Electron shuttles enhance anaerobic oxidation of methane coupled to iron(III) reduction

Science of the Total Environment 688 (2019) 664–672

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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv

Electron shuttles enhance anaerobic oxidation of methane coupled to iron(III) reduction Qiuxiang He a,1, Linpeng Yu a,⁎,1, Jibing Li b, Dan He a, Xixi Cai a, Shungui Zhou a a b

Fujian Provincial Key Laboratory of Soil Environmental Health and Regulation, College of Resources and Environment, Fujian Agriculture and Forestry University, Fuzhou 350002, China Guangzhou Institute of Geochemistry, Chinese Academy of Sciences, Guangzhou 510640, China

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

• Ferrihydrite was reduced to siderite using methane as microbial electron donor. • Methanobacterium conducted partial oxidation of methane but did not assimilate it. • Electron shuttles enhanced both AOM and ferrihydrite reduction. • The enhancement extents were linearly related with the ETCs of electron shuttles.

a r t i c l e

i n f o

Article history: Received 20 May 2019 Received in revised form 19 June 2019 Accepted 19 June 2019 Available online 20 June 2019 Editor: Frederic Coulon Keywords: Anaerobic methane oxidation Electron shuttle Iron reduction DNA-based stable isotope probing Metagenomics

a b s t r a c t Anaerobic oxidation of methane (AOM) has recently been coupled with the reduction of insoluble electron acceptors such as iron minerals. However, effects of electron shuttles (ESs) on this process and the underlying coupling mechanisms remain not well understood. Here, we evaluated AOM-coupled ferrihydrite reduction by a mixed culture in the absence and presence of ESs. The results showed that ESs (AQS, flavin, HA and AQDS) significantly enhanced the rate (up to 7.4 times) of AOM-dependent ferrihydrite reduction compared with the control. The enhancements were linearly related with the electron transfer capacity of ESs. Illumina high-throughput sequencing and DNA-based stable isotope probing revealed that the AOM-coupled iron reduction depended on the syntrophic interaction of Methanobacterium and the partner bacteria. Methanobacterium as the dominant microorganism, did not assimilate methane into its biomasses. However, it played a crucial role in the partial oxidation of methane into an intermediate (i.e. propionate), which was then assimilated by the partner bacteria (e.g. Cellulomonas, Desulfovibrio, Actinotalea, etc.) for ferrihydrite reduction. This work suggests that ESs in natural environments can mitigate the methane emissions by facilitating the AOM process and biogeochemical cycles of iron. © 2019 Elsevier B.V. All rights reserved.

1. Introduction

⁎ Corresponding author. E-mail address: [email protected] (L. Yu). 1 These authors contributed equally to this work.

https://doi.org/10.1016/j.scitotenv.2019.06.299 0048-9697/© 2019 Elsevier B.V. All rights reserved.

Anaerobic oxidation of methane (AOM) catalyzed by microorganisms is one of the main processes that regulate the methane emission to the atmosphere (Bar-Or et al., 2017). Anaerobic methanotrophs (ANME), known as the first typical anaerobic methane-oxidizing

