New microbial electrosynthesis system for methane production from carbon dioxide coupled with oxidation of sulfide to sulfate.

Microbial electrosynthesis system (MES) is a promising method that can use carbon dioxide, which is a greenhouse gas, to produce methane which acts as an energy source, without using organic substances. However, this bioelectrical reduction reaction can proceed at a certain high applied voltage when coupled with water oxidation in the anode coated with metallic catalyst. When coupled with the oxidation of HS- to SO42-, methane production is thermodynamically more feasible, thus implying its production at a considerably lower applied voltage. In this study, we demonstrated the possibility of electrotrophic methane production coupled with HS- oxidation in a cost-effective bioanode chamber in the MES without organic substrates at a low applied voltage of 0.2 V. In addition, microbial community analyses of biomass enriched in the bioanode and biocathode were used to reveal the most probable pathway for methane production from HS- oxidation. In the bioanode, electroautotrophic SO42- production accompanied with electron donation to the electrode is performed mainly by the following two steps: first, incomplete sulfide oxidation to sulfur cycle intermediates (SCI) is performed; then the produced SCI are disproportionated to HS- and SO42-. In the biocathode, methane is produced mainly via H2 and acetate by electron-accepting syntrophic bacteria, homoacetogens, and acetoclastic archaea. Here, a new eco-friendly MES with biological H2S removal is established.

Biological methane production coupled with sulfur oxidation in a microbial electrosynthesis system without organic substrates.

Methane is produced in a microbial electrosynthesis system (MES) without organic substrates. However, a relatively high applied voltage is required for the bioelectrical reactions. In this study, we demonstrated that electrotrophic methane production at the biocathode was achieved even at a very low voltage of 0.1 V in an MES, in which abiotic HS- oxidized to SO42- at the anodic carbon-cloth surface coated with platinum powder. In addition, microbial community analysis revealed the most probable pathway for methane production from electrons. First, electrotrophic H2 was produced by syntrophic bacteria, such as Syntrophorhabdus, Syntrophobacter, Syntrophus, Leptolinea, and Aminicenantales, with the direct acceptance of electrons at the biocathode. Subsequently, most of the produced H2 was converted to acetate by homoacetogens, such as Clostridium and Spirochaeta 2. In conclusion, the majority of the methane was indirectly produced by a large population of acetoclastic methanogens, namely Methanosaeta, via acetate. Further, hydrogenotrophic methanogens, including Methanobacterium and Methanolinea, produced methane via H2.

Multiple organic substrates support Mn(II) removal with enrichment of Mn(II)-oxidizing bacteria.

Three different organic substrates, K-medium, sterilized activated sludge (SAS), and methanol, were examined for utility as substrates for enriching manganese-oxidizing bacteria (MnOB) in an open bioreactor. The differences in Mn(II) oxidation performance between the substrates were investigated using three down-flow hanging sponge (DHS) reactors continuously treating artificial Mn(II)-containing water over 131 days. The results revealed that all three substrates were useful for enriching MnOB. Surprisingly, we observed only slight differences in Mn(II) removal between the substrates. The highest Mn(II) removal rate for the SAS-supplied reactor was 0.41 kg Mn⋅m-3⋅d-1, which was greater than that of K-medium, although the SAS performance was unstable. In contrast, the methanol-supplied reactor had more stable performance and the highest Mn(II) removal rate. We conclude that multiple genera of Comamonas, Pseudomonas, Mycobacterium, Nocardia and Hyphomicrobium play a role in Mn(II) oxidation and that their relative predominance was dependent on the substrate. Moreover, the initial inclusion of abiotic-MnO2 in the reactors promoted early MnOB enrichment.

Stormwater inflow loading of polycyclic aromatic hydrocarbons into urban domestic wastewater treatment plant for separate sewer system.

Polycyclic aromatic hydrocarbons (PAHs) are common contaminants present in wastewater, and determination of their sources is important for their management in the environment. In this study, stormwater loading of PAHs during rainfall periods was evaluated for sewage inflow into a wastewater treatment plant (WWTP) for a separate sewer system. To accomplish this, sewage inflow volumes, suspended solid concentrations, and PAH concentrations were measured during eight rainfall events and on two no-rainfall days at the inlet of the plant. Based on a comparison between the rainfall and no-rainfall loading quantified by the measurements, excess PAH loadings with stormwater were evaluated for the rainfall events. The relationship between rainfall intensity and stormwater loading was then used to evaluate long-term stormwater loadings of water and PAHs. Their contributions to the sewage inflow were 0.7% and 1.0% for 1 year for water and the sum of 16 measured PAHs, respectively. Our measurements and estimates demonstrate that direct stormwater inflow is not a primary source of PAHs to the plant for this separate sewer system.

