Impact of Antibiotics Pretreatment on Bioelectrochemical CH4

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Impact of antibiotics pretreatment on bioelectrochemical CH production Heng Xu, Abdulmoseen Segun Giwa, Cuiping Wang, Fengmin Chang, Quan Yuan, Kaijun Wang, and Dawn E. Holmes ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.7b00923 • Publication Date (Web): 01 Sep 2017 Downloaded from http://pubs.acs.org on September 10, 2017

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ACS Sustainable Chemistry & Engineering

Impact of antibiotics pretreatment on bioelectrochemical CH4 production Heng Xua, b, Abdulmoseen Segun Giwaa, Cuiping Wanga, Fengmin Changa, Quan Yuana, Kaijun Wanga, *, Dawn E. Holmesb, c a

State Key Joint Laboratory of Environment Simulation and Pollution Control,

School of Environment, Tsinghua University, No. 30 Shuangqing Road, Beijing, 100084, P.R. China b

Department of Microbiology, University of Massachusetts, Amherst, Massachusetts

01003, USA c

Department of Physical and Biological Sciences, Western New England University,

1215 Wilbraham Road, Springfield, Massachusetts, 01119, USA

* Corresponding Author: [email protected], 861062789411

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ACS Paragon Plus Environment


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ABSTRACT Methane (CH4)-producing bioelectrochemical systems (BES) are an attractive way to store excess renewable electricity and captured CO2. Studies have suggested that methanogenesis via direct electron uptake from a biocathode is more energetically efficient than hydrogenotrophic methanogenesis. However, mechanisms and key microorganisms involved in direct electron uptake remain unclear, primarily because of H2 produced by bacteria or extracellular hydrogenases in the system. In an attempt to minimize biological H2 production and enrich for methanogens that could efficiently convert electrons from the cathode surface to CH4, cathode chambers were pretreated with antibiotics targeting bacteria. We found that antibiotics pretreatment effectively reduced the proportion of H2-producing bacteria and H2-utilizing methanogens associated with the biocathode biofilm, and significantly promoted growth of acetoclastic methanogens from the genera Methanosarcina and Methanosaeta, several of which are known to participate in direct interspecies electron transfer. This shift in microbial community structure corresponded with 14%-36% higher cathode capture efficiencies. These results suggest that suppression of H2 production by antibiotics pretreatment could be a promising way to enrich for methanogens that can efficiently transform electrons from a biocathode into CH4. KEYWORDS: bioelectrochemical systems; methane; biocathode; electromethanogenesis; suppression; CO2 capture; direct electron transport INTRODUCTION Recently, the use of bioelectrochemical systems (BES) for production of fuels or other 2


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chemical commodities has been recognized as an attractive way to store excess renewable electricity and captured CO2.1-6 Among these renewable commodities, bioelectrochemical methane (CH4) production shows great promise, as it can easily be integrated into transportation, storage and utilization systems currently being used for natural gas.7 The performance of bioelectrochemical CH4 production by both pure culture and mixed culture systems has been described in several studies.7-12 However, the proportion of CH4 produced by either direct or indirect electron transport mechanisms in mixed-culture BES has yet to be determined. In general, bioelectrochemical CH4 production is driven by two groups of methanogenic archaea (namely, electromethanogenic and hydrogenotrophic methanogens).3 During electromethanogenesis, methanogens directly accept electrons from cathodes and transfer them to CO2, forming CH4 via the following reaction: CO2+8H++8e-→ CH4+2H2O.7, 13-15 Methanogens carrying out hydrogenotrophic CH4 production, on the other hand, use H2 abiotically produced by the cathode or produced by bacteria or extracellular enzymes present in the cathode chamber as an electron donor for methanogenesis (CO2+4H2→ CH4+2H2O).16-21 Some CH4 can also be attributed to activity by acetoclastic methanogens that utilize acetate produced by acetogenic bacteria to drive methanogenesis.3 Theoretically, in CH4-producing BES, CH4 produced by the direct uptake of electrons from biocathodes during electromethanogenesis is energetically more efficient than hydrogenotrophic or acetoclastic methanogenesis .15, 22-23 A greater proportion of electrons from an electron-donating cathode should be converted to CH4 3



