Comparison of Anodic Community in Microbial Fuel Cells with Iron Oxide-Reducing Community

Abstract

The group of Fe(III) oxide-reducing bacteria includes exoelectrogenic bacteria, and they possess similar properties of transferring electrons to extracellular insoluble-electron acceptors. The exoelectrogenic bacteria can use the anode in microbial fuel cells (MFCs) as the terminal electron acceptor in anaerobic acetate oxidation. In the present study, the anodic community was compared with the community using Fe(III) oxide (ferrihydrite) as the electron acceptor coupled with acetate oxidation. To precisely analyze the structures, the community was established by enrichment cultures using the same inoculum used for the MFCs. High-throughput sequencing of the 16S rRNA gene revealed considerable differences between the structure of the anodic communities and that of the Fe(III) oxide-reducing community. Geobacter species were predominantly detected (>46%) in the anodic communities. In contrast, Pseudomonas (70%) and Desulfosporosinus (16%) were predominant in the Fe(III) oxide-reducing community. These results demonstrated that Geobacter species are the most specialized among Fe(III)-reducing bacteria for electron transfer to the anode in MFCs. In addition, the present study indicates the presence of a novel lineage of bacteria in the genus Pseudomonas that highly prefers ferrihydrite as the terminal electron acceptor in acetate oxidation.

Introduction

Microbial fuel cells (MFCs) are environmentally friendly bioreactors that simultaneously perform sustainable bioenergy production and wastewater treatment [11]. Bacteria decompose the organic matter in wastewater to CO2 through redox reactions under anaerobic conditions in the reactors. The electrons generated in these redox reactions are transferred to the anode by bacteria, where they react with electron acceptors such as O2 and K3[Fe(CN)6] via a circuit in the cathodes. The anode serves as an insoluble extracellular electron acceptor in these redox reactions, called electrode respiration. Many bacteria, including Geobacter, Shewanella, Desulfuromonas, and Rhodopseudomonas, can mediate electron transfer to the anode [13]. However, the mechanism of formation of an electrochemically active biofilm on the anode is not well understood. Elucidating this mechanism would provide vital clues to improving the power output of MFCs.

In anoxic natural environments, bacteria decompose acetate to CO2 using soluble and insoluble electron acceptors, such as sulfate, nitrate, and Fe(III) oxides. The mechanism of electron transfer to the anode is suggested to be similar to the transfer to insoluble Fe(III) oxides. In fact, exoelectrogenic bacteria comprise many Fe(III) reducers, such as Geobacter species [2]. Given that the anode acts as a substitute for Fe(III) oxide, it is predicted that the anodic community (AC) and Fe(III)-reducing community (FRC) would have similar structures. In the present study, to test this hypothesis, ACs in three MFCs and a FRC were established by enrichment cultures, and the structure of the bacterial communities was analyzed using high-throughput sequencing. For comparison, a sulfate-reducing community (SRC) was also analyzed.

 

Materials and Methods

Bacterial Culture

MFC32 was a two-chambered reactor comprising two glass bottles (500 ml) and a Nafion 117 membrane (Dupont Japan, Tokyo, Japan). The anode and cathode used were a carbon cloth (5 cm × 5 cm) and a carbon rod (diameter, 0.5 cm; length, 15 cm), respectively. The anolyte was a basal medium containing (per liter of distilled water) 0.98 g potassium acetate, 0.6 g NaH2PO4·2H2O, 2 g NaHCO3, 2.9 g NaCl, 0.1 g KCl, 0.2 g NH4Cl, 1 mg resazurin, 41 mg 2-bromoethanesulfonate sodium salt (a methanogenesis inhibitor), 50 mg Na2S·9H2O, and 10 ml of trace mineral and vitamin solutions. The catholyte was composed of 16.5 g K3[Fe(CN)6], 0.6 g NaH2PO4·2H2O, 2 g NaHCO3, 2.9 g NaCl, 0.1 g KCl, and 0.2 g NH4Cl. MFC35 and MFC36 were cubic air-cathode single-chambered reactors (125 ml) fed with the basal medium. The carbon-cloth anode (5 cm × 5 cm) was placed at the opposite side to the carbon-cloth cathode containing 0.5 mg/cm2 of a Pt catalyst fused with the membrane. Activated sludge collected at the NARO Institute of Livestock and Grassland Science, Tsukuba, Japan, was inoculated into MFC32 and MFC35 as seed sludge, whereas cattle feces was inoculated into MFC36. The MFCs were connected to an external resistor and were operated at 30℃ in the fed-batch mode. The external resistor was adjusted such that the MFCs generated a voltage of 0.5–0.6 V. The FRC and SRC were established by enrichment culture in 15 ml test tubes containing a gas phase of 100% N2. The test tubes were filled with 10 ml of the basal medium supplemented with poorly crystalline Fe(III) oxyhydroxide (ferrihydrite; 15 mM) or Na2SO4 (20 mM) for the FRC and SRC, respectively. Ferrihydrite was prepared by titrating a FeCl3 solution against 10% NaOH [19], and it was stored in a glass bottle with a gas phase of nitrogen until use. Activated sludge (1 ml), identical to that used for the MFC, was inoculated into the test tubes, and the test tubes were statically incubated at 30℃. The culture medium (1 ml) was transferred into fresh medium at an interval of 1–4 weeks, and the transfer was repeated 10 times. Coulombic efficiency was estimated from the amount of electron flow and decrease in acetate concentration [14]. The Fe(II) concentration was determined photometrically using the phenanthroline method [6].