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archaea, can couple AOM to sulfate reduction when sulfate-reducing bacteria is present (Hoehler et al., 1994; Knittel and Boetius, 2009). Sulfate was first deemed as the electron acceptor for AOM since 1976 (Scheller et al., 2016). However, recent studies have showed that AOM can be coupled to the reduction of a variety of environmentally wide3+ − spread electron acceptors such as NO− and Mn4+ (Haroon 3 , NO2 , Fe et al., 2013; Ettwig et al., 2010; Beal et al., 2009; Ettwig et al., 2016; Shi et al., 2016). Iron is the fourth most widespread component in the earth's crust. AOM-driven iron oxide reduction is also a thermodynamically favorable process (Cai et al., 2018). So AOM coupled to iron oxide reduction has received increasing attentions than other electron acceptors. For example, Cai et al. (2018) have found a novel archaeon within the Candidatus Methanoperedenaceae family could conduct AOM-coupled ferrihydrite reduction. They hypothesized that the “reverse methanogenesis” pathway and multi-heme c-type cytochromes were the main approach for the novel archaeon to conduct dissimilatory Fe(III) reduction. Bar-Or et al. (2017) suggested a new route for AOM-coupled iron reduction in which methanogens may generate AOM intermediates via “reverse methanogenesis” pathway, which were transferred to methanotrophic bacteria for Fe(III) reduction. Similarly, Fu et al. (2016) explored a coculture of S. oneidensis MR-1 and denitrifying anaerobic methane oxidation (DAMO) microbes for ferrihydrite reduction with methane as an electron donor. They showed that the co-culture system could result in more ferrihydrite reduction than those for DAMO microbes and Shewanella alone. However, the specific DAMO microbes that catalyze AOM and the coupling mechanism between DAMO microbes and Shewanella remain enigmatic. Although insoluble iron oxides are widespread in natural environments, the bioavailability of iron oxides poses an important restriction on iron reduction. Electron shuttles (ESs) are redox-active substances that have been widely used to mediate iron reduction and electricity generation. ESs can facilitate the long-range electron transfer from dissimilatory iron-reducing bacteria to insoluble iron oxides (Wu et al., 2014; Hernandez and Newman, 2001). ESs have also been shown to be an electron sink of AOM. For example, Scheller et al. (2016) proved that artificial ESs (e.g. AQDS) could serve as electron accepters of AOM when decoupling AOM from sulfate reduction. Natural organic matter (i.e. humic substances) was reported to work as electron accepters in promoting AOM (Valenzuela et al., 2017). Moreover, they reported that humic substances functioned as ESs to stimulate AOM-coupled iron reduction (Valenzuela et al., 2019). Previously, Wu et al. (2014) and Li et al. (2014) explored the effects of various ESs on the electricity generation and iron reduction rates, and a linear relationship between iron reduction rates and the electron transfer capacity (ETC) of ESs was presented. This suggests that redox potential (E0′) and ETC of ESs are important parameters that may affect the mediation efficiencies of ESs. However, the effect of ESs with different redox properties (E0′ and ETC) on AOM-coupled Fe(III) reduction remain not well understood. In this work, we investigated systematically how ESs would affect the process of AOM-coupled ferrihydrite reduction by mixed cultures. DNA-based stable isotope probing (SIP) technique and metagenomics were used to probe functional microorganisms responsible for AOMcoupled ferrihydrite reduction. The coupling mechanisms of AOM and ferrihydrite reduction between the AOM microorganisms were interpreted. 2. Materials and methods 2.1. Materials AQDS (9,10-anthraquinone-2,6-disulfonate) was purchased from J&K Scientific Ltd. Flavin, humic acid (HA), 9,10-anthraquinone-2-sulfonic acid (AQS) and resazurin (RZ) were purchased from Aladdin. Fulvic acid (FA) and other chemicals were of analytical grade and purchased from Macklin Chemical Co., China. Stock solutions (500 mM) of

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each ES were prepared in deionized water and filter-sterilized (0.22 μm). 12CH4 (99.99%) and 13C-labeled methane (99% 13C atom) were purchased from Sigma-Aldrich. Ferrihydrite was synthesized according to a previously described method (McCormick and Adriaens, 2004). 2.2. Experimental setup Batch experiments were conducted in serum bottles that contained 80 mL of culture medium and a 38-mL headspace. The medium contained (per L): KH2PO4, 0.075 g; MgCl2·7H2O, 0.165 g; CaCl2·2H2O, 0.3 g; 1 mLof acidic trace element solution; 1 mL of alkaline trace element solution. The alkaline trace element solution (10 mM NaOH) was comprised of (per L): SeO2, 0.067 g; Na2WO4·2H2O, 0.050 g; and Na2MoO4, 0.242 g. The acidic trace element solution (100 mM HCl) included (per L): MnCl2·4H2O, 0.5 g; ZnSO4·7H2O, 0.068 g; FeSO4·7H2O, 2.085 g; CuSO4, 0.32 g; CoCl2·6H2O, 0.12 g; H3BO3, 0.014 g; and NiCl2·6H2O, 0.095 g (Ettwig et al., 2009; Yu et al., 2019). Ferrihydrite was provided to the culture medium at an initial concentration of 10 mM (iron atom). All culture mediums in the serum bottles were purged with O2-free N2 for 45 min and then sealed with gas-tight rubber stoppers. The AOM inoculum was obtained from microbial fuel cells in our previous study (Yu et al., 2019). Cells (4 mL, OD600 = 0.3) were centrifuged, washed three times, and then transferred into the serum bottle. Eight treatments were conducted in triplicates, including the experimental treatment with CH4, six treatments amended with each ES (0.05 mM) and the CH4-free control (Table S1). 10 mL of CH4 was injected into the headspace to act as the electron donor. To validate AOM and investigate the effects of ESs on AOM, 10 mL of 13CH4 was incubated with the inoculum in the presence and absence of AQS (0.05 mM), respectively. All the serum bottles were shaken at 200 rpm and 37 °C in the dark. 2.3. High-throughput sequencing After 40-day incubation, DNA samples were extracted using a soil DNA isolation kit (MoBio Laboratories, Solana Beach, CA) and amplified with a pair of primers 341b4-F (5’-CTAYGGRRBGCWGCAG-3′) and 806R (5′-GGACTACNNGGGTATCTAAT-3′) (Lu et al., 2015). Reaction mixtures for PCR contained: 10 ng of template DNA, 0.8 μL of each primer (5 μM), 4 μL dNTPs (2.5 mM), 0.4 μL of Fastpfu polymerase, 4 μL of Fastpfu buffer, 0.2 μL of bovine serum albumin (BSA) and ultrapure H2O to complement to 20 μL. The PCR procedure was: initial denaturation at 95 °C for 3 min, followed by 32 cycles of 95 °C for 30 s, 55 °C for 30 s and 72 °C for 45 s; final extension at 72 °C for 10 min. The PCR products were sequenced on the Illumina MiSeq platform (Shanghai Majirbio Technology Co., Ltd., China). The operational taxonomic units (OTU) of the identified 16S rRNA gene sequences were analyzed by the Majorbio I-Sanger Cloud online platform (www.i-sanger.com), using RDP classifier Bayesian Algorithms (3% difference of the sequence as classification standards). 2.4. DNA-based stable isotope probing (DNA-SIP) To identify which microorganisms are responsible for AOM-coupled iron reduction, an experimental microcosm with 13CH4 (10 mL) and a control microcosm with 12CH4 (10 mL) were incubated without electron shuttles under identical conditions (the same amount of inoculum, ferrihydrite concentration, temperature, etc.), respectively. After incubation for 40 days, all the cultures (80 mL) from the microcosms of 13 CH4 and 12CH4 were harvested via centrifugation (8000 rpm for 10 min). DNA from the two treatments (designated as 13CH4-DNA and 12 CH4-DNA, respectively) were extracted with a soil DNA isolation kit (MoBio Laboratories, Solana Beach, CA). The DNA concentrations were determined by a ND-2000 UV–vis spectrophotometer (NanoDrop Technologies, Wilmington, DE). The same amount of DNA samples (3 μg) were loaded onto a CsCl solution in Tris-EDTA buffer (pH 8.0, final