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because fewer electrons can be lost by intermediates (i.e. H2, acetate, formate) diffusing away from the cathode into the cathode chamber. Direct electron uptake by methanogens has only been documented in a few instances. For example, direct interspecies electron transfer (DIET) occurs in wastewater digester aggregates,24, 25 and in co-cultures with Methanosaeta harundinaceaea or Methanosarcina barkeri and Geobacter metallireducens.24, 26, 27 It has also been proposed that hydrogenotrophic methanogens from such genera as Methanobacterium and Methanococcus species can accept electrons from biocathodes or from zero-valent iron (Fe0).8-9,14-15, 28 Although these studies showed that cathodic H2 abiotically produced by biocathodes and Fe0 was unlikely to provide enough electrons for methanogenesis, it has been shown that some of the CH4 being produced in these studies could be attributed to utilization of intermediates formed by interactions between extracellular enzymes secreted by hydrogenotrophic methanogens and cathodic surfaces.17 In order to better understand the mechanisms involved in the unique metabolism that enables direct electron uptake by a methanogen from such surfaces as biocathodes and to identify electromethanogenic microorganisms that could potentially be involved in direct electron uptake from the cathode, intermediates such as H2 formed bioelectrochemically by bacteria need to be reduced or eliminated from the BES chamber. Previous studies have shown that the majority of H2-producing organisms found in BES taxonomically cluster within the domain Bacteria, while only Archaea are capable of methanogenesis.11, 29 Therefore, it was hypothesized that the addition of 4


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antibiotics targeting bacteria would inhibit growth of organisms catalyzing most of the bioelectrochemical H2 production while promoting the growth of microorganisms able to efficiently transform electrons taken up from the biocathode surface into CH4. In this work, an exploratory feasibility study was conducted to investigate the effect of antibiotics pretreatment on the performance and microbial community structure of CH4-producing BES. MATERIALS AND METHODS Experimental Set-up. The effect that antibiotics pretreatment might have on bioelectrochemical CH4 production was investigated in the H-cell devices (see Figure S1 in Supporting Information). An identical set-up without antibiotics pretreatment was run as a control. Duplicate antibiotic-pretreated (Anti1, Anti2) and control (Con1, Con2) H-cells were incubated at 37±2 oC with continuous mixing on magnetic stir plates (Thermo Scientific Variomag Poly 15) at a speed of 130 rpm. H-cells were connected to a potentiostat (Gamry G-350 and multiplexer) and cathode potentials were poised at -700 mV (versus standard hydrogen electrode, SHE). Non-inoculated H-cells were also monitored as controls. Results from these un-inoculated controls showed that daily H2 production rates were below 0.03 mmold-1 and average current densities was below 0.01 A/m2, suggesting that the contribution of electrochemical H2 production to hydrogenotrophic methanogenesis was negligible in this study. Inoculum, Medium and Antibiotics. The cathode chamber from each H-cell was anaerobically inoculated with 0.4g (based on total solids, TS) rice paddy sediment (collected from Vermont) that had been incubated at 37±2 oC for ~60 days 5



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and was stably producing CH4. The medium used in this study was bubbled with N2/CO2 gas (80/20; v/v) to remove oxygen before being autoclaved in bottles sealed with butyl rubber stoppers. The composition of this medium (per liter) consisted of the following: NaHCO3, 2.5 g; KH2PO4• H2O, 0.6 g; NH4Cl, 0.25 g; KCl, 0.1 g; trace mineral solution,30 10 ml; vitamin solution,30 10 ml. The same medium without vitamins was added to the anode chamber and it was continuously bubbled with a slow stream of N2-CO2 (80:20). Antibiotics, namely ampicillin (targeting bacterial cell wall synthesis) and kanamycin (targeting bacterial protein synthesis) were purchased from Sigma. Operation. During the start-up period, both antibiotic-pretreated and control H-cells were operated in batch mode without medium replacement for 30 days (results from the start-up period will not be discussed in this study). In contrast to the control H-cells, two antibiotics were added simultaneously to antibiotic-pretreated cathode chambers with a sterile syringe at a concentration of 200 mg/L, with a ratio of ampicillin to kanamycin of 1:1. In order to account for potential degradation of antibiotics, the same antibiotics at the same concentrations were added to the antibiotic-pretreated cathode chambers on a weekly basis. These specific antibiotics were selected because studies have shown that they do not inhibit growth of methanogenic archaea.31, 32 In addition, when pure cultures of Methanobacterium palustre were exposed to the same concentrations of antibiotics added to cathodic chambers, growth was not impaired (data not shown). The experimental period then followed to investigate the effect of antibiotics 6


