Enrichment of specific microbial communities by optimum applied voltages for enhanced methane production by microbial electrosynthesis in anaerobic digestion

Journal Pre-proofs Enrichment of specific microbial communities by optimum applied voltages for enhanced methane production by microbial electrosynthesis in anaerobic digestion Carla Flores-Rodriguez, Booki Min PII: DOI: Reference:

S0960-8524(19)31854-1 https://doi.org/10.1016/j.biortech.2019.122624 BITE 122624

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Bioresource Technology

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18 October 2019 10 December 2019 12 December 2019

Please cite this article as: Flores-Rodriguez, C., Min, B., Enrichment of specific microbial communities by optimum applied voltages for enhanced methane production by microbial electrosynthesis in anaerobic digestion, Bioresource Technology (2019), doi: https://doi.org/10.1016/j.biortech.2019.122624

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Enrichment of specific microbial communities by optimum applied voltages for enhanced methane production by microbial electrosynthesis in anaerobic digestion

Carla Flores-Rodriguez and Booki Min* Department of Environmental Science and Engineering, Kyung Hee University, Seocheondong, Yongin-si, Gyeonggi-do 446-701, Republic of Korea

*Corresponding author: Phone: +82-31-201-2463; Fax: +82-31-202-8854. E-mail address: [email protected]

Abstract This study investigates the distribution of microbiome in microbial electrosynthesis systems at different applied voltages (0.5, 1.0, and 1.5 V) for methane production. Results revealed that more favorable conditions for methane production were observed with 1.0 V applied voltage. In Venn plots, the bioelectrodes at 1.0 V had higher numbers of unique operational taxonomic units compared to those at 0.5 and 1.5 V. Hierarchical cluster, non-metric multidimensional scaling, and principal component ordinate analyses revealed that the biocathode at 1.0 V clustered separately from the rest of the biofilms mainly because of the quantitative differences in the microbial distribution. Taxonomically, exoelectrogens (Geobacter spp.) dominated the bioanode at 1.0 V, while the syntrophic assemblages of hydrogen-producing bacteria (i.e., Bacteroidetes and Firmicutes) and hydrogen-consuming methanogens (i.e., Methanobacterium sp.) existed in the biocathode. These results suggest that the optimum applied voltage enriched specific microbial communities on the anode and cathode for enhanced methane production. Keywords: Microbial electrosynthesis (MES); anaerobic digestion (AD); applied voltage; Geobacter; Methanobacterium; hydrogenotrophic methanogenesis.

1. Introduction Anaerobic digestion (AD) is a technology comprising resource recycling and energy production aspects. Various types of organic matter (e.g., wastes, residues, and energy crops) are utilized in anaerobic digesters to produce methane (Yu et al., 2018). The process involves four coupled stages (i.e., hydrolysis, fermentation, acid-acetogenesis, and methanogenesis) mediated by synergistic interaction of functionally distinct microorganisms (Blasco-Gomez et al., 2017). Its efficiency and performance strongly depend on the cooperation between these communities. Any unbalance results in the accumulation of volatile fatty acids (VFAs), pH depression, and toxicity (Franke-Whittle et al., 2014). Improving the VFA consumption is considered one of the main strategies for stable operations in AD (Franke-Whittle et al., 2014). In recent years, bioelectrochemical systems (BES) approach have been developed to treat a wide range of organic substrates (e.g., defined VFAs, food waste, raw sludge), while it harvests value-added products from CO2 reduction by the integration of microbial electrosynthesis system and anaerobic digestion (MES-AD) ( Yu et al., 2018; Blasco-Gomez et al., 2017). Understanding the community assembly can guide and optimize the MES implementation in AD. Studies focusing on anode biofilms are generally performed in double-chamber MES and microbial fuel cells (MFCs) to prevent parasitic processes that lead to lower substrate-tocurrent efficiency (Semenec and Franks, 2015). The microbial communities in these systems are predominantly composed of fermentative bacteria and anodophilic exoelectrogens with a general enrichment of Proteobacteria, Firmicutes, Bacteriodetes, Acidobacteria and Actinobacteria (Cotterill et al., 2018). Among the various Proteobacteria, Geobacter spp. dominate the anodophilic microbial community at different operational conditions (Cotterill et al., 2018). For example, the most dominant genera detected in Rao Hari et al. (2016) were Geobacter, followed by Smithella and Syntrophobacter; however, their relative abundance

