Enhancement of methanogenesis by electric syntrophy with biogenic iron‐sulfide minerals

Abstract Recent studies have shown that interspecies electron transfer between chemoheterotrophic bacteria and methanogenic archaea can be mediated by electric currents flowing through conductive iron oxides, a process termed electric syntrophy. In this study, we conducted enrichment experiments with methanogenic microbial communities from rice paddy soil in the presence of ferrihydrite and/or sulfate to determine whether electric syntrophy could be enabled by biogenic iron sulfides. Although supplementation with either ferrihydrite or sulfate alone suppressed methanogenesis, supplementation with both ferrihydrite and sulfate enhanced methanogenesis. In the presence of sulfate, ferrihydrite was transformed into black precipitates consisting mainly of poorly crystalline iron sulfides. Microbial community analysis revealed that a methanogenic archaeon and iron‐ and sulfate‐reducing bacteria (Methanosarcina, Geobacter, and Desulfotomaculum, respectively) predominated in the enrichment culture supplemented with both ferrihydrite and sulfate. Addition of an inhibitor specific for methanogenic archaea decreased the abundance of Geobacter, but not Desulfotomaculum, indicating that Geobacter acquired energy via syntrophic interaction with methanogenic archaea. Although electron acceptor compounds such as sulfate and iron oxides have been thought to suppress methanogenesis, this study revealed that coexistence of sulfate and iron oxide can promote methanogenesis by biomineralization of (semi)conductive iron sulfides that enable methanogenesis via electric syntrophy.

In this study, methanogenic microbial communities were enriched in the presence of sulfate and/or nonconductive, amorphous iron oxides (ferrihydrite). We then tested the hypothesis that biogenic iron sulfides enabled methanogenesis by mediating electric syntrophy. The experiments involved measurements of methanogenic activity, determination of mineral species formed, and investigation of microbial community structure in each enrichment culture.

| Enrichment cultures
Methanogenic microbial communities were enriched in vials (68 ml in capacity) filled with 20 ml of an inorganic medium (PSN medium) supplemented with 20 mmol/L sodium acetate as described previously (Kato et al., 2012b). Fifty milligrams (wet weight) of rice paddy field soil was inoculated as a source of microorganisms. Ferrihydrite was prepared as described elsewhere (Lovley & Phillips, 1986) and was used to supplement +Fer and +Fer/SO 2− 4 cultures to give a final Fe concentration of 20 mmol/L. Sodium sulfate (final concentration, 10 mmol/L) was used to supplement +SO 4 and +Fer/SO 2− 4 cultures. For the cultures of methanogenesis inhibition, bromoethane sulfonate (BES) (final concentration, 5 mmol/L) was added as a specific inhibitor of methanogenic archaea to the same medium used for the enrichment cultures. All cultures were incubated at 30°C without shaking under an atmosphere of N 2 /CO 2 (80/20). CH 4 and H 2 in the gas phases were measured, using a gas chromatograph (GC-2014, Shimadzu, Kyoto, Japan) as described previously (Kato, Sasaki, Watanabe, Yumoto, & Kamagata, 2014). At the early stationary phases, iron minerals were collected by centrifugation and were dried under N 2 atmosphere in an anaerobic chamber (Vinyl Anaerobic Chambers Type C, Coy Laboratory Products, Grass Lake, MI). The X-ray diffraction (XRD) and the X-ray fluorescence (XRF) spectra of the iron minerals were obtained, using an X-ray diffractometer (Ultima X, Rigaku, Tokyo, Japan) and X-ray fluorescence spectrometer (SEA5120A, Hitachi High-Technologies, Tokyo, Japan), respectively.
The culture experiments were conducted in triplicate, and Student's t-test was used to determine the significance of treatment effects.

| Microbial community analysis
After five successive subcultures, enriched microorganisms were collected by centrifugation. DNA was extracted using the FAST DNA Spin Kit for soil (MP Biomedicals, Irvine, US) according to the manufacturer's instructions. Partial 16S rRNA gene fragments were amplified by PCR with a primer pair of 27F and 533R for bacteria and A25F and A958R for archaea, as described previously . PCR products were purified using a

| Quantitative PCR
Quantitative real-time PCR was performed using a LightCycler 96 real-time PCR system (Roche, Basel, Switzerland) and THUNDERBIRD SYBR qPCR Mix (Toyobo, Osaka, Japan) according to the manufacturer's instructions. Table S3 lists the primers used for the qPCR analyses. After an initial denaturation at 95°C for 1 min, targets were amplified by 40 cycles of denaturation for 15 s at 95°C followed by annealing and extension for 45 s at 60°C.
Fluorescence was measured at the end of the extension step.
The PCR amplicons were assessed via a melting-curve analysis to check for successful amplification. At least two separate trials were conducted for each DNA sample. Standard curves were generated with serially diluted PCR products amplified using the respective primer sets.