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buoyant density (BD) 1.77 g/mL). Then the mixed medium was transferred to Quick-Seal polyallomer tubes (Beckman Coulter, Pasadena, CA) and centrifuged (Beckman Optima XPN-100) at 20 °C, 53000 g for 48 h. DNA was then fractioned and collected with a fraction recovery system (Beckman Coulter). Each fraction of DNA was approximately 320 μL. The BD was measured using a refractive index meter (AR200, Leica Microsystems Inc.). Afterward, the fractionized DNA was purified to removal the CsCl using our previously described method (Li et al., 2017). The DNA concentrations of each fraction are shown in Fig. S1. Microbial community structures of DNA fractions were analyzed by highthroughput sequencing as mentioned above. 2.5. Electrochemical measurement Chronoamperometry (CA) was used to determine the electron transfer capacities (ETC) of ESs with an electrochemical workstation (CHI 660D, CH Instrument Inc., China) according to Ye et al. (2018). The working electrode, reference electrode and counter electrode were graphite plate (1 × 2 cm), saturated calomel electrode (SCE) and platinum mesh, respectively. To measure the electron donor capacity (EDC) and electron accepting capacity (EAC) of ESs, CA measurements were performed in a phosphate buffer (0.1 M with 0.1 M KCl, pH = 7.0) at applied electrode potentials of +0.37 and −0.73 V, respectively. All solutions were purged with pure N2 gas (99.99%) for 30 min prior to use. The amount of ES added to the electrochemical cell at each time point was 50 μM. The EDC and EAC of ESs were obtained by integrating the oxidation and reduction peak areas in the chronoamperometry tests, respectively (Fig. S2) (Huang et al., 2010; Li et al., 2013). ETCs were calculated from the sum of EDC and EAC (Table S2). 2.6. Analytical methods Fluorescence in situ hybridization (FISH) was performed with our previously described method (Yu et al., 2019). After 40-day incubation with ferrihydrite and methane, the enrichment cultures (1 mL) were collected by centrifugation. Then the biomasses were resuspended, fixed, and hybridized with the general archaeal probe Ar915 and the general bacterial probe EUB338 (Raghoebarsing et al., 2006). Microorganisms were visualized under a confocal laser scanning microscope (Zeiss, LSM880, Germany). The methane concentration in the headspace was determined with a gas chromatography (SHIMADZU-GC 2014C, Japan). Nitrogen was used as carrier at a flow rate of 20 mL/min and the injection volume of gas samples was 100 μL. 13CO2 were measured with gas chromatography coupled to mass spectroscopy (Aglient, 7890A/5975C, USA) using our previously described methods (Yu et al., 2019). The AQH2DS concentration in the medium was measured at 450 nm on a spectrophotometer (Cervantes et al., 2000). Total Fe(II) in the samples were extracted with 0.5 M HCl overnight, mixed with ferrozine, and then measured at 562 nm on a spectrophotometer (Zhou et al., 2014a). 3. Results and discussion 3.1. AOM-coupled ferrihydrite reduction The reduction of ferrihydrite by the enrichment cultures with methane as the electron donor was tested in the absence of ESs. After a 35day incubation, 0.29 ± 0.03 mM of Fe (II) was produced from ferrihydrite (Fig. 1a). However, ferrihydrite was not reduced when CH4 was omitted in the system, suggesting the AOM-dependent ferrihydrite reduction (Fig. 1a). To validate the occurrence of AOM during ferrihydrite reduction, 13CH4 was used as the substrate and the production of 13CO2 were monitored in the presence and absence of inoculum. As shown in Fig. 1b, the amount of 13CO2 in the headspace increased slightly from 1.42 ± 0.04% at day 0 to 1.50 ± 0.15% at day 10, and then reached 8.05 ± 0.50% at day 30. This was close to that of a previous study