varied among the different potentials tested (−0.25, 0, and 0.25 V) (Hari et al., 2016). Geobacter has also been identified as the largest anodophilic bacteria overtime at low (0.5 g·L−1) and high (4 g·L−1) concentrations of acetate and propionate (Hari et al., 2017). This behavior in Geobacter spp. and their syntrophic interactions with fermenters enable them the possible role of maintaining the functional stability of MES-AD (Hari et al., 2017, 2016). While exoelectrogens mainly dominate the anodes, the communities that develop in cathodic biofilms can be more varied and undefined depending on the type of reaction occurring (Siegert et al., 2014; Speers and Reguera, 2012). When methane is the target product, a single chamber MES-AD is generally set to decrease the pH gradients between the electrodes (anode and cathode), ohmic resistances, and overall costs (Xafenias and Mapelli, 2014). Current investigations have given some insights into the microbial community structure at the biocathodes, where hydrogenotrophic methanogens have been considered the essential actors in the process at different operational conditions (Blasco-Gomez et al., 2017). For example, in a single chamber, despite the use of acetate, hydrogenotrophic methanogens (i.e., Methanobacterium and Methanobrevibacter) are commonly developed on the cathodes rather than acetoclastic methanogens (i.e., Methanosarcina and Methanosaeta), which are generally the exclusive archaea in mixed culture inocula (Lee et al., 2009; Siegert et al., 2014). Moreover, Methanobacterium spp. have been found to enrich the biocathodes in both double and single chambers ran with different cathode materials (i.e., carbon brushes, plain graphite blocks, or blocks coated with carbon black and platinum, stainless steel, nickel, magnetite, iron sulfide, or molybdenum disulfide) (Siegert et al., 2015). In a wide range of applied voltage (0.3 to 1.8 V), hydrogenotrophic and acetoclastic methanogens have been identified. For example, In Feng et al. (2015), the highest methane production was observed at an applied voltage of 0.3 V and it was related to the enhanced diversity of Methanosphaerula palustris and Methanosarcina barkeri in iron–graphite biocathode (Feng

et al., 2015). In Chen et al. (2016), the best performance was obtained at 0.6 V with enhanced growth of Methanoregula sp. on activated carbon fiber textile biocathode (Chen et al., 2016). In Xiao et al. (2018), the enhanced methane production and growth of an uncultured Methanobacterium sp. was determined at 1.8 V (Xiao et al., 2018). The enrichment of these hydrogen-dependent methanogens can be attributed to the hydrogen (H2) produced and the electrons directly derived from the cathode and bacterial interactions (Blasco-Gomez et al., 2017). In some investigations, the direct electron transfer has been evidenced

in

Methanobacterium-like

strain

IM1,

Methanococcus

maripaludis,

Methanosarcina sp., and Methanosaeta sp. (Beese-Vasbender et al., 2015; Blasco-Gomez et al., 2017; Lohner et al., 2014). The syntrophic interactions between bacteria and methanogens might also have an important role in methane production in MES-AD. For example, some bacteria can potentially recycle cell lysis products into H2 and CO2, bioelectrochemically catalyze the H2 production (i.e., Hydrogenophaga caeni and Desulfovibrio putealis), transfer electrons to produce reduced compounds (i.e., hydrogen or formate), and consume oxygen (i.e., Hydrogenophaga caeni, Methylocystis sp., and Acidovorax caeni), which is toxic to methanogens (Blasco-Gomez et al., 2017). In all of the aforementioned MES-AD investigations, the studies on microbial community structure have been based only on the individual function of anode, suspension, or cathode. Therefore, in this study, the comprehensive microbial community structure (anode, cathode, and suspended biofilm) was investigated in a single chamber MES-AD system at different applied voltages. The MES-AD systems were operated at various voltages ranging from 0.5 to 1.5 V with acetate as initial substrate for methane production (Flores-Rodriguez et al., 2019). The microbial communities of the attached (anode and cathode) and suspended biomass in

MES-AD systems at different input voltages were characterized based on

Illumina MiSeq sequencing analysis, and the possible bioelectrochemical routes were suggested based on the community analysis. 2. Materials and methods 2.1. Inoculum and MES cell setup The anaerobic digester sludge was collected from the effluent of anaerobic digestion at the Yongin Wastewater Treatment Facility (Yongin-si, South Korea) and used to enrich the functional biofilm on the carbon brush electrodes of three MES-AD (300 ml total volume and 270 ml working volume). The anode and the cathode were inoculated by mixing preincubated sludge with growth media at 1:1 (v/v) and adding 2 g·L−1 sodium acetate as an electron donor The synthetic growth medium contained 50 mM phosphate buffer solution (KH2PO4 3.4 g/L and K2HPO4 4.3 g·L−1; pH 7), NH4Cl, 0.53 g·L−1, CaCl2 2H2O 0.08 g·L−1, MgCl2 2H2O, 0.1 g.L−1, and 1 ml of trace elements (HCl 36%, 5.1 ml·L−1; FeCl2·4H2O, 1500 mg·L−1; H3PO3, 60 mg·L−1; MnCl2·4H2O, 10 mg·L−1; CoCl2·6H2O, 120 mg·L-1, ZnCl2, 70 mg·L−1; NiCl2·6H2O, 25 mg·L−1; CuCl2·2H2O, 15 mg·L−1; NaMoO4.2H2O, 25 mg·L-1) (Flores-Rodriguez et al., 2019; Siegert and Banks, 2005). All systems were operated in a fedbatch mode at varying supplemental voltages (0.5, 1.0, and 1.5 V) for 8 days (one cycle) (Flores-Rodriguez et al., 2019). At each condition, the externally applied voltage was supplied using a DC power supply unit (Hwasung Electronics Co., South Korea). The operational conditions were set up at an agitation rate of 235 RPM (ATL-4200, Anytech Co., Republic of Korea) and a constant temperature of 35 ± 2 °C (MIR-553, Sanyo Electric Co., Japan). An analogous control (without electrodes; AD) was set up to identify the microbial community shifts in the MES-AD. 2.2. Sampling, DNA isolation, and sequencing After eight operational cycles, the samples were collected from the anode, cathode, and