| Enrichments of methanogenic microbial communities with ferrihydrite and/or sulfate
Enrichment culture experiments were conducted to determine whether or not electric syntrophy could be mediated by biogenic iron sulfide minerals derived from simultaneous reduction in Fe(III) and sulfate. Cultures of methanogenic microbial communities from rice paddy field soil enriched with 20 mmol/L acetate as the sole energy and carbon sources and supplemented with both ferrihydrite (20 mmol/L as Fe) and sulfate (10 mmol/L) are hereafter referred to as the +Fer/SO 2− 4 enrichments. Enrichment cultures with no supplements or supplemented with either ferrihydrite or sulfate, but not both, were also conducted as controls. They are hereafter referred to as the Non, +Fer, and +SO 2− 4 enrichments, respectively. After five successive subcultures, the rate of CH 4 production by each culture was measured ( Figure 1). In the Non enrichment, approximately 17.6 mmol/L of CH 4 was produced from 20 mmol/L acetate (Figure 1a), suggesting that methanogenesis was the major electron sink in the enrichment culture. Rates of CH 4 production in the +Fer and +SO 2− 4 enrichments were significantly suppressed compared to the Non enrichment ( Figure 1b).
This observation is consistent with previous reports; the explanation is that DIRBs and SRBs outcompete methanogenic archaea for common substrates (e.g., acetate) (Achtnich, Bak, & Conrad, 1995;Chidthaisong & Conrad, 2000;Kato et al., 2012b;Lueders & Friedrich, 2002;Yamada, Kato, Ueno, Ishii, & Igarashi, 2014). In contrast, a significant enhancement of methanogenesis was observed in the +Fer/SO 2− 4 enrichment. These results clearly demonstrated that the presence of ferrihydrite and sulfate together enhanced methanogenesis, whereas the presence of either substrate alone did not. This result supports the hypothesis that electric syntrophy was mediated by biogenic iron sulfide minerals.

| Analyses of the biomineralization products
During the +Fer/SO 2− 4 enrichment, ferrihydrite particles that were reddish brown in color turned into black precipitates (Figure 2a).
The precipitates were collected from the +Fer/SO 2− 4 enrichment before and after cultivation, dried under anoxic conditions, and subjected to the XRF and the XRD analyses to identify the Fe species contained in the black precipitates. The XRF analysis showed that the black precipitates generated during the +Fer/SO 2− 4 enrichment contained more phosphorous and much more sulfur than the minerals recovered from samples before cultivation (Figure 2b

| Dominant microorganisms in the enrichment cultures
Clone library analysis of both the archaeal and bacterial 16S rRNA genes was conducted to determine the dominant microorganisms in the enrichment cultures. A phylotype was defined as a unique clone or a group of clones with sequence similarity >98%. All phylotypes obtained in this study are summarized in Tables S1 and S2. Only one archaeal phylotype (FeAcA001, 99% identity to Methanosarcina barkeri) was recovered from all the enrichment cultures (Table S1), and no other archaeal phylotype was recovered from any of the enrichment cultures. These results suggest that Methanosarcina spp. generated CH 4 in all the enrichment cultures.
A total of 26 phylotypes were obtained from the clone library analysis targeting bacteria (Table S2).   (Junier et al., 2009;Lovley, 1993), a difference in the availability of Fe(III) might have caused the changes in the SRB communities. Other plausible factors include a change in redox potential due to the presence of ferrihydrite, toxicity of ferrous ions, and a decrease in availability of acetate due to coexistence of Geobacter spp.