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pretreatment on bioelectrochemical CH4 production and further enrich for key microorganisms retained on biocathodes. All operations during the experimental period were carried out under strictly anaerobic and sterile conditions to minimize the risk of introducing exogenous microorganisms into the H-cells. The experimental period consisted of three phases. In phase P1 (0-12 days), H-cells were operated in batch mode and 20% of the medium in the cathode chambers was replaced with fresh and sterile medium every 6 days. In phase P2 (12-20 days), cathode chambers were continuously bubbled with a slow stream of N2/CO2 gas (80/20; v/v) in order to supply enough CO2 for bioelectrochemical CH4 production; and 20% of the medium in the cathode chambers was replaced with fresh medium every day. On day 21, all biocathodes were transferred to newly constructed H-cells. In phase P3 (22-30 days), H-cells were operated in flow-through mode in which both fresh medium (0.1 mL/min) and a slow stream of N2/CO2 (80/20; v/v) were continuously flushed through the cathode chambers. The continuous bubbling of N2/CO2 gas in the cathode chambers during phase P2 and P3 made it difficult to monitor CH4 and H2 production directly. Therefore, batch tests were done by temporarily pausing bubbling on days 20, 21 and 30 to accurately measure gas production rates. Organic acids were also monitored during batch tests. Analysis and Calculation. Both H2 and CH4 concentrations were measured by gas chromatography with TCD detectors (G1530A, Agilent and GC-8A, Shimadzu) as previously described.10 The concentrations of organic acids were determined in both the antibiotic-pretreated and control cathode chambers by high-performance liquid 7



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chromatography (HPLC) (Shimadzu) as previously described.25 Active biomass associated with biofilms that formed on the surface of biocathodes was determined by protein measurements made with the bicinchoninic acid method (Pierce, Rockford, IL, USA) as prevciously described.10 Microbial community structure was analyzed by 16S rRNA gene cloning and sequencing (Supporting Information). Cathode capture efficiencies (ηCCE, %)20 and Shannon-Wiener indices (H') (for bacterial diversity analysis) 33 were also calculated (Supporting Information). RESULTS AND DISCUSSION Performance of Antibiotic-pretreated and Control H-cells. The impact of antibiotics pretreatment on current densities, CH4 production and cathode capture efficiencies was determined in antibiotic-pretreated and control H-cells during phase P1 (Figure 1). As shown in Figure 1A, current densities were significantly lower in the antibiotic-pretreated H-cell, particularly after medium replacement. Maximum current density seen in the antibiotic-pretreated H-cell reached only 0.3 A/m2, ca. 5 times lower than that observed in the control H-cell. Consistent with the fact that bioelectrochemical CH4 production is directly associated with current density, CH4 production was also lower in the antibiotic-pretreated H-cell (Figure 1B). For example, accumulated CH4 production on day 12 in the control H-cell was around 0.6 mmol, compared to only 0.3 mmol produced by the antibiotic-pretreated H-cell. Analysis of headspace gas composition showed that H2 was undetectable in both the control and antibiotic-pretreated H-cells during the P1 phase. It is likely that any H2 being generated by H2-producing bacteria in the control chamber was rapidly 8