suspended liquid inside an anoxic chamber. The electrodes were gently rinsed with deionized water to remove the residual sludge (Lu et al., 2012), submerged in a vial, and washed with a buffer solution until the entire biofilm was removed. All samples were centrifuged at 500 g to remove the supernatant. The total DNA from the solid biomass was extracted using PowerMax Soil DNA Isolation Kits (MoBio Laboratories) according to the manufacturer's instructions. The DNA quality was measured by PicoGreen and Nanodrop. Each sample was prepared to amplify the V3 and V4 regions of the 16S rRNA gene following the Illumina 16S Metagenomic Sequencing Library protocols. Amplicons were sequenced by MiSeq Illumina platforms to reconstruct phylogenies. Raw tags were assembled using the Fast Length Adjustment of Short Reads (FLASH; V1.2.11) software (Magoc and Salzberg, 2011). Quality control filtering (denoizing) and clustering (cutoff: 97%) were performed using CD-HIT-Otu (Li et al., 2012) and rDNATools (Schloss et al., 2009), respectively. The diversity analysis (taxonomy composition and alpha-diversity) for each sample was processed with the Quantitative Insights into Microbial Ecology Pipeline (Bhute and Al., 2011). The data analyses encompassed the assessment of specific and shared operational taxonomic units (OTUs) across the sample groups via the estimates of symbiont richness (Chao1) and diversity (Shannon's and Simpson’s index). The visualization tools included Venn diagrams for shared and unique OTUs (Wang et al., 2016). 2.3. Statistical tests All samples were subjected to non-metrical multidimensional analysis to identify the systematic differences among the samples. The software package Plymouth Routines in Multivariate Ecological Research (PRIMER) V7 was used to study the nMDS, principal component ordinate (PCO), and hierarchical cluster analysis based on the Bray–Curtis matrices calculated from the square root transformed data (Clarke and Gorley, 2015).

3. Results and discussion 3.1. Microbial community diversity The numbers of reads sequenced and OTUs and the richness and diversity of the communities were summarized to understand how the microbial community was distributed at the electrode and suspension biofilm (Table 1). Biofilms were noted regarding their sampling site, followed by the voltage applied. A total of 510,599 reads were optimized from quality control and clustering. At least 43,845 reads were conducted for each sample site in the MES-AD and control operation (control). The number of OTUs and the Chao1 index estimated the microbial richness. Chao1, which is a measure of the expected species richness based on abundance data), showed values above the OTUs (observed species), implying the existence of additional species. However, their Good's coverage value reached more than 0.99, which indicated that most of the OTUs in each of the samples were sequenced. The Shannon diversity index (H: 3.867–4.663) and the Simpson diversity index (D; 0.851– 0.8951) showed high diversity across samples. The indices in the bioanodes, biocathodes, and suspended biofilms were slightly lower compared to those in the community in the control operation. In previous studies, lower community diversity values (Shannon index of 2.0) were found in the biocathode when an acetate-fed MES-AD was set up with an inoculum having a highly diverse community (H: 4.7–4.9). These results suggest that the electrodes probably developed microenvironments that could host-specific communities (Siegert et al., 2014). Similarly, Liu et al. found that the highest diversity (H: 6.96) in raw samples was reduced in acetate-fed reactors (H; 5.39) (Liu et al., 2016). Table 1. Venn diagrams were used to quantify the enrichment of specific OTUs (Fig. 1). Strikingly, 1.0 V stimulated a specific growth of 8% (30 OTUs) of the total OTUs in the A1.0

anode biofilm. Alongside the other conditions, it represented only 4% of the growth. Among the biocathodes, 1.0 V stimulated a specific growth of 11% (47 OTUs), while those at 0.5 V and 1.5 V were 6% (29 OTUs) and 8% (36 OTUs), respectively. Fig. 1. Multivariate ordination methods were applied to reduce the multiple dimensions in the datasets, visualize patterns and identify the systematic differences among the total OTU composition of each sample. All samples were subjected to non-metrical multidimensional (nMDS), PCO, and hierarchical cluster analyses based on the Braye Curtis index (Fig. 2). The nMDS and PCO plots revealed observable variations among the samples (ANOSIM, p = 0.009, R = 0.377) (Figures 2A and B). The nMDS plot stress value below 0.1 indicated that the two-dimensional representation is ideal for the data interpretation (Joyce et al., 2018). The microbial community from all the MES-AD operations was different from the control inoculum. In addition, the microbial community structures in MES-AD substantially differed based on the location (anode, cathode, and suspension), except for the anode and the cathode biofilm at 1.5 V. The community structure at 1.0 V was also very different from the communities at 0.5 and 1.5V. The PCO identified 30.6% and 23.6% of the variance caused by the first and second coordinates, respectively. The variation in the first component was more strongly related compared to the other components for all sample biofilms, except for the 1.0 V biocathode, which greatly contributed to the variations in the second component. The differences might be associated with the changes in the predominant microbial community of each sample. The hierarchical cluster plot (Figure 2C) confirmed the observations of the nMDS and PCO plots. Two clusters were initially identified. The first cluster grouped only the 1.0 V biocathode, while the 1.0 V bioanode grouped independently to two sub-clusters in the second cluster. These results show that the C1.0 biofilm clustered separately from the rest of the biofilms and confirmed that the applied voltage induced different enrichments in