where a coculture of denitrifying AOM microbes and S. oneidensis MR1 produced approx. 5 ± 2% of 13CO2 from AOM-coupled ferrihydrite reduction after a 130-d operation (Fu et al., 2016). Nevertheless, the 13CO2 production rate was relatively slower in their system, which was probably due to different microorganisms involved in the process. By contrast, the amount of 13CO2 for the abiotic control remained nearly constant (1.40 ± 0.05%) during the entire experiment. This indicated the occurrence of microbial AOM. During the reduction process, the color of the suspension changed gradually from red brown to reseda with times except the CH4-free control (Fig. S3). The XRD analysis demonstrated the production of siderite in the reactors (Fig. 1c). This indicated that ferrihydrite were reduced to siderite by the enrichment culture. 3.2. Microorganisms involved in AOM-coupled ferrihydrite reduction FISH was used to analyze the microbial compositions in the reactors after 40 days of incubation with methane and ferrihydrite. As shown in Fig. 1d, both the archaea (red fluorescence) and bacteria (green fluorescence) were enriched in the culture. The bacteria seemed to be more abundant than archaea. This suggested that both archaea and bacteria were probably involved in AOM coupled to ferrihydrite reduction. To reveal the mechanisms of AOM-coupled ferrihydrite reduction, microbial community structures of the inoculum and enriched culture were determined by high-throughput sequencing. The inoculum was mainly comprised of Ignavibacterium (19.97%), Methanobacterium (16.86%), Desulfovibrio (5.13%), Cellulomonas (6.04%), Geobacter (2.84%), Actinotalea (3.94%) (Fig. 2a). After the 40-day incubation, microorganisms in the reactors were dominated by Methanobacterium (30.16%), Ignavibacteriae (10.81%), Geobacter (2.63%), Desulfovibrio (1.8%), Actinotalea (8.13%), unclassified_Rhodocyclaceae (4.55%) (Fig. 2b). The relative abundances of Ignavibacterium, Geobacter, Cellulomonas, Desulfovibrio decreased slightly compared with those in the inoculum. However, the relative abundances of Methanobacterium were almost one-fold higher than that of the inoculum. Methanobacterium have previously been shown to be capable of trace AOM (Moran et al., 2005). The increased abundance of Methanobacterium suggested that it could have played a major role in AOM-dependent ferrihydrite reduction. Stable isotope probing (SIP) technique was employed to identify which microorganism members had assimilated methane into biomass. Both the 13CH4-DNA and 12CH4-DNA samples (from the microcosm with 13CH4 and the control with 12CH4, respectively) produced seven fractions after density gradient centrifugation (Fig. S1). Compared with 12CH4-DNA, the concentrations of 13CH4-DNA were lower at the light buoyant density (BD) (1.69 g/ml) in the 13CH4-DNA sample, but were much higher at the high buoyant density (BD) (1.75 g/ml) (Fig. S1). This suggested that the labeled carbon of 13CH4 had been assimilated into the genomes of AOM microorganisms. The relative abundances of specific microbes as a function of BD were compared between 13 CH4-DNA and 12CH4-DNA. Two methanogens (Methanobacterium and Methanolobus) were detected from 13CH4-DNA and 12CH4-DNA. Methanolobus showed a lower relative abundance at the light BD (1.71 g/ml) in 13CH4-DNA (0.21%) than in 12CH4-DNA (0.40%) but a higher relative abundance at the heavier BD (1.76 g/ml) in 13CH4-DNA (0.23%) than in 12CH4-DNA (0.02%) (Fig. 3). This indicated that Methanolobus had participated in AOM and assimilated 13CH4 into its biomasses. By contrast, the relative abundance of Methanobacterium was higher at the light BD (1.69 g/ml) in 13CH4-DNA (19.85%) than in 12 CH4-DNA (8.52%), but was basically the same at the heavier BDs (N1.73 g/ml) for 13CH4-DNA and 12CH4-DNA (Fig. 3). Therefore, although the high abundance of Methanobacterium suggested that it was responsible for the methane oxidation, it did not assimilate the CH4 carbon. This is consistent with a previous report that the methanogens participated in the partial oxidation of 13CH4 without uptake of 13C (Bar-Or et al., 2017). This indicated that Methanobacterium

