Methylomusa anaerophila gen. nov., sp. nov., an anaerobic methanol-utilizing bacterium isolated from a microbial fuel cell.

Abacterial strain, designated MMFC1T, was isolated from a methanol-fed microbial fuel cell that had been inoculated with sludge obtained from a wastewater-treatmentfacility in a chemical plant. The strain grows by fermenting methanol to produce acetate under anaerobic conditions, while homoacetogenic growth is not observed. MMFC1T also grows on pyruvate and lactate but not on sugars and other organic acids. Cells are curved rods and motile, have peritrichous flagella, and form endospores. The genome sequence of strain MMFC1T supports the physiological data. Phylogenetic analysis based on the 16S rRNA gene sequence shows that strain MMFC1T is affiliated with the family Sporomusaceae, while the closest relative is Sporomusa ovata with nucleotide-sequencesimilarity of 93.5 %. Major fatty acids are iso-C13 : 0 3-OH, C16 : 1ω9 and iso-C17 : 0. On the basis of its physiological, genomic and phylogenetic features, a novel genus and species are proposed to accommodate strain MMFC1T, with the name Methylomusa anaerophila gen. nov., sp. nov. The type strain of Methylomusa anaerophila is MMFC1T (=JCM 31821T = KCTC 15592T).

Comparative evaluation of wastewater-treatment microbial fuel cells in terms of organics removal, waste-sludge production, and electricity generation.

Microbial fuel cells (MFCs) are devices that exploit living microbes for electricity generation coupled to organics degradation. MFCs are expected to be applied to energy-saving wastewater treatment (WWT) as alternatives to activated-sludge reactors (ASRs). Although extensive laboratory studies have been performed to develop technologies for WWT-MFCs, limited information is available for comparative evaluation of MFCs and ASRs in terms of organics removal and waste-sludge production. In the present study, laboratory WWT experiments were performed using cassette-electrode MFCs and ASRs that were continuously supplied either with artificial domestic wastewater (ADW) containing starch and peptone or with artificial industrial wastewater (AIW) containing methanol as the major organic matter. We found that these two types of WWT reactors achieved similar organics-removal efficiencies, namely, over 93% based on chemical oxygen demands for the ADW treatment and over 97% for the AIW treatment. Sludge was routinely removed from these reactors and quantified, showing that amounts of waste sludge produced in MFCs were approximately one-third or less compared to those in ASRs. During WWT, MFCs continuously generated electricity with Coulombic efficiencies of 20% or more. In reference to ASRs, MFCs are demonstrated to be attractive WWT facilities in terms of stable organics removal and low waste-sludge production. Along with the unnecessity of electric power for aeration and the generation of power during WWT, the results obtained in the present study suggest that MFCs enable substantial energy saving during WWT.

Anode macrostructures influence electricity generation in microbial fuel cells for wastewater treatment.

Microbial fuel cells (MFCs) are devices that exploit microbes for generating electricity from organic substrates, including waste biomass and wastewater pollutants. MFCs have the potential to treat wastewater and simultaneously generate electricity. The present study examined how anode macrostructure influences wastewater treatment, electricity generation and microbial communities in MFCs. Cassette-electrode MFCs were equipped with graphite-felt anodes with three different macrostructures, flat-plate (FP), vertical-fin (VF), and horizontal-fin (HF) structures (these were composed of a same amount of graphite felt), and were continuously supplied with artificial wastewater containing starch as the major organic constituent. Polarization analyses revealed that MFCs equipped with VF and HF anodes generated 33% and 21% higher volumetric power densities, respectively, than that of MFCs equipped with FP anodes. Organics were also more efficiently removed from wastewater in MFCs with VF and HF anodes compared to reactors containing FP anodes. In addition, pyrosequencing of PCR-amplified 16S rRNA gene fragments from microbial samples collected from the anodes showed that the presence of fins also affected the bacterial compositions in anode biofilms. Taken together, the findings presented here suggest that the modification of anodes with fins improves organics removal and electricity generation in MFCs. The optimization of anode macrostructure therefore appears to be a promising strategy for improving MFC performance without additional material costs.