Integrated biological-physical process for biogas purification effluent treatment.

Biogas purification via water scrubbing produces effluent containing dissolved CH4, H2S, and CO2, which should be removed to reduce greenhouse gas emissions and increase its potential for water regeneration. In this study, a reactor built with air supplies at the top and bottom was utilized for the treatment of biogas purification effluent through biological oxidation and physical stripping processes. Up to 98% of CH4 was removed through biological treatment at a hydraulic retention time of 2 hr and an upper airflow rate of 2.02 L/day. Additionally, a minimum CH4 concentration of 0.04% with no trace of H2S gas was detected in the off gas. Meanwhile, a white precipitate was captured on the carrier showing the formation of sulfur. According to the developed mathematical model, an upper airflow rate of greater than 2.02 L/day showed a small deterioration in CH4 removal performance after reaching the maximum value, whereas a 50 L/day bottom airflow rate was required to strip the CO2 efficiently and raise the effluent pH from 5.64 to 7.3. Microbiological analysis confirmed the presence of type 1 methanotroph communities dominated by Methylobacter and Methylocaldum. However, bacterial communities promoting sulfide oxidation were dominated by Hyphomicrobium.

Pollutants removal from synthetic wastewater by the combined electrochemical, adsorption and sequencing batch reactor (SBR).

Wastewater filtration is considered the main solution to water shortages. Here, we treated synthetic wastewater by combining treatment techniques, namely, electrochemical oxidation and adsorbent added sequencing batch reactor (SBR). One beaker with a working value of 1500 mL was applied in this contemporary study. In the upper part of the beaker, an anode and a cathode (Ti/RuO2-IrO2) were arranged in parallel for the electrochemical oxidation process. Sodium sulfate (Na2SO4) with a concentration of 2.5 g/L was added as the electrolyte. The voltage and current were set to 7.50 V and 0.40 A, respectively. Aeration was conducted at the bottom of the beaker. Then, 15% working value of the reactor was filled by activated sludge, and 85% working value of the reactor was added with synthetic wastewater. In addition, 1.50 g/L of powdered cockleshell was added in the reactor. Response surface methodology was used for statistical analysis. In synthetic wastewater, concentrations of COD, ammonia, phenols and chromium were 2500 mg/L, 2500 mg/L, 100 mg/L and 100 mg/L, respectively. pH and reaction time (h) were considered as independent factors. A total of 2430 mg/L biochemical oxygen demand, 2500 mg/L ammonia, 90.0 mg/L phenols, and 84.0 mg/L chromium were eliminated at the optimum reaction time (72.9 min) and pH (6.5). The energy consumption value was 6.5 (kWh kg-1) at the optimum operating conditions. This study indicated that this combined treatment system exhibited high performance.

Biotic manganese oxidation coupled with methane oxidation using a continuous-flow bioreactor system under marine conditions.

Biogenic manganese oxides (BioMnOx) can be applied for the effective removal and recovery of trace metals from wastewater because of their high adsorption capacity. Although a freshwater continuous-flow system for a nitrifier-based Mn-oxidizing microbial community for producing BioMnOx has been developed so far, a seawater continuous-flow bioreactor system for BioMnOx production has not been established. Here, we report BioMnOx production by a methanotroph-based microbial community by using a continuous-flow bioreactor system. The bioreactor system was operated using a deep-sea sediment sample as the inoculum with methane as the energy source for over 2 years. The BioMnOx production became evident after 370 days of reactor operation. The maximum Mn oxidation rate was 11.4 mg L-1 day-1. An X-ray diffraction analysis showed that the accumulated BioMnOx was birnessite. 16S rRNA gene-based clone analyses indicated that methanotrophic bacterial members were relatively abundant in the system; however, none of the known Mn-oxidizing bacteria were detected. A continuous-flow bioreactor system coupled with nitrification was also run in parallel for 636 days, but no BioMnOx production was observed in this bioreactor system. The comparative experiments indicated that the methanotroph-based microbial community, rather than the nitrifier-based community, was effective for BioMnOx production under the marine environmental conditions.

Evaluation of the resilience of a full-scale down-flow hanging sponge reactor to long-term outages at a sewage treatment plant in India.