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Fig. 1. The production of HCl-extractable Fe2+ (a) from ferrihydrite (10 mM) in the presence and absence of methane; the production of 13CO2 from 13CH4 and ferrihydrite (10 mM) with and without of inoculum (c); the ratio of 13CO2 is calculated from 13CO2/(13CO2 + 12CO2) and data are mean ± SD of three independent replicates (n = 3). The XRD spectra of the iron oxides in the reactors at day 0 and day 40 as well as the standard spectrum of pure siderite (c). Fluorescence in situ hybridization (FISH) image of the archaea and bacteria incubated with methane and ferrihydrite at day 40 (d). The red fluorescence and green fluorescence indicate the archaea and bacteria, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 2. Relative abundances of the main microorganisms in the inoculum (a) and after 40-day incubation with methane and ferrihydrite (b) at the genus level.

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Fig. 3. Shift tendency of archaea and bacteria in the DNA-SIP experiments. The relative abundances of the OTU_149, OTU_694, OTU_25, OTU_292, OTU_300, OTU_178, OTU_868, OTU_304 and OTU_239 fragments in the buoyant density-fractionized DNA that were extracted from the samples amended with 12CH4 or 13CH4 at day 40.

was similar to ANME-1, which only uses dissolved inorganic carbon as the sole carbon source during AOM (Kellermann et al., 2012). The dissimilatory iron reducer Geobacter showed lower relative abundances at the heavier BD (N1.74 g/mL) in 13CH4-DNA than in 12 CH4-DNA (Fig. 3). This suggested that it did assimilate the carbon of methane into its biomass as well. Such a phenomenon was in agreement with that of Bar-Or et al. (2017) who demonstrated the absence of 13CH4 enrichment into a fatty acid biomarker of Geobacter during iron-coupled AOM. However, five bacterial genera, namely Desulfovibrio (OUT_25), unclassified Rhodocyclaceae (OUT_178), Stenotrophomonas (OUT_292) Actinotalea (OUT_300) and Cellulomonas (OUT_239) showed higher relative abundances at the heavier BD (1.75 g/ml) in 13 CH4-DNA (5.52%, 7.28%, 2.57%, 36.91% and 41.03%, respectively) than in 12CH4-DNA (0.37%,0.08%, 0.02%, 0.02% and 14.41%, respectively). These indicated that these bacteria had assimilated the CH4 carbon and participated in the AOM process. Both Cellulomonas and Desulfovibrio were previously reported to be capable of dissimilatory Fe(III) reduction (Kleindienst et al., 2014; Park et al., 2008). It was therefore speculated that Cellulomonas and Desulfovibrio linked the AOM process to ferrihydrite reduction. Cellulomonas and Desulfovibrio might survive in the reactors by forming a syntrophic relationship with methanogens. Such a syntrophic interaction may be similar to the syntrophy of ANME and Desulfovibrio, which is a widespread phenomenon in the natural sediments (Valentine and Reeburgh, 2000). Two distinct syntrophic AOM pathways, i.e., direct interspecies electron transfer (DIET) and intermediate-mediated interspecies electron transfer (MIET) have been proposed up to date between archaea and bacteria during AOM