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being used by hydrogenotrophic methanogens to reduce CO2 to CH4. In contrast, methanogens in antibiotic-pretreated cathode chambers might not have been using as much H2 as an intermediate source of electrons as methanogens in control cathode chambers. Evidence for this assumption comes from the observation that cathode capture efficiencies from the antibiotic-pretreated H-cell (81.8%~85.3%) were significantly higher than that of the control H-cell (66.4%~73.0%) (Figure 1B). These results show that while antibiotics pretreatment seemed to have an inhibitory effect on overall CH4 and current production, it resulted in much higher cathode capture efficiencies (Figure 1). Two possible explanations for these seemingly contradictory effects are that (1) the antibiotics pretreatment effectively inhibited biological H2 production, greatly reducing its contribution to overall current densities and CH4 production, and that (2) enhanced biofilm formation associated with antibiotics treatment allowed cells to be in closer proximity to electrons being generated directly or via intermediates on the biocathode surface.34 This was further supported by biomass comparisons between antibiotic-pretreated and control biocathodes discussed later in the study. Current densities in the P1 phase, as shown in Figure 1A, decreased gradually as a result of intermittent CO2 feeding. In order to avoid this drop in current densities during phase P2 of the experiment, medium was replaced daily, and CO2 was continuously bubbled into the cathode chambers to provide a constant supply of electron acceptor for methanogenesis and to maintain a stable pH (7.0 to 7.8). Current dropped in the initial days of phase P2 in the control reactor likely because continuous 9



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bubbling initially disturbed previously established cathode biofilms (Figure 2A). However, Figure 2A illustrates the increasing trend of current densities during the P2 phase (days 12-20). At the end of P2, current densities from the antibiotic-pretreated and control H-cells increased to around 0.45 and 0.7 A/m2, respectively. This increase in current densities likely resulted from maturation of the cathode biofilm as thick mixed-species biofilms have been shown to produce higher current densities than thinner biofilms.35 At the beginning of phase P3, biocathodes were transferred to new H-cells in order to eliminate any effect that planktonic microorganisms might have on CH4 production. Fresh anaerobic medium was supplied to these new cathode chambers in flow-through mode instead of the daily media replacement approach used during the P2 phase. As shown in Figure 2B, trends in current densities generated by antibiotic-pretreated and control biocathodes were quite different. While the antibiotic-pretreated biocathode was relatively stable (~1.0 A/m2) throughout phase P3, current densities generated by the control biocathode increased from 1.0 to 3.0 A/m2. When the same antibiotics used in the start-up period (200mg/L) were added again to cathode chambers during phase P3, they appeared to have no effect on current densities generated by antibiotic-pretreated H-cells (Figure 2B), suggesting that microorganisms that had established a biofilm on the biocathode by phase P3 of the experiment were resistant to ampicillin and kanamycin. It is also possible that some of the bacteria encased within the polysaccharide and protein matrix associated with cathode biofilms were protected from contact with these antibiotics.36 Further 10


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investigation into this possibility is necessary. One-day Batch Tests. Cathode capture efficiencies and accumulated CH4 and H2 production by the antibiotic-pretreated and control biocathodes were monitored and compared at the end of phase P2 (day 20) and at the beginning (day21) and end of phase P3 (day30) using one-day batch tests (Figure 3). As demonstrated in Figure 3, H2 accumulated in control H-cells throughout phases P2 and P3 but was only detected at the beginning of P3 in the antibiotic-pretreated H-cell. These results differ from phase P1 when negligible concentrations of H2 were detected in all cathode chambers, and can be explained by the fact that the HRT (hydraulic retention time) was ~30 days for all cathode chambers in the P1 phase but decreased to 5 days in phase P2. While this lower HRT is beneficial for biofilm formation on the biocathode, it also accelerates removal of planktonic microorganisms from the cathode chamber.37 Therefore, the accumulation of H2 could be attributed to the fact that planktonic hydrogenotrophic methanogens were “washed out” of the control cathode chamber during phase P2. This speculation was further supported by results seen on day 21, which was the only day when H2 was detected in the antibiotic-pretreated chamber. In addition, in the control chamber, the amount of H2 produced on day 21 were 10 and 5 times higher than those measured on days 20 and 30. These results suggest that immediately following the transfer of biocathodes into new H-cells, planktonic methanogens were not yet available to oxidize H2 accumulated in the chamber and transfer those electrons to CO2 to form CH4. At the end of phase P3 (day 30), H2 concentrations in 11