High-Throughput Sequencing

Next-generation sequencing was performed with the MiSeq Illumina sequencing platform (Illumina Inc., CA, USA) using the V4 region of the 16S rRNA gene [10]. The biofilms developed on the anodes were extensively washed with distilled water for removing the bacteria loosely attached to the anodes. The genomes were extracted from the washed biofilms with an UltraClean Soil DNA Isolation kit (MO BIO Laboratories, Carlsbad, CA, USA). To prepare the genomes of the FRC and SRC, the cultured medium was centrifuged at 12,000 ×g for 15 min. The precipitates were washed with distilled water, and the genomes were extracted from the precipitates using the kit. Nearly the full length of the 16S rRNA gene fragment was amplified by polymerase chain reaction (PCR) with the 27F and 1492R primers using the KAPA HiFi HotStart ReadyMix PCR Kit (Kapa Biosystems, MA, USA). Subsequently, the second round of PCR was conducted using 563F and 802R primers, including the Illumina overhang adapter sequences, according to the manufacturer’s instructions. The libraries were sequenced on a 300PE MiSeq run, and image analysis, base calling, and data quality assessment were performed with the MiSeq Reporter software (Illumina). Paired-end read data exported in the FASTQ format were processed with the Quantitative Insights Into Microbial Ecology (QIIME software ver. 1.8) pipeline [4]. The read sequences were joined, quality-checked, and clustered into operational taxonomic units (OTUs) using the Uclust method [5]. Representative sequences were aligned using PyNAST [3], and a phylogenetic tree was constructed. After a chimera check, the taxonomic classification and alpha and beta diversities were computed using the QIIME tool. The taxonomic assignment of the major OTUs was checked using Classifier [18]. The beta diversity was calculated using an unweighted UniFrac distance matrix [15], and the result was visualized using a principal coordinate (PCo) plot.

The sequencing data were deposited in DDBJ under the accession numbers LC071702-LC071715 and DRR040632-DRR040636 (Sequence Read Archive).

 

Results and Discussion

MFC Operation and Enrichment Cultures of FRC and SRC

To precisely compare the community structures, all communities, except MFC36, were cultured using the same inoculum (activated sludge) and the same medium containing acetate as the sole carbon and energy source; cattle feces were inoculated into MFC36. The MFCs were operated for 8 months. The profiles of electricity generation by the MFCs are shown in Fig. 1. Usually, higher electricity generation is obtained with the use of K3[Fe(CN)6] than with O2 as an electron acceptor in the cathode. Air-cathode single-chambered MFCs generally exhibit lower Coulombic efficiency than double-chambered MFCs because of O2 intrusion from the membrane. Consistent with these previous observations, MFC32 (two-chambered MFC with K3[Fe(CN)6]) generated a higher current (1.0–1.5 mA) than the air-cathode single-chambered MFC35 and MFC36 (0.2–0.3 mA). The Coulombic efficiency of MFC32 was approximately 71–85%, which was higher than that of MFC35 (32–33%). The FRC decomposed acetate coupled with reduction of Fe(III) oxide. The stoichiometry was acetate:Fe(III) = 1:6.8, which was close to the expected ratio of 1:8. The SRC consumed acetate and sulfate with the stoichiometry of acetate:sulfate = 1:1.1. This value was also close to the expected ratio of 1:1. These values of stoichiometry verified the integrity of the FRC and SRC activities.

Fig. 1.Time course of electricity generation by MFC32 (top), MFC35 (middle), and MFC36 (bottom).

Community Structure

Next-generation sequencing technology is a powerful tool for analyzing the structure of microbial communities at extremely high resolution. The numbers of reads sequenced and the OTUs and alpha diversity of the communities are summarized in Table 1. The Chao1 richness was 12,000–18,000 for the ACs and 10,000–11,000 for the FRC and SRC. Although Good’s coverage was more than 0.99 in all the communities, none of the rarefaction curves reached a plateau (Fig. 2A). In the beta diversity analysis, the ACs formed a cluster in the PCo plot (Fig. 2B), suggesting that the AC structures were similar to each other. Surprisingly, the distance of the cluster to the FRC was almost identical to that to the SRC. These results show that the AC structures were considerably different from that of the FRC, and that the degree of the difference between the AC and FRC structures was similar to that between the AC and SRC structures. The physical and electrochemical properties of the anode surface would differ from that of the ferrihydrite surface.