(McGlynn et al., 2015). However, the DIET between Methanobacterium and the partner bacteria was probably absent in our reactors because Methanobacterium seems not capable of DIET due to lack of OMCs (Gao et al., 2017). Therefore, Methanobacterium could solely utilize the intermediate-mediated pathway (i.e. MIET) to share electrons with the bacteria partners. Such a MIET syntrophy via the transfer of intermediates may account for the observation that Methanobacterium did not assimilate the 13CH4 into its biomasses. Metagenomics was performed to further probe the AOM mechanisms by detecting the distributions of functional genes in the consortium. The genes that are possibly related with the methane metabolism and electron transport are of special interest. Fig. 4a shows the relative abundances of methanogenic genes, which have recently been hypothesized to be involved in the reverse methanogenesis for the AOM catalyzed by methanogens (Hallam et al., 2004). Two genes encoding methyl coenzyme M reductase (mcr) and methenyltetrahydromethanopterin (methenyl-H4MPT) reductase (mer) were exclusively present in Methanobacterium. Soo et al. (2016) reported that an engineered methanogen Methanosarcina catalyzed reverse methanogenesis for AOM efficiently when the mcr gene of ANME-1 was cloned into it. In view of the great importance of mcr genes, Methanobacterium probably played a major role for the AOM in our reactors. Methanobacterium also showed the highest relative abundances in the gene fmd (39.62%, encoding formylmethanofuran dehydrogenase), mtr (74.78%, encoding methyl-H4MPT coenzyme M methyltransferase) and ftr (35.01%, encoding formylmethanofuran-H4MPT formyltransferase) among the consortium. However, Methanobacterium

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(Davis and Yarbrough, 1966), it was also likely that Desulfovibrio coupled the oxidation of methane directly with ferrihydrite reduction. 3.3. Effect of ESs on AOM-coupled ferrihydrite reduction

Fig. 4. Relative abundances of genes for the methanogenesis enzymes (a) and genes for the redox enzymes or EET-related enzymes (b) at day 40.

missed three methanogenic genes mtd, mch and hmd, which encode methylene-H4MPT dehydrogenase, methenyl-H4MPT cyclohydrolase and methylene-H4MPT cyclohydrolase, respectively. By contrast, the mtd gene was solely detected in the methanotrophic bacteria Methylomonas. The lack of three methanogenic genes in Methanobacterium indicated that it could only perform a partial oxidation of methane when it reversed the methanogenesis pathway. In other words, Methanobacterium must have oxidized methane into an AOM intermediate, which was then used as an electron source by the partner bacteria for ferrihydrite reduction. Six genes (i.e. fdh, hya, hdr, MTHFR, cyc and pil) were used to investigate the coupling mechanisms between AOM and EET. These genes encode formate dehydrogenase (FDH), hydrogenase, CoB-S-S-CoM heterodisulfide reductase (Hdr), methylenetetrahydrofolate reductase (MTHFR), c-type cytochromes and pili proteins, respectively. The genes fdh and MTHFR were selected because FDH and MTHFR can catalyze the C1 (i.e. formate and methylene groups, respectively) metabolisms. Methanobacterium possessed the genes hya and hdr but lacked the genes fdh, cyc, pil and MTHFR (Fig. 4b). Previous studies suggested that hydrogenase and Hdr on the surface of methanogens functioned as terminal electron carriers for EET and iron-dependent AOM, respectively (Deutzmann et al., 2015; Yan et al., 2018). For example, the HdrDE of Methanosarcina acetivorans were suggested to donate the AOM derived electrons to extracellular Fe3+ (Yan et al., 2018). On the other hand, the surface-associated hydrogenase of methanogens could mediate an apparent direct electron uptake from Fe(0) granules (Deutzmann et al., 2015). Thus, it was likely that Methanobacterium utilized the surface-associated hydrogenase and/or Hdr for EET and ferrihydrite reduction. Geobacter and Desulfovibrio possessed all the six genes (Fig. 4b), suggesting their great potential in conducting EET and C1 metabolisms. Desulfovibrio as a sulfate-reducing bacterium (SRB), was previously shown to be capable of the reduction of metal oxides (Kleindienst et al., 2014; Valenzuela et al., 2017). Thus, the DNA-SIP results in combination with metagenomic data indicated that Desulfovibrio used the AOM intermediates for EET-based ferrihydrite reduction. Since pure cultures of Desulfovibrio can also catalyze AOM alone