| The effects of a methanogenic inhibitor on DIRBs and SRBs
In electric syntrophy-facilitated methanogenesis, chemoheterotrophic bacteria and methanogenic archaea are in an interdependent F I G U R E 2 Biomineralization of iron sulfides in the enrichment culture supplemented with both ferrihydrite and sulfate. (a) Changes in color of minerals before and after cultivation. The X-ray fluorescence (XRF) spectra (b) and the powder X-ray diffraction (XRD) patterns (c) of the minerals taken before (orange line) and after (black line) cultivation. In (c), the reflection pattern of pure siderite (FeCO 3 ) is shown under the sample patterns relationship. The growth of chemoheterotrophic bacteria, which accompanies electric syntrophy, was therefore indirectly suppressed by a specific inhibitor of methanogenesis (Kato et al., 2012b). To identify microorganisms involved in electric syntrophy, each enrichment was further cultivated in the presence or absence of BES, a specific inhibitor of methanogens, and the abundances of total bacteria, archaea, and dominant DIRB and SRB species were measured via the quantitative PCR (qPCR) method ( Figure 4).
Methane production was completely suppressed in the cultures supplemented with 5 mmol/L of BES (data not shown).
F I G U R E 3 Phylogenetic distribution of bacterial 16S rRNA gene clones recovered from the enrichment cultures supplemented with no additives (Non), ferrihydrite (+Fer), sulfate (+SO 2− 4 ), and both ferrihydrite and sulfate (+Fer/SO 2− 4 ). The dominant phylotypes (>10% in at least one enrichment) and their closest relatives (sequence identity, %) are shown in the legends. The phylotypes closely related to Fe(III)-reducing and sulfate-reducing bacteria are highlighted in red and green, respectively F I G U R E 4 The abundances of archaea (a), bacteria (b), Geobacteraceae (c), Desulfovibrio (d), and Desulfotomaculum (e) in the enrichment cultures assessed via the qPCR method. (c-e) The relative abundances (%) were calculated as a relative ratio to the sum of total bacteria and total archaea. Asterisks represent a significant difference (p < .05) between results in the presence or absence of a methanogenesis inhibitor (5 mmol/L BES). Data are presented as the means of three independent cultures, and error bars represent standard deviations Supplementation of BES reduced the abundance of total archaea to less than 5% of those in the BES-free cultures (Figure 4a), suggesting that the growth of archaea was completely suppressed by BES, considering the inoculation size (5% v/v). In the absence of BES, the total bacterial abundances were significantly higher in the enrichments supplemented with ferrihydrite and/or sulfate than in the Non enrichment (Figure 4b).

| Estimation of metabolic and electron flow in each enrichment culture
Based on the results obtained in this study, we estimated the meta-  (Figures 3 and 4). Similar to the results of the +Fer enrichment, competition between SRBs and methanogens could have caused the rate of CH 4 production to decrease (Figure 1).
In contrast, the metabolic flow and the microbial species involved dramatically changed when the medium was simultaneously supplemented with ferrihydrite and sulfate. Unlike the case of the +Fer and +SO 2− 4 enrichments, CH 4 production was significantly enhanced in the +Fer/SO Methanosarcina spp. then performed electric syntrophy with the biogenic iron sulfides as conduits to generate CH 4 . Methanogenesis based on electric syntrophy has been reported to be more efficient than the usual methanogenic processes (Cruz Viggi et al., 2014;Kato et al., 2012b;Li et al., 2015;Liu et al., 2015;Yamada et al., 2015;Zhuang et al., 2015), which could explain the increased rate of CH 4 production in the +Fer/SO 2− 4 enrichment.

| Conclusion
This study demonstrated for the first time that biogenic iron sulfide minerals can enable methanogenesis based on electric syntrophy.
It has been reported that the existence of electron acceptor compounds such as sulfate and ferrihydrite inhibit methanogenesis (Achtnich et al., 1995;Chidthaisong & Conrad, 2000;Kato et al., 2012b;Lueders & Friedrich, 2002;Yamada et al., 2014). The results of this study suggest that methanogenesis can be promoted when sulfate and ferrihydrite coexist. Because iron sulfide minerals are ubiquitous in anaerobic environments, it is highly possible that electric syntrophy with iron sulfides contributes greatly to methanogenesis and also to carbon cycles in natural environments.
Further investigation on the effects of iron sulfide minerals in natural environments and also experiments using model co-cultures of Geobacter spp. and Methanosarcina spp. with synthetic iron sulfide species will shed light on the importance of iron sulfide minerals for the global methane flux.

ACK N OWLED G M ENTS
We thank Hiromi Ikebuchi and Asuka Tanaka for technical assistance. The XRD and XRF analyses were performed at the Innovation Booting Equipment Common (IBEC) Center of the National Institute of Advanced Industrial Science and Technology. This work was supported by Japan Society for the Promotion of Science KAKENHI Grant Number 16H06191.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.