Table 1.The OTUs and alpha diversity indices of the anodic communities (MFC32, MFC35, and MFC36) and Fe(III)- and sulfate-reducing communities (FRC and SRC, respectively) were calculated using QIIME.

Fig. 2.Rarefaction curves (A) and PCo plot (B) showing the relationship between the anodic communities (MFC32, MFC35, and MFC36) and Fe(III)- and sulfate-reducing communities (FRC and SRC, respectively).

Taxonomic Assignment

Deltaproteobacteria were detected at high frequency (50–91%) in the ACs, whereas Gammaproteobacteria (71%) were predominant in the FRC (Fig. 3). Deltaproteobacteria (38%), Bacteroidia (31%), and Gammaproteobacteria (23%) were abundant in the SRC. At the genus level, Geobacter accounted for 46–90% of the ACs. Geobacter species were more abundant in the AC of MFC32 (90%) than in the AC of MFC35 and MFC36 (46–67%) because the double-chambered MFC32 was more anaerobic than the single-chambered MFC35 and MFC36. Approximately 90% of the reads affiliated to Geobacter species in the ACs showed the highest similarity to Geobacter chapellei among all the Geobacter species (Fig. 4A). In the FRC, Geobacter species were detected at a very low frequency of 0.7%, which is inconsistent with the results of previous studies [8,12]. This inconsistency may have resulted from differences in the type of inocula, medium composition, and ferrihydrite preparation. For example, Geobacter-predominant communities (>50%) have been obtained by enrichment cultures with ferrihydrite and acetate using anoxic rice paddy soil [8] and the sediments of a pond [12] as the inocula.

Fig. 3.Class (A) and genus (B) distributions of the anodic communities (MFC32, MFC35, and MFC36) and Fe(III)- and sulfatereducing communities (FRC and SCR, respectively) based on the 16S rRNA gene sequences.

Fig. 4.Neighbor-joining phylogenetic trees showing the relationship between OTUs and Geobacter (A), Pseudomonas (B), or Desulfitobacterium (C) species. The percentages represent the number of reads assigned to the OTUs per number of reads assigned to the genus in the anodic communities in the MFCs or Fe(III)-reducing community (FRC). OTUs with a frequency higher than 1% are shown. Numbers on major branch points indicate bootstrap values. The scale bars represent a 0.5% difference in the DNA sequences.

Unexpectedly, Pseudomonas was the most predominant genus (70%) in the FRC. The high prevalence of Pseudomonas in the FRC is a rare case. Pseudomonas comprises diverse species, including denitrifying bacteria. Although the Fe(III)-reducing or electricity-generating activity for most Pseudomonas species has not been examined, Pseudomonas sp. strain 200 has been reported to reduce Fe(III) [1]. OTU813-FRC was detected at the highest frequency (73%) among the OTUs assigned to Pseudomonas. This implies that bacteria affiliated with OTU813-FRC efficiently obtain energy to grow from ferrihydrite reduction coupled with acetate oxidation, as acetate and ferrihydrite were added as the sole electron donor and acceptor, respectively, in the medium. OTU813-FRC displayed 93% identity to Pseudomonas aeruginosa, which is known to produce electricity in an MFC (Fig. 4B) [17]. Desulfitobacterium was the second predominant genus (16%) in the FRC. Desulfitobacterium hafniense, an exoelectrogen [16], was one of the most closely related species to the OTUs assigned to Desulfitobacterium (Fig. 4C). Some Desulfitobacterium species can reduce Fe(III) citrate and Mn(IV) oxide in addition to chlorinated compounds [7]. Desulfovibrio species were detected in both communities; the prevalence was 2.5% in the FRC and 0.6–5.9% in the ACs (Fig. 3). Desulfovibrio contains the exoelectrogenic species Desulfovibrio desulfuricans [9].

Based on these observations, potent exoelectrogenic bacteria and Fe(III)-reducing bacteria are abundantly included in the FRC, although the community structure was markedly different between the ACs and FRC. The anode is an artificial material that is not present in the natural environment. Nevertheless, Geobacter species can use the anode as the terminal electron acceptor. Bacteria likely possess the plasticity to use various extracellular electron acceptors, including the artificial material of the anode, to adapt to their environment. The present study has demonstrated that Geobacter species are the most specialized among Fe(III)-reducing bacteria for electron transfer to the anode coupled with acetate oxidation. In addition, this study indicates the presence of a novel lineage of bacteria (OTU813-FRC) in the genus Pseudomonas that highly prefers ferrihydrite as the terminal electron acceptor.