Electricity generation from rice bran in microbial fuel cells.

BACKGROUND: Rice bran is a by-product of the rice milling process and mostly discarded in Japan. Although many studies have shown that microbial fuel cells (MFCs) are able to generate electricity from organic wastes, limited studies have examined MFCs for generating electricity from rice bran. FINDINGS: Laboratory-scale single-chamber MFCs were inoculated with paddy field soil and supplied with rice bran for examining electricity generation. Power outputs and microbiome compositions were compared between MFCs containing pure water as the liquid phase (MFC-W) and those containing mineral solution (MFC-M). Polarization analyses showed that both MFCs successfully generated electricity with the maximum power densities of 360 and 520 mW m-2 (based on the projected area of anode) for MFC-W and MFC-M, respectively. Amplicon-sequencing analyses revealed that Trichococcus and Geobacter specifically occurred in anode biofilms in MFC-W and MFC-M, respectively. CONCLUSIONS: The results suggest that rice bran is a feasible fuel by itself for generating electricity in MFCs.

Poly iron sulfate flocculant as an effective additive for improving the performance of microbial fuel cells.

Laboratory microbial fuel cells were supplied with artificial wastewater and used to examine how supplementation with poly iron sulfate, an inorganic polymer flocculant widely used in wastewater-treatment plants, affects electricity generation and anode microbiomes. It is shown that poly iron sulfate substantially increases electric outputs from microbial fuel cells. Microbiological analyses show that iron and sulfate separately affect anode microbiomes, and the increase in power output is associated with the increases in bacteria affiliated with the families Geobacteraceae and/or Desulfuromonadaceae. We suggest that poly iron sulfate is an effective additive for increasing the electric output from microbial fuel cells. Other utilities of poly iron sulfate in microbial fuel cells are also discussed.

Sodium chloride concentration determines exoelectrogens in anode biofilms occurring from mangrove-grown brackish sediment.

Single-chamber microbial fuel cells (MFCs) were inoculated with mangrove-grown brackish sediment (MBS) and continuously supplied with an acetate medium containing different concentrations of NaCl (0-1.8M). Different from MFCs inoculated with paddy-field soil (high power outputs were observed between 0.05 and 0.1M), power outputs from MBS-MFCs were high at NaCl concentrations from 0 to 0.6M. Amplicon-sequence analyses of anode biofilms suggest that different exoelectrogens occurred from MBS depending on NaCl concentrations; Geobacter occurred abundantly below 0.1M, whereas Desulfuromonas was abundant from 0.3M to 0.6M. These results suggest that NaCl concentration is the major determinant of exoelectrogens that occur in anode biofilms from MBS. It is also suggested that MBS is a potent source of microbes for MFCs to be operated in a wide range of NaCl concentrations.

Evaluation of microbial fuel cells for electricity generation from oil-contaminated wastewater.

Large quantities of oils and fats are discharged into wastewater from food industries. We evaluated the possibility of using microbial fuel cells (MFCs) for the generation of electricity from food-industry wastewater containing vegetable oils. Single-chamber MFCs were supplied with artificial wastewater containing soybean oil, and oil removal and electric output were examined under several different conditions. We found that MFC performance could be improved by supplementing wastewater with an emulsifier, inoculating MFCs with oil-contaminated soil, and coating the graphite-felt anodes with carbon nanotubes, resulting in a power output of more than 2 W m-2 (based on the projected area of the anode). Sequencing of polymerase chain reaction (PCR)-amplified 16S rRNA gene fragments detected abundant amount of Burkholderiales bacteria (known to include oil degraders) in the oil-contaminated soil and anode biofilm, whereas those affiliated with the genus Geobacter were only detected in the anode biofilm. These results suggest that MFCs can be used for energy recovery from food industry wastewater containing vegetable oils.

Genomic features of uncultured methylotrophs in activated-sludge microbiomes grown under different enrichment procedures.