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the control and antibiotic-pretreated cathode chambers dropped to 0.79 and 0 mmol, while CH4 concentrations increased (Figure 3). It is likely that some of this increase in CH4 levels in both the antibiotic-pretreated and control H-cells can be attributed to planktonic hydrogenotrophic methanogens. Therefore, the role of planktonic microorganisms needs to be considered in future studies. As shown in Figure 3, significantly more H2 accumulated in the control chamber than the antibiotic-pretreated chamber, particularly on day 21 (~4.07 versus ~0.06 mmol). The reduced accumulation of H2 in the antibiotic-pretreated chamber could either be caused by suppression of biological production of H2 and/or by stimulation of hydrogenotrophic methanogenesis. However, if the antibiotics pretreatment process enhanced hydrogenotrophic methanogenesis, one would expect higher CH4 production rates. However, CH4 produced by antibiotic-pretreated H-cells was consistently lower or equal to that in control H-cells (Figure 3). Therefore, antibiotics pretreatment appeared to be effective at reducing bioelectrochemical H2 production, which minimized its use as an electron donor for hydrogenotrophic methanogenesis. Other potential electron donors for methanogenesis, like acetate or formate, were also not detected in the cathode chamber. These results suggest that more of the electrons available from the antibiotic-treated biocathode were being converted to CH4 and that electromethanogenesis may have played an important role in CH4 production in antibiotic-pretreated H-cells. It is also noteworthy that similar concentrations of CH4 were observed in antibiotic-pretreated and control H-cells at the end of phase P3 12


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(Figure 3). Similar to results observed during phase P1 of the experiment, cathode capture efficiencies calculated during phase P3 were significantly higher in the antibiotic-pretreated H-cell (81.3%~84.5%) than the control H-cell (48.3%~70.8%) (Figure 3A and 3B). In addition, cathode capture efficiencies of control H-cells gradually decreased with time while those of antibiotic-pretreated H-cells were quite stable. These results suggest that microorganisms enriched on biocathodes from antibiotic-pretreated chambers were more efficient at capturing electrons and converting CO2 to CH4 than those enriched in the non-amended control. One explanation for these results is that the antibiotic-treated cathodes were colonized by organisms that could directly accept electrons from the cathode surface. This meant that they were not dependent on electron donors like H2, acetate or formate that could diffuse from the cathode surface or leak from the cathode chamber.38-39 In order to determine whether loss of these intermediates accounted for much of the reduction in cathode capture efficiencies, future studies will need to compare the build-up of intermediates in antibiotic-treated and non-treated cathodic chambers that have been amended with a methanogenic inhibitor like 2-bromoethanesulfonate (BES). It was also interesting to see that the cathode capture efficiencies from the control chambers decreased with each phase; phase P1 had the highest efficiencies while P3 had the lowest efficiencies. This could be attributed to the fact that planktonic cells and/or intermediates were being removed from the system most rapidly during phase P3. Another possible explanation for the decrease in cathode 13



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capture efficiencies observed in the control H-cells over time is that pressure built up in the control chamber resulting from the production of gases caused H2 to leak from the system. Biocathode Characterization. Although pretreatment of biocathode chambers with antibiotics significantly altered the physiology of the antibiotic-pretreated biofilm, biomass of the control and antibiotic-pretreated biofilms was comparable. The total protein-based biomass of the antibiotic-pretreated biocathode was around 25.2 mg (0.42 mg/cm2 of electrode surface area), slightly higher than that of the control biocathode (~23.0 mg; 0.38 mg/cm2) (Figure S3). These results showed that “survivors” of the antibiotic pretreatment process were able to colonize the biocathode just as effectively as microorganisms in the non-pretreated control chamber. The physiological differences observed between the two conditions can be attributed to significant differences in microbial community structures associated with biocathode biofilms recovered from antibiotic-pretreated and control H-cells (Figure 4). Not only were bacterial communities dramatically different (Figure 4A), bacterial diversity was also greater on the control than the antibiotic-pretreated biocathode (Shannon-Weiner index H' of 1.90 and 2.50 on the antibiotic-pretreated and control electrodes, respectively). The majority (31%) of bacterial sequences found on the control biocathode clustered with the genus Desulfotomaculum, while Desulfovibrio (9.5%), Sporomusa (16.7%), Aquabacterium (7.1%) and Thiobacillus (11.9%) also accounted for a large 14