Six ESs (AQS, flavin, AQDS, HA, RZ, FA) with different redox potentials were selected to evaluate their effects on AOM-coupled ferrihydrite reduction. The Fe (II) concentrations in the reactors amended with AQS, flavin, AQDS and HA at day 35 were 1.77 ± 0.12, 1.48 ± 0.10, 1.12 ± 0.05, 1.15 ± 0.01 mM, which were 5.1, 4.1, 2.9, 3.0 times higher than the control without ESs (0.29 ± 0.03 mM), respectively (Fig. 5a). These results indicated that AQS, flavin, AQDS and HA could accelerate ferrihydrite reduction by the enrichment consortia. By contrast, the addition of RZ or FA did not show a promotion effect. Thus the enhancement extents of ferrihydrite reduction by ESs at day 40 followed the order of AQS N flavin N HA N AQDS N RZ ≈ FA ≈ Control. Microbial reduction of the insoluble electron accepters depends on electron transfer from the inside of cells to extracellular solids. This process, termed microbial extracellular respiration (MER) (Zhou et al., 2015), generally needs a direct contact between extracellular solids and microbes (Gralnick and Newman, 2007). This differs from aerobic respiration in which oxygen enters cells and is reduced in the cytoplasm. As an insoluble electron accepter, ferrihydrite cannot diffuse into the cell membrane or the cytoplasm. Thus microbial reduction of ferrihydrite with methane as the electron donor is also a process of MER. However, in the presence of soluble ESs (e.g. AQS), it was no longer necessary for microorganisms to contact with ferrihydrite directly because the AOM microorganisms could first reduce ESs around them. Then the reduced ESs transferred electrons to ferrihydrite that was far away from cells. This may account for the promotion effects of ESs observed here. In other words, the AOM microorganisms shifted the ferrihydrite utilization from a direct reduction to an ESs-mediated reduction, which increased microbial availability of ferrihydrite via the long-range electron transfer. In order to analyze the effects of ESs on AOM during ferrihydrite reduction, 13CH4 was used as the electron donor and the production of 13 CO2 were monitored in the presence of the most efficient ES (i.e. AQS). The amendment of AQS (2 mM) led to a higher amount of 13CO2 (2.10 ± 0.10% of total CO2) at day 10 compared to the control without AQS (1.50 ± 0.15%) (Fig. 5b). Moreover, the amount of 13CO2 increased dramatically to 13.29 ± 0.98% at day 30 for the reactors amended with AQS, which was significantly higher than that of the control without AQS (8.05 ± 0.50%) (Fig. 5b). This phenomenon is in agreement with their respective extents of ferrihydrite reduction. Therefore, both AOM and the ferrihydrite reduction were enhanced by the electron shuttle AQS. Compared with the control, the presence of ESs could facilitate the release of electrons from certain redox enzymes or cytochromes in the cell membrane or on the cellular surface (Gralnick and Newman, 2007). This allowed a rapid regeneration of these electron carrier proteins, thus enhancing the rates of both AOM metabolisms and electron transfer from AOM to extracellular ferrihydrite. 3.4. Relationships with the redox potential and electron transfer capacity of ESs The rate constants (k) of the ferrihydrite reduction process were calculated by linear fitting of the Fe2+ data (Li et al., 2013). The ferrihydrite reduction in all treatments followed a zero-order reaction kinetics (R2 N 0.87) (Table S3). This meant that the amounts of Fe2+ increased linearly with time during the 40-day incubation. For the treatments of AQS, AQDS, flavin and HA, the rate constants (k) were 7.4, 4.3, 5.8 and 4.6 times higher than that of the control without ES (5.5 ± 0.2 μM d−1), respectively. However, the k values for FA (5.8 ± 0.8 μM d−1) and RZ (8.0 ± 0.5 μM d−1) treatments were close to that of the control without ES. In the presence of AQS, the reduction rate of ferrihydrite (46.1 ± 0.1 μM d−1) was comparable with that of soluble Fe3+-citrate (approx. 56 μM d−1) by the enrichment culture of Candidatus Methanoperedens

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Fig. 5. The production of HCl-extractable Fe2+ from ferrihydrite in the presence of different ESs (a). The production of 13CO2 from 13CH4 with and without AQS during ferrihydrite reduction (b); the 13CO2 data of Fig. 1b are repeated to aid in comparing results for the treatment with AQS and the control without AQS. Data are mean ± SD of three independent replicates (n = 3).

nitroreducens (Ettwig et al., 2016). This indicated that AQS eliminated maximally the difficulty of microbial access to the insoluble ferrihydrite. To reveal how the redox potentials of ESs affect AOM-dependent ferrihydrite reduction, we analyzed the relationship between kES/k0 and their standard redox potentials (E0′). The results showed that the reaction rate ratios (kES/k0) increased linearly with the decreased E0′ from −51 to −225 mV (R2 = 0.9315) (Fig. 6a). The addition of AQS (E0′ = −225 mV) resulted in the highest ferrihydrite reduction rates than other tested ESs. However, further decrease of the E0′ to −478 mV (for FA) reduced kES/k0 to the background value. This suggested that the optimal redox potential of ESs for AOM-dependent ferrihydrite reduction

Fig. 6. The relationships between the ratios of iron reduction rates (kES/k0) and the redox potentials of ESs (a), and between kES/k0 and the electron transfer capacities of ESs (b). The lines are the fitted results.