Methylotrophs are organisms that are able to grow on C1 compounds as carbon and energy sources. They play important roles in the global carbon cycle and contribute largely to industrial wastewater treatment. To identify and characterize methylotrophs that are involved in methanol degradation in wastewater-treatment plants, methanol-fed activated-sludge (MAS) microbiomes were subjected to phylogenetic and metagenomic analyses, and genomic features of dominant methylotrophs in MAS were compared with those preferentially grown in laboratory enrichment cultures (LECs). These analyses consistently indicate that Hyphomicrobium plays important roles in MAS, while Methylophilus occurred predominantly in LECs. Comparative analyses of bin genomes reconstructed for the Hyphomicrobium and Methylophilus methylotrophs suggest that they have different C1-assimilation pathways. In addition, function-module analyses suggest that their cell-surface structures are different. Comparison of the MAS bin genome with genomes of closely related Hyphomicrobium isolates suggests that genes unnecessary in MAS (for instance, genes for anaerobic respiration) have been lost from the genome of the dominant methylotroph. We suggest that genomic features and coded functions in the MAS bin genome provide us with insights into how this methylotroph adapts to activated-sludge ecosystems.

Effects of NaCl concentration on anode microbes in microbial fuel cells.

Understanding of how operational parameters affect the composition of exoelectrogenic microbes is an important step in the development of efficient microbial fuel cells (MFCs). In the present study, single-chamber MFCs were inoculated with rice paddy-field soil and continuously supplied with an acetate medium containing different concentrations of NaCl (0-1.8 M). Polarization analyses showed that power output increased as the NaCl concentration increased to 0.1 M, while it was markedly diminished over 0.3 M. The increase in power output was associated with an increased abundance of anode microbes as assessed by protein assays. Notably, the power increase was also accompanied by an increase in the abundance ratio of Geobacter bacteria to total anode bacteria as assessed by pyrosequencing of 16S rRNA gene amplicons and specific quantitative PCR. Although most Geobacter species are known to exhibit high growth rates in freshwater media without NaCl, the present study shows that 0.1 M NaCl facilitates the growth of Geobacter in MFC anode biofilms. This result suggests that the optimum salt concentration in MFC is determined by the balance of two factors, namely, the solution conductivity and salt tolerance of exoelectrogens.

Conversion of activated-sludge reactors to microbial fuel cells for wastewater treatment coupled to electricity generation.

Wastewater can be treated in microbial fuel cells (MFCs) with the aid of microbes that oxidize organic compounds using anodes as electron acceptors. Previous studies have suggested the utility of cassette-electrode (CE) MFCs for wastewater treatment, in which rice paddy-field soil was used as the inoculum. The present study attempted to convert an activated-sludge (AS) reactor to CE-MFC and use aerobic sludge in the tank as the source of microbes. We used laboratory-scale (1 L in capacity) reactors that were initially operated in an AS mode to treat synthetic wastewater, containing starch, yeast extract, peptone, plant oil, and detergents. After the organics removal became stable, the aeration was terminated, and CEs were inserted to initiate an MFC-mode operation. It was demonstrated that the MFC-mode operation treated the wastewater at similar efficiencies to those observed in the AS-mode operation with COD-removal efficiencies of 75-80%, maximum power densities of 150-200 mW m(-2) and Coulombic efficiencies of 20-30%. These values were similar to those of CE-MFC inoculated with the soil. Anode microbial communities were analyzed by pyrotag sequencing of 16S rRNA gene PCR amplicons. Comparative analyses revealed that anode communities enriched from the aerobic sludge were largely different from those from the soil, suggesting that similar reactor performances can be supported by different community structures. The study demonstrates that it is possible to construct wastewater-treatment MFCs by inserting CEs into water-treatment tanks.

Effectiveness of heat treatment to protect introduced denitrifying bacteria from eukaryotic predatory microorganisms in a pilot-scale bioreactor.

Bioaugmentation of bioreactor systems with pre-cultured bacteria has proven difficult because inoculated bacteria are easily eliminated by predatory eukaryotic-microorganisms. Here, we demonstrated an intermediate thermal treatment was effective for protecting introduced denitrifying bacteria from eukaryotic predators and consequently allowed the inoculated bacteria to survive longer in a denitrification reactor.