microbiological biofilms. Fig. 2. 3.2. Microbial community taxonomy The sequence reads of each sample and their relative abundance were taxonomically allocated by phylum level (Fig. 3). The relative abundance in each sample was calculated as the quotient between the number of sequences affiliated with each taxon and the total number of obtained sequences in the sample multiplied by 100. The phylum allocation was greater than 1% in all samples. The unassigned readings were detected at a very high frequency of 38.8%–55.1% possibly because of the differences in the nature of the inoculum, enrichment process, and sampling procedure. Among the assigned communities, the most predominant phylum in all niches was Firmicutes (11.9%–20.6%) except the anode biofilm at 1.0 and 0.5 V, in which the dominant phyum was Proteobacteria (28.0% and 17.7%. respectively). The Euryarchaeota phylum was dominantly enriched on the biocathode operated at 1.0 V (10.9%) compared to other samples (0.3-0.9%). This result may suggest that the highly enrichment of methanogens on the cathode electrode is important to enhance methane production in MESAD system. Interestingly, the Thermotogae phylum was dominantly presented from suspended biomass (8.8-10.3%) in all applied voltages compared to anode and cathode biofilms, and this phyum is known to utilize different complex carbohydrate for hydrogen production. The rest of the enriched phyla were Bacteroidetes (2.8%–9.2%), Spirochaetes (0.5%–6.5%), Chloroflexi (2.8%–6.1%), Caldiserica (2.3%–5.6%), and Synergistetes (1.3%– 3.2%). Previous studies also reported that these phyla predominated in anaerobic digester sludge (Guo et al., 2015), and their presence on the electrodes may have originated from the inoculum and formed symbiotic relationships. Fig. 3.

The overall assigned microbial compositions of each MES-AD and control operations were analyzed at the phylum level (Fig 4.). The Firmicutes was the most abundant phylum in all applied voltages and also control condition. The Proteobacteria phylum was dominantly present at MES-AD at 1.0 V followed by 0.5 and 1.5V conditions. The methane production in MES-AD was in the same order of applied voltages as shown for the enrichment of Proteobacteria. The maximum yield and percentage of methane production were 0.35 L CH4 g-1 COD and 66%, respectively, at 1.0V, followed by 0.5V (0.29 CH4 g-1 COD; 58%) and 1.5 V (0.29 CH4 g-1 COD; 56%) (Flores-Rodriguez et al., 2019). In addition, different cathode potentials of -1.11, -1.27, and -1.16 V (vs.Ag/AgCl) were reported at 0.5, 1.0, and 1.5 applied voltages. It was also suggested that 1.0 V might promote mechanisms of electron transfer in the electrode biofilms (Croese, 2012). Additionally, Euryarchaeota was mostly enriched in 1.0V MES-AD system compared to other systems. Euryarchaeota and Proteobacteria are evolutionarily, geologically, and environmentally important group of microorganisms. Methanogenesis is the main metabolic process in autotrophs from Euryarchaeota, while several metabolic processes are carried out by Proteobacteria. Geobacter, a delta-Proteobacter have been well known to have the ability to use solid-state electron acceptors such as metal oxides and electrodes with oxidation of a variety of organic carbons (Poole, 2019; Zhang et al., 2019a, 2019b, 2018). Fig. 4. The class and genus level identification of the microbial communities were determined at Figure 5 and 6, respectively. Illumina sequencing detected 36 classes among all biofilms. The archaea

sequences

belonged

to

the

three

following

classes:

Methanobacteria,

Methanomicrobia, and Thermoplasmata (Fig. 5A). The archaeal microbial community structure revealed that the most dominant methanogenic archaea were the Methanobacteria and Methanomicrobia, and their relative abundances varied among the tested samples.