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proportion of the sequences (Figure 4A). Species from the genera Desulfotomaculum and Desulfovibrio are able to produce H2 in organic poor environments.40, 41 Desulfovibrio vulgaris has also been shown to utilize electrons from a cathode to reduce H+ to molecular H2,42,43 and Desulfovibrio species were dominant members of the biocathode community in a H2-producing MEC.44 These results suggest that Desufotomaculum and/or Desulfovibrio might have been responsible for a significant proportion of H2 being produced in the control biocathode chamber. However, further investigation into this possibility is required. Sequences that clustered with acetogens from the genus Sporomusa and chemolithoautotrophic metal-oxidizing bacteria from the genus Thiobacillus were also only enriched on the control cathode (Figure 4A). Studies have shown that species from both of these genera can accept electrons directly from the cathode or from the oxidation of H2 and transfer these electrons to other electron acceptors.45-47 In the case of Sporomusa, it seems likely that electrons accepted from the cathode and/or H2 were being transferred to CO2 forming acetate that could then be used by acetoclastic methanogens.2, 48 However, Sporomusa is also capable of incomplete interspecies H2 transfer with hydrogenotrophic methanogens,49 suggesting that this organism could have also been interacting with hydrogenotrophic methanogens in the cathode chamber. Neither acetate nor any other organic acids were detected as intermediates, suggesting that any acetate generated by Sporomusa was being completely oxidized. Most Thiobacillus species are facultative anaerobes50 and could have been using any oxygen present in the cathode chamber as an electron acceptor for growth. 15



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However, it is also possible that they were interacting with methanogens in the cathode chamber as studies have shown that the presence of conductive materials can facilitate interspecies electron transfer with Thiobacillus.51 It has also been shown that Thiobacillus species are able to accept electrons from biocathodes52 and could be transferring these electrons to electron accepting syntrophic partners. The ability for Sporomusa and Thiobacillus species to utilize electrons from H2 or biocathodes to reduce CO2 or to form syntrophic partnerships with other “non-methanogenic” organisms could help account for H2 losses and low cathode capture efficiencies observed in control cathode chambers. Sequences most similar to species from the genus Aquabacterium were enriched on the surface of both the antibiotic-pretreated and control cathodes and accounted for 8.8% and 7.1% of the bacterial sequences, respectively. Aquabacterium species are microaerophilic and could be acting as oxygen scavengers in both H-cell conditions, creating anoxic conditions needed for anaerobic microorganisms.11, 53 As shown in Figure 4A, the dominant sequence (35.3% of the bacterial sequences) detected on the antibiotic-pretreated biocathode was most similar to Paludibacter propionicigenes (90% identical), a strictly anaerobic bacterium that produces propionate and acetate as major fermentation products.54 Little is known about the genus Paludibacter, as only one species, P. propionicigenes, has been characterized to date. However, Paludibacter-like sequences have also been found to be significant members of microbial communities associated with other biocathodes or microbial electrolysis cells.2, 55 It is also possible that the organism detected by 16S 16


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rRNA gene analysis that is most similar to P. propionicigenes has an entirely different metabolism from P. propionicigenes as it only has 90% sequence identity, enough to be classified as a separate genus. Antibiotics pretreatment also seemed to shift the microbial community to one dominated by species from the phylum Bacteroidetes. No Bacteroidetes were detected on the control biocathode, while Bacteroidetes species (Alistipes, Paludibacter, and Proteiniphilum) accounted for 53% of the bacterial community on the antibiotic-pretreated cathode. This increase in Bacteroidetes can be explained by the fact that many Bacteroidetes species are known to harbor a number of antibiotic resistance genes and are resistant to multiple antibiotics.56 Analysis of archaeal 16S rRNA gene sequences showed that Methanobacterium was the only archaeal genus associated with the control biocathode, which was consistent with results from other published studies11, 19 (Figure 4B). Methanobacterium species that have been described thus far use H2 as an electron donor for methanogenesis,2, 11 but it has been suggested that some Methanobacterium species can accept electrons directly from a cathode.9, 15 A much lower percentage of Methanobacterium sequences (30.0%) were detected in the antibiotic-pretreated biofilm, and another hydrogenotrophic methanogen from the genus Methanoculleus accounted for 24.5% of the sequences. The results showed that the proportion of hydrogenotrophic methanogens was significantly reduced in the antibiotic-pretreated chamber. The remaining (almost half) of the sequences in the antibiotic-pretreated cathode 17