Electricity generation from cattle manure slurry by cassette-electrode microbial fuel cells.

Cassette-electrode microbial fuel cells (CE-MFCs) are efficient and scalable devices for electricity production from organic waste. Previous studies have demonstrated that CE-MFCs are capable of generating electricity from artificial wastewater at relatively high efficiencies. In this study, a single-cassette CE-MFC was constructed, and its capacity for electricity generation from cattle manure suspended in water (solid to water ratio of 1:50) was examined. The CE-MFC reactor was operated in batch mode for 49 days; electricity generation became stable 2 weeks after initiating the operation. The maximum power density was measured at 16.3 W m⁻³ on day 26. Sequencing analysis of PCR-amplified 16S rRNA gene fragments obtained from the original manure and from anode biofilms suggested that Chloroflexi and Geobacteraceae were abundant in the anode biofilm (29% and 18%, respectively), whereas no Geobacteraceae sequences were detected in the original manure sample. The results of this study suggest that CE-MFCs can be used to generate electricity from water-suspended cattle manure in a scalable MFC system.

Bioaugmentation of a wastewater bioreactor system with the nitrous oxide-reducing denitrifier Pseudomonas stutzeri strain TR2.

In bioaugmentation technology, survival of inoculant in the treatment system is prerequisite but remains to be a crucial hurdle. In this study, we bioaugmented the denitrification tank of a piggery wastewater treatment system with the denitrifying bacterium Pseudomonas stutzeri strain TR2 in two pilot-scale experiments, with the aim of reducing nitrous oxide (N(2)O), a gas of environmental concern. In the laboratory, strain TR2 grew well and survived with high concentrations of nitrite (5-10 mM) at a wide range of temperatures (28-40°C). In the first augmentation of the pilot-scale experiment, strain TR2 inoculated into the denitrification tank with conditions (30°C, ~0.1 mM nitrite) survived only 2-5 days. In contrast, in the second augmentation with conditions determined to be favorable for the growth of the bacterium in the laboratory (40-45°C, 2-5 mM nitrite), strain TR2 survived longer than 32 days. During the time when the presence of strain TR2 was confirmed by quantitative real-time PCR, N(2)O emission was maintained at a low level even under nitrite-accumulating conditions in the denitrification and nitrification tanks, which provided indirect evidence that strain TR2 can reduce N(2)O in the pilot-scale system. Our results documented the effective application of growth conditions favorable for strain TR2 determined in the laboratory to maintain growth and performance of this strain in the pilot-scale reactor system and the decrease of N(2)O emission as the consequence.

Use of cassette-electrode microbial fuel cell for wastewater treatment.

Cassette-electrode microbial fuel cells (CE-MFCs) have been developed for the conversion of biomass wastes into electric energy. The present study modified CE-MFC for its application to wastewater treatment and examined its utility in a long-term (240 days) experiment to treat a synthetic wastewater, containing starch, yeast extract, peptone, plant oil, and a detergent (approximately 500 mg of total chemical oxygen demand [COD] per liter). A test MFC reactor (1 l in capacity) was equipped with 10 cassette electrodes with total anode and cathode projection areas of 1440 cm(2), and the operation was initiated by inoculating with rice paddy-field soil. It was demonstrated that CE-MFC achieved COD removal rates of 80% at hydraulic-retention times of 6 h or greater, and electricity was generated at a maximum power density of 150 mW m(-2) and Coulombic efficiency of 20%. Microbial communities established on anodes of CEs were analyzed by pyrosequencing of PCR-amplified 16S rRNA gene fragments, showing that Geobacter, Clostridium, and Geothrix were abundantly detected in anode biofilms. These results demonstrate the utility of CE-MFC for wastewater treatment, in which Geobacter and Geothrix would be involved in the electricity generation.

Survival of the aerobic denitrifier Pseudomonas stutzeri strain TR2 during co-culture with activated sludge under denitrifying conditions.