Methanobacteria were majorly identified in the reactor at 1.0 V with an apparent enrichment in the biocathode (10.3%), which was approximately 158 and 295 times higher than abundance in the bioanode (0.06%) and suspended samples (0.03%), respectively. This similar low distribution was detected in the electrode biofilms (0.002%–0.03%) at 0.5 and 1.5 V operations and null in their suspended biofilms. Methanomicrobia were ubiquitous within all samples in the 0.5, 1.0, and 1.5 V reactors, although they were in low abundance, accounting for only 0.9% to 0.2% while representing 2.1% in the control. Thermoplasmata were the least abundant archaea in all the samples (0.006%–0.03%). Further identification at the genus level of archaea (Fig. 5B) showed that Methanobacteria only contained Methanobacterium, and were dominantly found at the 1.0 cathode in comparison to other samples. The absence of this hydrogenotrophic methanogen in the suspended and control samples, except for the suspended biofilm at 1.0 V that probably spread from the electrodes during sampling, indicated that this genus was a carbon-electrodeassociated microorganism, and hydrogen–electron transfer occurred to contribute to methane production, especially at the 1.0 V supply (Blasco-Gomez et al., 2017; Siegert et al., 2015). The presence of hydrogenotrophic methanogens in the anode biofilm implied that the direct interspecies electron transfer may occur between these methanogenic archaea and exoelectrogenic bacteria as previously reported (Lee et al., 2016; Liu et al., 2012; Rotaru et al., 2014a). For example, Lee et al. (2016) found the enrichment of exoelectrogens (e.g., Geobacter) and hydrogenotrophic methanogens (e.g., Methanospirillum and Methanolinea) from the biomass attached to GAC (Lee et al. 2016). However, the putative interspecies electron transfer between Methanobacteria and Geobacter should be further proven based on a co-culture test. Following the main distributions, Methanomicrobia and Methanosaeta were identified in

all the samples. Their presence on the anodes suggested that the Methanosaeta species might be another sink to accept the electrons from the electrogenic bacteria (Geobacter spp.) in MES-AD. The direct interspecies electron transfer (DIET) from the Geobacter species to the Methanosaeta species was confirmed in the defined co-culture of G. metallireducens and M. harundinacea in aggregates from brewery wastewater digesters (Morita et al., 2011; Rotaru et al., 2014a). Ametatranscriptomic analysis revealed that the genes for the CO2 reduction pathway in M. harundinacea were highly expressed, causing the Methanosaeta species to have the capacity to directly accept the electrons from the Geobacter species to reduce CO2 to CH4 (Rotaru et al. 2014b). Thus, with the coexistence of the Geobacter and Methanosaeta species in the 0.5 and 1.0 V operations, the DIET is expected to be another important method of producing methane. Biochemically, the coexistence of Methanosaeta with bacterial communities at the bioanodes also suggests competitiveness for the electron donors (acetate) for methane generation (Vrieze, 2014). The presence of Methanosarcina was mainly identified in the bioanode of the 0.5 V reactors. The low energy yield from the acetate to methane conversion (−75.7 kJ mol−1 methane) and the ability of Methanosarcina to produce methane with electrons derived from DIET with bacteria (Geobacter spp.) may add a competitive advantage for methane production (Rotaru et al., 2014a, 2014b). Growing evidence of Geobacter species being able to form syntrophic associations with Methanosarcina that function via DIET was found. The promoting mechanism of DIET was well understood in ethanol metabolism with a co-culture of Geobacter metallireducens and Methanosarcina barker (Liu et al., 2012; Rotaru et al., 2014a). In accordance with our previous bioelectrochemical study, 0.5 V operations only contributed 12% to the Coulombic efficiency. Furthermore, the methane production via cathodic reduction accounted for a small portion (18 mL) of the methane increment (i.e., 106.24 mL) (Flores-Rodriguez et al., 2019). These results indicate that the chemical and

electrochemical reactions at the anode biofilm were also responsible for the substrate consumption and methane production in these conditions. In the control sample, Methanosaeta mostly prevailed over the other hydrogenotrophic methanogens, Methanolinea, and an unclassified Thermoplasmata. This result confirms that the acetoclastic pathway prevailed over hydrogenotrophic in conventional anaerobic digesters (Guo et al., 2015). The Methanolinea growth was stimulated in all samples with respect to the control and only decreased in the cathode at 1.0 V, suggesting that an H2 sink was effectively controlled by the hydrogenotrophic methanogens in all the samples, mainly in the biocathode at 1.0 V that was performed by the cathodephilic Methanobacterium (Blasco-Gomez et al., 2017; Lu et al., 2012). Fig. 5. Among the bacteria, sequences were identified for 32 classes, of which 23 accounted for less than 1% of the bacterial 16S rRNA gene sequences recovered in each biofilm. Biofilms were generally enriched with the Bacteroidia, Caldisericia, Anaerolineae, Clostridia, b- and d- Proteobacteria, and Synergistia classes as well as unclassified Spirochaetes and Thermotogae (Fig. 6A). Marked changes in bacterial composition were also observed with added voltage. Shifts in class level correlated well with both bioelectrogenic activity and substrate degradation. Interestingly, an apparent enrichment of the delta-Proteobacteria was found in the anode biofilms at 1.0 V (27.39%), 0.5 V (15.89%), and 1.5 V (1.62%), while its abundance ranged from 0.76% to 0.44% in the remaining biofilms. Compared with the low abundance in the control sample (0.01%.), the externally applied voltage promoted the establishment of exo-electroactive biofilms on the carbon electrode-anodes in all the MES-AD reactors and affected the acetate oxidation and electron transport. The reduced populations at 0.5 and 1.5 V might have been generated by processes, such as substrate competition

