 using its membrane-

Fig. 6. TEM images of microbial cells from hollow fibers. Typical Methanosarcina-like cells are circled.

associated methane mono‑oxygenase, Fu et al. (2017) reported the relative abundance of these genera decreased along with enrichment in a AnMO-D system without O2. We also analyzed biofilm samples at the end of experiment using TEM. As shown in Fig. 6-A and B, marked bacteria had shapes similar to Methanosarcina (Croese et al., 2013): Aggregates of irregular sphere-like cells with a diameter of 2–4 μm (Ganzert et al., 2014). This reinforces our hypothesis that these Methanosarcina–like cells, which were distributed throughout the inner layer of the biofilm, were responsible for AnMO. 4. Conclusions

Fig. 5. Relative abundances of high-throughput bacterial sequences from the biofilms at the levels of class (A) and genus (B) for all stages.

Using an anaerobic MBBR, we documented AnMO-PR. Fig. 7A illustrates the synergistic pathway that we propose allowed AnMO-PR. Multiple lines of evidence support that the archaeon Methenosarcina was responsible for AnMO via reverse methanogenesis. Quantitative realtime PCR documented the archaeal 16S rRNA genes, AnMO-related mcrA genes were enriched after the batch tests, the predominant archaeon in the biofilm was Methanosarcina, and Methanosarcina-like cells were evident from TEM. The main PRB appeared to be Denitratisoma and Azospira, which were increasingly enriched in the biofilm. Carrying out reverse methanogenesis, the archaeon Methanosarcina oxidized methane anaerobically and provided electron donor (such as acetate or H2) for the PRB to reduce perchlorate. Bacterial methanotrophs—Methylococcus, Methylomonas, and Methylocystis—were present in the inoculum, but decreased as AnMOPR increased its rate. We cannot rule out that they also were involved in AnMO-PR, as shown in Fig. 7B. They must have received from the PRB O2 generated by disproportionation of ClO2−, and the PRB must have received electron donors from the methanogens. Since we never

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Fig. 7. The proposed pathways of AnMO-PR in the CH4-MBBR. (A) Methanosarcina carry out reverse methanogenesis and provide electron donor (acetate, H2) to the PRB who reduce ClO− 4. (B) Aerobic methanotrophs do aerobic activation of CH4 using O2 generated by disproportionation of ClO− 2 by the PRB and electron donor from the methanogens.

detected O2, saw a decline in the methanotrophs over time, and saw increases in all markers of Methanosarcina, the pathway in Fig. 7A seems more likely. Acknowledgments Authors greatly thank the “The National Key Technology R&D Program (2017ZX07206-002)”, the “National Natural Science Foundation of China (Grant No. 21577123, 21377109)”, and the “Natural Science Funds for Distinguished Young Scholar of Zhejiang Province (LR17B070001)” for their financial support. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.scitotenv.2019.02.330. References Anantharaman, K., Hausmann, B., Jungbluth, S.P., Kantor, R.S., Lavy, A., Warren, L.A., 2018. Expanded diversity of microbial groups that shape the dissimilatory sulfur cycle. ISME J. 12, 1715–1728. Beal, E.J., House, C.H., Orphan, V.J., 2009. Manganese- and iron- dependent marine methane oxidation. Science 325 (5937), 184–187.

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