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chamber clustered within the order Methanosarcinales (30.3% Methanosaeta and 15.2% Methanosarcina). All Methanosaeta species grow via acetoclastic methanogenesis and are unable to utilize H2 as an electron donor for growth.57 In addition, species from the genus Methanosaeta can accept electrons from bacterial species by direct interspecies electron transfer (DIET) and transfer these electrons to CO2 to form CH4.24, 58 Their ability to perform DIET might allow them to accept electrons from a cathode that could fuel CH4 production via electromethanogenesis. While most Methanosarcina species are capable of hydrogenotrophic methanogenesis, several can only grow via acetoclastic methanogenesis and have been shown to participate in DIET with bacteria.26, 58-59 It is possible that Methanosarcina and Methanosaeta were using acetate formed by acetogenic or fermentative microorganisms present in the cathode chamber. Acetogens such as Sporomusa were not detected on the antibiotic-pretreated cathode; however, the biocathode was enriched with Paludibacter and Proteiniphilum, both species that produce acetate as major fermentation end-products.54, 60 While this acetate likely served as an energy and carbon source for some of the CH4 produced, it might not have been a major contributor as acetate was not detected in the cathode chamber at any point in the experiment. However, further studies are required to exclude the possibility of high acetate turnover between acetogenesis and acetoclastic methanogenesis in these systems. In addition, until Methanosaeta and Methanosarcina are isolated from these antibiotic-treated BES for pure culture studies, one can only speculate about the role that they might be playing in electromethanogenesis in these BES. 18


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CONCLUSIONS This study showed that antibiotics pretreatment significantly altered the microbial community structure of biocathode biofilms from methanogenic BES. H2 concentrations in the headspace of antibiotic-pretreated cathode chambers and the proportion of H2-producing bacteria in biocathode biofilms were significantly lower than the controls, suggesting that biological H2 production was suppressed by the antibiotics pretreatment. Antibiotic pretreatment also appeared to reduce the proportion of hydrogenotrophic methanogens (i.e. Methanobacterium species) on the biocathode and to enrich for strictly acetoclastic methanogens (Methanosaeta) that are known to be capable of direct electron uptake from bacteria. We also found that biocathodes pretreated with antibiotics had higher cathode capture efficiencies, suggesting that more electrons provided by the biocathode were converted to CH4 in antibiotic pretreated chambers. While this approach shows promise as a way to study direct electron uptake from the biocathode, the lower CH4 yields and current densities in the antibiotics pretreated reactors suggest that hydrogenotrophic methanogenesis plays a major role in CH4-producing BES.

SUPPORTING INFORMATION Additional information regarding experimental procedures and calculations is provided in Supporting Information. Figure S1 is a diagram of the H-cell device used for bioelectrochemical CH4 production in these studies. Figure S2 shows current densities of Anti2 and Con1 during phase P1. Figure S3 shows total protein recovered 19