(fermentation), methanogenesis, and respiration (if oxygen intrudes), that avoid the inherent conversion of a fraction of the substrate into anodophilic biomass (Pham et al., 2009; Speers and Reguera, 2012). Another identifiable change was the increment of beta-proteobacteria only on the suspended biofilm (5.02%) in the 1.0 V reactors. This class is the most dominant group in propionate-, butyrate-, and acetate-utilizing microbial communities in anaerobic digesters (Yu et al., 2014). Accordingly, the high abundance of beta-proteobacteria might lead to VFA consumption. A higher Spirochaetes growth was also observed in bioanodes (2.55%– 6.49%), biocathodes (2.57%–4.34%), and suspended biofilm (1.19%–1.48%) in comparison to that of the control (0.51%). Spirochaetes utilized carbohydrates and produced H2 and CO2 along with other fermentation products, such as acetate, lactate, and ethanol. Spirochaetes are normally low in abundance in most anaerobic reactors, but persist under low-substrate conditions because of several mechanisms, including nutrient-seeking chemotaxis and the ability to derive energy from polymeric storage molecules, amino acids, or intracellular RNA (Dykstra and Pavlostathis, 2017). Thermotogae also showed increased abundance in all the MES-AD biofilms (5.39%–10.39%) compared to the control biofilm (4.46%), except for the C1.0 biocathode (3.64%). These bacteria ferment a wide array of carbohydrates and amino acids known to produce H2 (Dykstra and Pavlostathis, 2017). The presence of Spirochaetes and Thermotogae may play a role in the oxidation of more complex substrates (i.e., EPS and dead cells) together with Deltaproteobacteria at the anode. Meanwhile, at the cathode, the resulting H2 and fermentation products could nourish methanogens and other bacteria (Dykstra and Pavlostathis, 2017). Caldisericia were present in the least abundance in all the MES-AD biofilms (2.67%–5.39%) with respect to the control (5.60%). Similarly, the relative abundances of Bacteroidia were reduced in all the MES-AD environments (2.33%–4.92%) with respect to growth on the control biofilm (8.43%). Anaerolineae predominantly populated the control biofilm (5.63%) instead of the MES-AD environment (2.67%–5.39%). Synergistia

was least predominant in all the electrode biofilms (1.26%–2.07%) than in the suspended (2.59%–3.21%) and control biofilms (3.01%). These decreased abundances in the MES-AD environments, especially in the electrodes, suggested microenvironments that could hostspecific communities. Furthermore, members of Caldisericia, Bacteroidia, Synergistia, and Anaerolineae were involved in syntrophic and synergistic interactions with exoelectrogen and methanogen communities in bioelectrochemical systems (Hari et al., 2017). Fig. 6. Evident changes were identified at the genus level (Fig. 6B). Optimal biofilm growth and current production were expected in all anode biofilms because acetate is the preferred substrate for Geobacter (Speers and Reguera, 2012). Nevertheless, these anode-respiring bacteria dominated at the A0.5 bioanode and the A1.0 bioanode. The total amount of coulombs was higher in bioelectrochemical operations at 1.0 V (6,385 C) by approximately 10 and 19 times, respectively than at 0.5 V (619 C) and 1.5 V (342 C) (Flores-Rodriguez et al., 2019). The MES-AD at 1.0 V produced the maximum volume (166.14 mL) of biomethane, which was higher by approximately 3.34, 2.98, and 1.56 than those of the control (49.69 mL), 1.5 V (55.76 mL) and 0.5 V (106.24 mL), respectively (Flores-Rodriguez et al., 2019). This result highlights the importance of the optimal applied voltage in manipulating the microbial and metabolic diversity of the anode biofilms for better-performing bioelectrochemical systems. Furthermore, the methanogen enrichment on the anode biofilms possibly contributed to the methane production enhancement by directly accepting the electrons from the Geobacter species (Rotaru et al., 2014a, 2014b). The bacterial compositions for the rest of the biofilms were similar, except for the biocathode operated at 1.0 V, thereby showing the presence of Decholoromonas spp., whose growth occurred with acetate and propionate as the electron donors (Wolterink et al., 2005). These were particularly

shown to dominate in the anode chambers of MFCs and MESs fed with propionate compared to acetate, which can be directly utilized by exoelectrogens (Fu et al., 2013; Hari et al., 2017). However, at the cathode, the presence of carbohydrates and proteins in the extracellular matrix and in the dead cells could act as a source of substrate for hydrolytic and fermentative bacteria, leading to the production of unknown metabolic intermediates that could act as a carbon source for Decholoromonas. In previous studies, members of the Bacteroidetes and Firmicutes phyla were found as H2-producing electroactive microorganisms in an autotrophic electroactive biofilm, in which only growth was sustained when using a biocathode as the sole electron source for the proton reduction to H2 (Jourdin et al., 2015). In this study, members of the Proteiniborus, Proteiniphilum, and Lutaonella phyla presented a noticeable growth on the C1.0 biocathode. They might have provided biochemical or bioelectrochemical H2 to hydrogenotrophic methanogens and other labile products for biofilm matrix maintenance (Arun et al., 2009; Dykstra and Pavlostathis, 2017). Considering their substantial presence in the C1.0 biocathode, they are noteworthy candidates for future study in terms of their role in the cathode biofilm community. The minor, but not less important microbiota (<1.0%) consisted of the genus Pelotomaculum, which was only present at the biocathodes (data not shown). This genus was found in syntrophic relationships with hydrogenotrophic methanogens. Ishi et al. (2005) demonstrated that P. thermopropionicum SI and M. thermautotrophicus has a co-aggregated relationship regarding the H2 flux during propionate oxidation (Ishii et al., 2005). These results suggest that bacteria control the H2 flux along with an H2-consumer on the C1.0 biofilm and imply a significant contribution to the biocathodic catalytic activity. Among the ubiquitous genera, fermenters, protein degraders, H2 producer bacteria, and acetate producer bacteria (Coprothermobacter, unclassified Bacteroidia, Porphyromonadaceae, Levilinea, and Thermotogae) could also play an important role in nourishing biofilms (Dykstra and Pavlostathis, 2017; Jourdin et al., 2015;