The aerobic denitrifier Pseudomonas stutzeri TR2 (strain TR2) has the potential to reduce nitrous oxide emissions during the wastewater treatment process. In this application, it is important to find the best competitive survival conditions for strain TR2 in complex ecosystems. To that end, we examined co-cultures of strain TR2 with activated sludge via five passage cultures in a medium derived from treated piggery wastewater that contained a high concentration of ammonium. The results are as follows: (i) The medium supported the proliferation of strain TR2 (P. stutzeri strains) under denitrifying conditions. (ii) Nitrite was a better denitrification substrate than nitrate for TR2 survival. (iii) Strain TR2 also demonstrated strong survival even under aerobic conditions. This suggests that strain TR2 is effectively augmented to the wastewater treatment process, aiding in ammonium-nitrogen removal and reducing nitrous oxide production with a partial nitrification technique in which nitrite accumulates.

Advenella faeciporci sp. nov., a nitrite-denitrifying bacterium isolated from nitrifying-denitrifying activated sludge collected from a laboratory-scale bioreactor treating piggery wastewater.

Strain M-07(T) was isolated from nitrifying-denitrifying activated sludge treating piggery wastewater. Phylogenetic analysis based on 16S rRNA gene sequences demonstrated that strain M-07(T) belonged to the genus Advenella. 16S rRNA gene sequence similarity between M-07(T) and Advenella incenata CCUG 45225(T), Advenella mimigardefordensis DPN7(T) and Advenella kashmirensis WT001(T) was 96.5, 97.3 and 96.9%, respectively. The DNA G+C content of strain M-07(T) was 49.5 mol%, which was approximately 5 mol% lower than the range for the genus Advenella (53.5-58.0 mol%). The predominant cellular fatty acids of strain M-07(T) were C(16:0), summed feature 3 (comprising C(16:1)ω7c and/or iso-C(15:0) 2-OH), C(17:0) cyclo and summed feature 2 (comprising one or more of C(14:0) 3-OH, iso-C(16:1) I, an unidentified fatty acid with an equivalent chain-length of 10.928 and C(12:0) alde). The isoprenoid quinone was Q-8. On the basis of phenotypic characteristics, phylogenetic analysis and DNA-DNA relatedness, strain M-07(T) should be classified as a novel species of the genus Advenella, for which the name Advenella faeciporci sp. nov. is proposed. The type strain is M-07(T) ( = JCM 17746(T)  = KCTC 23732(T)).

Population dynamics and current-generation mechanisms in cassette-electrode microbial fuel cells.

Cassette-electrode microbial fuel cells (CE-MFCs) have been demonstrated useful to treat biomass wastes and recover electric energy from them. In order to reveal electricity-generation mechanisms in CE-MFCs, the present study operated a bench-scale reactor (1 l in capacity; approximately 1,000 cm(2) in anode and cathode areas) for treating a high-strength model organic wastewater (comprised of starch, peptone, and fish extract). Approximately 1 month was needed for the bench reactor to attain a stable performance, after which volumetric maximum power densities persisted between 120 and 150 mW/l throughout the experiment (for over 2 months). Temporal increases in the external resistance were found to induce subsequent increases in power outputs. After electric output became stable, electrolyte and anode were sampled from the reactor for evaluating their current-generation abilities; it was estimated that most of current (over 80%) was generated by microbes in the electrolyte. Cyclic voltammetry of an electrolyte supernatant detected several electron shuttles with different standard redox potentials at high concentrations (equivalent to or more than 100 µM 5-hydroxy-1,4-naphthoquinone). Denaturing gradient gel electrophoresis and quantitative real-time PCR of 16S ribosomal RNA gene fragments showed that bacteria related to the genus Dysgonomonas occurred abundantly in association with the increases in power outputs. These results suggest that mediated electron transfer was the main mechanism for electricity generation in CE-MFC, where high-concentration electron shuttles and Dysgonomonas bacteria played important roles.

Potential of aerobic denitrification by Pseudomonas stutzeri TR2 to reduce nitrous oxide emissions from wastewater treatment plants.