was likely close to −225 mV. This is consistent with the observations of Wolf et al. (2009), who showed that ESs could accelerate dissimilatory ferrihydrite reduction by Geobacter when the E0′ of ESs fell in the range of −137 to −225 mV (vs. SHE). The distinct enhancement effect by each ES may be explained by the reason that the E0′ of ESs affects the thermodynamical energy gains of microorganisms and thus the electron transfer pathways across cell membranes (Levar et al., 2017). Microorganisms generally use outer membrane c-type cytochrome proteins (OMCs) to reduce ESs and the E0′ of OMCs ranges typically from −320 to −15 mV (Wolf et al., 2009; Liu et al., 2011; Wu et al., 2014). Thus ESs with more positive E0′ values than those of OMCs would be more easily reduced by the AOM consortia. Compared with the E0′ of the CO2/methane couple (−244 mV) (Li et al., 2017), the E0′ of FA (−478 mV) is too negative to be reduced by microorganisms, resulting in its inability in mediating ferrihydrite reduction. The electron transfer capacity (ETC) of ESs is another important parameter that influences the kinetic rates of electron transfer between microorganisms and ferrihydrite. The relationship between kES/k0 and ETC of ESs are shown in Fig. 6b. A positive and linear correlation was observed between ETC and kES/k0 (R2 = 0.7745). AQS had the highest ETC value (131.86 ± 7.75 mmol e− mol−1) among these ESs, whereas FA had the lowest one (61.40 ± 5.69 mmol e− mol−1) (Fig. 6b, Table S2). These were in agreement with their promotion extents of ferrihydrite reduction. This indicated that the ETC of ESs played an important role in mediating electron transfer from AOM microorganisms to ferrihydrite. This phenomenon is consistent with previous studies in which microbial iron reduction rates were linearly related with the ETCs of ESs (Li et al., 2013; Wu et al., 2014; Wolf et al., 2009). Therefore, ESs with higher ETC values could mediate AOM-coupled ferrihydrite reduction more efficiently. GC analysis of the culture medium showed that propionate was produced as an AOM intermediate up to 26 mg/L (Fig. 7a), while acetate and formate were not detected. Thus, the propionate-degrading bacteria such as Desulfovibrio could have utilized propionate for ferrihydrite reduction. However, the presence of AQS eliminated the accumulation of propionate, suggesting AQS had stimulated its degradation. Propionate is not a feasible substrate for some Geobacter speices (Zhou et al., 2014b), which may account for the finding that Geobacter did not assimilate the methane carbon (Fig. 3). So the role of Geobacter in ironcoupled AOM is unclear and needs a further investigation. Based on the results of DNA-SIP, metagenomics and the intermediates, a syntrophic mechanism for AOM-coupled ferrihydrite reduction and the mediation of ESs in this process are proposed and presented in Fig. 7b. Methanobacterium oxidized CH4 to the intermediates with the generation of electrons. These electrons were likely transferred to ferrihydrite directly (possibly via hydrogenase and/or Hdr) or captured first by ESs (Lovley et al., 2000). The reduced ES (ESred) finally donated electrons to ferrihydrite to regenerate oxidized ESs (ESox). Meanwhile, the

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Fig. 7. Changes of the propionate concentrations over time in the experimental treatment (ferrihydrite + CH4, circle symbol) and the AQS treatment (ferrihydrite + CH4 + AQS, square symbol) (a). Data are expressed as mean ± SD of three replicates (n = 3). The proposed mechanisms for the mediation of ESs in AOM-coupled ferrihydrite reduction (b).

methanotrophic bacteria utilized the AOM intermediates for direct or ESs-mediated ferrihydrite reduction. 4. Conclusions This study has demonstrated that the enrichment cultures could catalyze AOM-coupled ferrihydrite reduction with simultaneous production of siderite. The presence of electron shuttles enhanced the rates of both the AOM process and ferrihydrite reduction. The rates of the latter were linearly related with the ETCs of electron shuttles. The mechanisms of AOM-coupled ferrihydrite reduction depended on the syntrophic cooperation of methanogens (dominated by Methanobacterium) and the partner bacteria (such as Desulfovibrio). Methanobacterium oxidized methane into an intermediate (e.g. propionate), which was assimilated by the partner bacteria for direct or ESsmediated ferrihydrite reduction. Acknowledgements This study was supported by the Natural Science Foundation of China (No. 41701270), the Natural Science Foundation of Fujian Province, China (No. 2019J01394), and Key Technologies R&D Program of Fujian Province (2017NZ0001-1). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.scitotenv.2019.06.299. References Bar-Or, I., Elvert, M., Eckert, W., Kushmaro, A., Vigderovich, H., Zhu, Q.Z., Ben-Dov, E., Sivan, O., 2017. Iron-coupled anaerobic oxidation of methane performed by a mixed bacterial-archaeal community based on poorly reactive minerals. Environ. Sci. Technol. 51 (21), 12293–12301. https://doi.org/10.1021/acs.est.7b03126.

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