Tandishabo et al., 2012). The reactors were not fed with any sulfur compound; hence, the presence of Caldisericum may have originated from the inoculum (Mori et al., 2019). Thus, considering the cathodic bioelectrochemical contributions for methane production at 0.5 V (17%), 1.0 V (112%), and 1.5 V (18%) (Flores-Rodriguez et al., 2019), we conclude herein that the growth of exoelectrogens at the anode and the synergistic relationships between bacteria and the methanogenic community at the cathode operated at 1.0 V could be the most beneficial for methane production. 3.3. Finding possible electromethanogenesis routes at the 1.0 V biocathode Further analysis was conducted using the dominant OTUs in the biocathode to explore the enhanced methanogenesis in this system. The enrichment of Methanobacterium at the biocathode could be attributed to both hydrogen evolution by hydrogen producers and the electrons derived from the cathode (Blasco-Gomez et al., 2017). The dominance of Methanobacterium alcaliphilum, which is an alkaliphilic methanogen, suggested that the microbial community composition was affected by the consumption of protons (H+), which increased the local pH and influenced the methane production and the microbial community structure at the cathode matrix (Cai et al., 2018). It also suggests that putative hydrogenproducing bacteria were enriched on the biocathode. The bacterial community analysis indicated that the phylotypes similar to Proteiniphilum acetatigenes and Proteiniborus ethanoligenes may have played an essential role in sustaining hydrogenotrophic methanogens (Dykstra and Pavlostathis, 2017). The other phylotype, Lutaonella sp., is known for utilizing organic and amino acids (Arun et al., 2009), but its role in the AD of methane is still unknown. Bacteroidete phylotypes have been involved in the hydrogen evolution on biocathodes (Croese et al., 2011; Jourdin et al., 2015); hence, the Lutaonella phylotype could be a noteworthy candidate for studying its possible role in sustaining hydrogenotrophic

methanogenic biofilms. Alkaliphilic bacteria are also expected to enrich the biocathode, as is the case of Alkalitalea saponilacus, a carbohydrate consumer that could produce labile products for propionate and acetate consumers (Zhao and Chen, 2012). Clostridia have generally been found in other biocathodes that bioelectrochemically produce hydrogen (Croese et al., 2011). Their presence in biocathodes, including in this study, suggests that they are capable of using an electrode as an electron source. Even though the other Geobacter spp. formed part of the minor species (< 1%), they might contribute to the transfer of electrons to Methanosaeta spp. At the anode, the Geobacter anodireducens dominated the bioanode, and this was accompanied by an increase in the abundance of methanogens (Methanosaeta thermophila, Methanolinea tarda, and Methanobacterium alcaliphilum). Studies have revealed that Geobacter spp. and Methanosaeta spp. can exchange electrons via a DIET because Methanosaeta possesses a complete complement of genes for the enzymes necessary for the reduction of carbon to methane (Rotaru et al., 2014a, 2014b). The presence of Methanolinea tarda and Methanobacterium alcaliphilum suggests that they likely played a role as the electron sink, although their specific role was not clearly understood herein. An increase of the bacterial phylotypes (Spirochaetes, Thermotogae, Synergistetes, and Firmicutes) was also observed. The members of these phylotypes are well-known fermenters that play an essential role in bioanodes in generating fermentation products that are later consumed by exoelectrogens, which can add methane production through syntrophic interactions (Hari et al., 2017, 2016). 3.4. Implications In addition to the previous report (Flores-Rodriguez et al., 2019), this study showed that the applied voltage to an MES-AD could affect not only electrochemical processes but also