In contrast to most denitrifiers studied so far, Pseudomonas stutzeri TR2 produces low levels of nitrous oxide (N(2)O) even under aerobic conditions. We compared the denitrification activity of strain TR2 with those of various denitrifiers in an artificial medium that was derived from piggery wastewater. Strain TR2 exhibited strong denitrification activity and produced little N(2)O under all conditions tested. Its growth rate under denitrifying conditions was near comparable to that under aerobic conditions, showing a sharp contrast to the lower growth rates of other denitrifiers under denitrifying conditions. Strain TR2 was tolerant to toxic nitrite, even utilizing it as a good denitrification substrate. When both nitrite and N(2)O were present, strain TR2 reduced N(2)O in preference to nitrite as the denitrification substrate. This bacterial strain was readily able to adapt to denitrifying conditions by expressing the denitrification genes for cytochrome cd(1) nitrite reductase (NiR) (nirS) and nitrous oxide reductase (NoS) (nosZ). Interestingly, nosZ was constitutively expressed even under nondenitrifying, aerobic conditions, consistent with our finding that strain TR2 preferred N(2)O to nitrite. These properties of strain TR2 concerning denitrification are in sharp contrast to those of well-characterized denitrifiers. These results demonstrate that some bacterial species, such as strain TR2, have adopted a strategy for survival by preferring denitrification to oxygen respiration. The bacterium was also shown to contain the potential to reduce N(2)O emissions when applied to sewage disposal fields.

Nitrous oxide emission from nitrifying activated sludge dependent on denitrification by ammonia-oxidizing bacteria.

Nitrous oxide (N(2)O) is emitted during the aerated nitrification process of wastewater treatment, but its mechanism is not understood. In this study, we employed a model system to clarify the mechanism of N(2)O emission, utilizing the activated sludge derived from a piggery effluent. Aerated incubation of the sludge with ammonium (NH(4)(+)) or hydroxylamine (NH(2)OH) resulted in the emission of a significant amount of N(2)O. The emission stopped when the nitrification substrate (NH(4)(+) or NH(2)OH) was exhausted. When NH(4)(+) was replaced with nitrate (NO(3)(-)) and nitrite (NO(2)(-)), no N(2)O was emitted. This result suggests that the N(2)O emission under nitrifying conditions did not depend on the oxidation of NO(2)(-) by nitrite-oxidizing bacteria (NOB) or denitrification by heterotrophic denitrifiers but depended on the oxidation of NH(4)(+) by ammonia-oxidizing bacteria (AOB). When NO(2)(-), the product of nitrification by AOB, was added to the NH(4)(+)-oxidizing system, N(2)O emission was enormously enhanced, suggesting that N(2)O was formed via denitrification. Diethyldithiocarbamate (DCD), an inhibitor of copper-containing nitrite reductase (NirK), strongly blocked N(2)O emission from NH(2)OH. Furthermore, the expression of the gene (nirK) encoding NirK of AOB was detected in the sludge exposed to the nitrifying conditions. The results showed that N(2)O emission during the nitrification process depends on denitrification by AOB that reside in the activated sludge. This study provides direct evidence for the cause of N(2)O emission from activated sludge (non-pure culture).

Streptomyces griseus enhances denitrification by Ralstonia pickettii K50, which is possibly mediated by histidine produced during co-culture.

Ralstonia pickettii K50 (strain K50) is a denitrifying bacterium that produces low levels of N(2)O under aerobic conditions. In this study, we found that co-culturing of strain K50 with Streptomyces griseus significantly enhanced the denitrification activity of strain K50 in an artificial wastewater (AWW) system. Most factors that enhance denitrification activity were in the high molecular weight fraction of the cell-free broth of S. griseus, and were suggested to be extracellular proteases. Further investigation revealed that the cultivation of strain K50 in protease-treated AWW medium fully enhanced denitrification, and that a shortage of amino acids in the medium limited it. Among the 20 standard amino acids tested, only histidine had a significant effect in inducing denitrification by strain K50. Our results indicate that histidine is a novel inducer of bacterial denitrification.