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from antibiotic-pretreated and control biocathode surfaces. ACKNOWLEDGEMENTS This work was supported by the National Key Technology Support Program of China (Grant No. 2014BAC27B01), China Postdoctoral Science Foundation (Grant No. 2016M600100) and Major Science and Technology Program for Water Pollution Control and Treatment of China (Grant No. 2015ZX07509-001). References 1. Lovley, D. R.; Nevin, K. P. Electrobiocommodities: powering microbial production of fuels and commodity chemicals from carbon dioxide with electricity. Curr. Opin. Biotech. 2013, 24 (3), 385−390. DOI: 10.1016/j.copbio.2013.02.012. 2. Marshall, C. W.; Ross, D. E.; Fichot, E. B.; Norman, R. S.; May, H. D. Electrosynthesis of commodity chemicals by an autotrophic microbial community. Appl. Environ. Microb. 2012, 78 (23), 8412−8420. DOI: 10.1128/aem.02401-12. 3. Logan, B. E.; Rabaey, K. Conversion of wastes into bioelectricity and chemicals by using microbial electrochemical technologies. Science 2012, 337 (6095), 686−690. DOI: 10.1126/science.1217412. 4. Siegert, M.; Yates, M. D.; Call, D. F.; Zhu, X.; Spormann, A.; Logan, B. E. Comparison of nonprecious metal cathode materials for methane production by electromethanogenesis. ACS Sustainable Chem. Eng. 2014, 2 (4), 910−917. DOI: 10.1021/sc400520x. 5. Van Eerten-Jansen, M. C. A. A.; Ter Heijne, A.; Grootscholten, T. I. M.; Steinbusch, K. J. J.; Sleutels, T. H. J. A.; Hamelers, H. V. M.; Buisman, C. J. N. Bioelectrochemical production of caproate and caprylate from acetate by mixed cultures. ACS Sustainable Chem. Eng. 2013, 1 (5), 513−518. DOI: 10.1021/sc300168z. 6. Cai, W.; Liu, W.; Yang, C.; Wang, L.; Liang, B.; Thangavel, S.; Guo, Z.; Wang, A. Biocathodic methanogenic community in an integrated anaerobic digestion and microbial electrolysis system for enhancement of methane production from waste sludge. ACS Sustainable Chem. Eng. 2016, 4 (9), 4913−4921. DOI: 10.1021/acssuschemeng.6b01221. 7. Van Eerten-Jansen, M. C. A. A.; Ter Heijne, A.; Buisman, C. J. N.; Hamelers, H. V. M. Microbial electrolysis cells for production of methane from CO2: long-term performance and perspectives. Int. J. Energ. Res. 2012, 36 (6), 809−819. DOI: 10.1002/er.1954. 8. Villano, M.; Aulenta, F.; Ciucci, C.; Ferri, T.; Giuliano, A.; Majone, M., Bioelectrochemical reduction of CO2 to CH4 via direct and indirect extracellular electron transfer by a hydrogenophilic methanogenic culture. Bioresour. Technol. 2010, 101 (9), 3085−3090. DOI: 10.1016/j.biortech.2009.12.077. 9. Beese-Vasbender, P. F.; Grote, J.-P.; Garrelfs, J.; Stratmann, M.; Mayrhofer, K. J. Selective microbial electrosynthesis of methane by a pure culture of a marine lithoautotrophic archaeon. Bioelectrochemistry 2015, 102, 50−55. DOI: 10.1016/j.bioelechem.2014.11.004. 20


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Figure captions Figure 1. Current densities (A), CH4 production and cathode capture efficiencies (B) of antibiotic-pretreated and control H-cells during phase P1. MR: medium replacement. The results from Anti1 and Con2 are shown in the present study as representatives of duplicate antibiotic-pretreated and control experiments, respectively, which followed the same overall pattern of performance throughout the experimental period. In addition, current densities of Anti2 and Con1 during phase P1 are included in the supporting information (Figure S2) for reference. Figure 2. Current densities of antibiotic-pretreated and control H-cells during phase P2 (A) and P3 (B). Figure 3. Performance of antibiotic-pretreated (A) and control (B) biocathodes during one-day batch tests on days 20, 21 and 30. Figure 4. Relative abundance of 16S rRNA gene sequences for Bacteria (A) and Archaea (B) at the genus level from antibiotic-pretreated and control biocathodes

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Figure 1. Current densities (A), CH4 production and cathode capture efficiencies (B) of antibiotic-pretreated and control H-cells during phase P1. MR: medium replacement. The results from Anti1 and Con2 are shown in the present study as representatives of duplicate antibiotic-pretreated and control experiments, respectively, which followed the same overall pattern of performance throughout the experimental period. In addition, current densities of Anti2 and Con1 during phase P1 are included in the Supporting Information (Figure S2) for reference.

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Figure 2. Current densities of antibiotic-pretreated and control H-cells during phase P2 (A) and P3 (B).

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Figure 3. Performance of antibiotic-pretreated (A) and control (B) biocathodes during one-day batch tests on days 20, 21 and 30.

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Figure 4. Relative abundance of 16S rRNA gene sequences for Bacteria (A) and Archaea (B) at the genus level from antibiotic-pretreated and control biocathodes

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Table of Contents (TOC)

Bioelectrochemical CH4 production could be used to store excess renewable electricity and captured CO2, providing a sustainable substitute for limited natural gas.

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Impact of Antibiotics Pretreatment on Bioelectrochemical CH4

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