microbial metabolic reactions at different locations in the rector. Additionally, the result allowed us to hypothesize that the input of voltage enhanced the specific enrichment of certain exoelectrogens, hydrophilic methanogens, and H2-producing bacteria. Enhanced abundance of Geobacter sp. was observed on the anode and Methanobacterium sp. on the cathode, which could be indicatives of an improved AD process in a single chamber MESAD. H2-producing bacteria has been previously studied to possibly take part in the electromethanogenesis process (Blasco-Gomez et al., 2017). Thereby, Bioaugmentation of these bacteria or the addition of H2-producing bacteria-methanogens conglomerates could be suggested for the enhancement of methane generation by the hydrogenotrophic pathway. The enhanced reductions of nitrate and vanadium were also observed with applied voltages, which promoted specific microbial growth and their metabolic activity (Zhang et al. 2014; Hao et al. 2015). Moreover, three are some insights for practical application of singlechambered MES-AD technology with culture adaptations at high concentrations (2000 mg.L−1), at which the inhibitions can be accompanied in AD processes (> 767 mg.L−1) (Ahring et al., 1995; Franke-Whittle et al., 2014; Wang et al., 2009). The microbial adaptation and growth at 1.0 V applied voltage could be applied for reinforcing electrode biofilm development and therefore stable and enhanced anaerobic digestion could be possible with high loading, high ammonia concentrations and other complex substrates (Florentino et al., 2019; Xu et al., 2019). Furthermore, with a better understanding of the functioning of this microbial community at an optimal input voltage, it could guide future reactor operation as well as the process of consortium adaptation and/or bioaugmentation of related microorganisms to significantly shorten the duration of substrate degradation and enhance the methane production. MES-AD approach is still in development, and as a new technology, it is required to consider reactor operations at different conditions to understand the essential functioning of the AD

microbiome over time, and to make biggest contributions to the AD process. Thus, a better understanding of the functioning microbial community, the closer is the gap to overcome the main limitations in the conventional AD processes. Reactor operation at a laboratory scale is required as the first step to understand and improve MES-AD technologies, and for their real application and commercialization, further scale-up operations with various substrates need to be assessed under real field conditions.

Conclusion This study expands our understanding that the application of voltages (0.5, 1.0, and 1.5 V) can enhance the growth of specific microbiome in bioelectrodes for improved methane production. The MES-AD operation at 1.0 V particularly resulted in more favorable environments for high methane production by the Geobacter enrichment on the anode and Methanobacterium species on the cathode. The coexistence of these communities in bioanodes may suggest a common phenomenon in methanogenic environments, and that might be relevant with respect to the methane production. Deeper investigations are clearly required to elucidate the importance and potential of bioelectrochemical reactions in both electrodes. Acknowledgments The study was carried out with research grants from Gyeonggi Green Environment Center (17-06-3-10-12), the National Research Foundation of Korea (2015R1D1A1A09059935, 2018R1A2B6001507) Appendix A. Supplementary E-supplementary data for this work can be found in e-version of this paper online.

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Figures

Figure 1. Venn diagram of the number of common/unique OTUs within anode-A, cathode-C and suspended-S biofilm of each MES-AD (0.5, 1.0 and 1.5 V) and Control biofilm. Figure 2. nMDS, PCO, and hierarchical cluster analysis based on the BrayeCurtis index of communities from anode-A, cathode-C and suspended-S biofilm of each MES-AD (0.5, 1.0 and 1.5 V) and Control biofilm. Figure 3. Microbial community classification by phylum-level from anode-A, cathode-C and

suspended-S biofilm of each MES-AD (0.5, 1.0 and 1.5 V) and Control biofilm. Figure 4. Overall microbial community classification by phylum-level from each MES-AD (0.5, 1.0 and 1.5 V) and Control biofilm. Figure 5. Class (A) and Genus (B) classification of Archaea 16S rRNA gene from anode-A, cathode-C and suspended-S biofilm of each MES-AD (0.5, 1.0 and 1.5 V) and Control biofilm. Figure 6. Class (A) and Genus (B) classification of Bacteria 16S rRNA gene from anode-A, cathode-C and suspended-S biofilm of each MES-AD (0.5, 1.0 and 1.5 V) and Control biofilm.

Figure 7.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

Tables Table 1. Comparison of diversity indices for bacterial 16S rRNA gene sequences retrieved from MES-AD anode -A, cathode -C, suspended biofilm -S and control -C0 biofilm.

Name of samples

No. of sequences

GoodsCoverage

No. Of

Chao1

Shannon

Simpson

421

502.4286

4.3991

0.8934

OTUs

reads

A1.0

53,892

0.9983

371

431.6818

3.8678

0.8611

A.1.5

45,600

0.9979

421

524.8372

4.4588

0.8903

C0.5

56,398

0.9982

456

541.8500

4.2892

0.8582

C1.0

59,960

0.9984

436

511.0968

4.3309

0.8510

C1.5

59,404

0.9985

474

537.4242

4.4586

0.8825

S0.5

46,285

0.9977

423

513.7797

4.3458

0.8745

S1.0

51,604

0.9980

402

478.1091

4.3942

0.8879

S1.5

43,845

0.9981

442

552.2500

4.4935

0.8895

Control

46,077

0.9980

408

479.9423

4.6634

0.8951

Highlights



Applied voltage significantly affected the microbiome distribution in MES-AD



Hydrogenotrophic methanogens was favorably enriched on the cathode at 1.0 V



Syntrophic growths was found on the cathode for enhanced methanogenic process

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The abundance of methanogen was observed on the anode with exoelectrogens

Author Contributions

1. Carla Flores-Rodriguez: Methodology, Formal analysis, Investigation, Writing – Original Draft, Revisions 2. Booki Min: Conceptualization, Writing-Review & Editing, Supervision