Resilience to process outages is an essential requirement for sustainable wastewater treatment systems in developing countries. In this study, we evaluated the ability of a full-scale down-flow hanging sponge (DHS) reactor to recover after a 10-day outage. The DHS tested in this study uses polyurethane sponge as packing material. This full-scale DHS reactor has been tested over a period of about 4 years in India with a flow rate of 500 m(3)/day. Water was not supplied to the DHS reactor that was subjected to the 10-day outage; however, the biomass did not dry out because the sponge was able to retain enough water. Soon after the reactor was restarted, a small quantity of biomass, amounting to only 0.1% of the total retained biomass, was eluted. The DHS effluent achieved satisfactory removal of suspended solids, chemical oxygen demand, and ammonium nitrogen within 90, 45, and 90 min, respectively. Conversely, fecal coliforms in the DHS effluent did not reach satisfactory levels within 540 min; instead, the normal levels of fecal coliforms were achieved within 3 days. Overall, the tests demonstrated that the DHS reactor was sufficiently robust to withstand long-term outages and achieved steady state soon after restart. This reinforces the suitability of this technology for developing countries.

Recovery and biological oxidation of dissolved methane in effluent from UASB treatment of municipal sewage using a two-stage closed downflow hanging sponge system.

A two-stage closed downflow hanging sponge (DHS) reactor was used as a post-treatment to prevent methane being emitted from upflow anaerobic sludge blanket (UASB) effluents containing unrecovered dissolved methane. The performance of the closed DHS reactor was evaluated using real municipal sewage at ambient temperatures (10-28 °C) for one year. The first stage of the closed DHS reactor was intended to recover dissolved methane from the UASB effluent and produce a burnable gas with a methane concentration greater than 30%, and its recovery efficiency was 57-88%, although the amount of dissolved methane in the UASB effluent fluctuated in the range of 46-68 % of methane production greatly depending on the temperature. The residual methane was oxidized and the remaining organic carbon was removed in the second closed DHS reactor, and this reactor performed very well, removing more than 99% of the dissolved methane during the experimental period. The rate at which air was supplied to the DHS reactor was found to be one of the most important operating parameters. Microbial community analysis revealed that seasonal changes in the methane-oxidizing bacteria were key to preventing methane emissions.

Nitrogen removal using an anammox membrane bioreactor at low temperature.

Membrane bioreactors (MBRs) have the ability to completely retain biomass and are thus suitable for slowly growing anammox bacteria. In the present study, an anammox MBR was operated to investigate whether the anammox activity would remain stable at low temperature, without anammox biomass washout. The maximum nitrogen removal rates were 6.7 and 1.1 g-N L⁻¹ day⁻¹ at 35 °C and 15 °C, respectively. Fluorescence in situ hybridization and 16S rRNA-based phylogenetic analysis revealed no change in the predominant anammox species with temperature because of the complete retention of anammox biomass in the MBR. These results indicate that the predominant anammox bacteria in the MBR cannot adapt to a low temperature during short-term operation. Conversely, anammox activity recovered rapidly after restoring the temperature from the lower value to the optimal temperature (35 °C). The rapid recovery of anammox activity is a distinct advantage of using an MBR anammox reactor.

PAH contents in road dust on principal roads collected nationwide in Japan and their influential factors.

Fifty-four road dust samples were collected from principal roads (n = 37) and residential roads (n = 17) nationwide in Japan from March 2010 to November 2012. Sixteen polycyclic aromatic hydrocarbons (PAHs) and ignition loss (IL) were determined. The total PAH contents ranged from 62 to 6,325 ng g(-1) with a geometric mean of 484 ng g(-1). The IL ranged from 0.8 to 17% with a mean of 6%. The PAH contents were correlated with the IL contents, and the IL contents were dependent on the population density. From the PAH pattern analysis, the PAHs from road dust are considered to be mainly from diesel emissions.

Physiological characterization of an anaerobic ammonium-oxidizing bacterium belonging to the "Candidatus scalindua" group.

The phylogenetic affiliation and physiological characteristics (e.g., Ks and maximum specific growth rate [µmax]) of an anaerobic ammonium oxidation (anammox) bacterium, "Candidatus Scalindua sp.," enriched from the marine sediment of Hiroshima Bay, Japan, were investigated. "Candidatus Scalindua sp." exhibits higher affinity for nitrite and a lower growth rate and yield than the known anammox species.

A method for evaluating the host range of bacteriophages using phages fluorescently labeled with 5-ethynyl-2'-deoxyuridine (EdU).

The evaluation of bacteriophage (phage) host range is a significant issue in understanding phage and prokaryotic community interactions. However, in conventional methods, such as plaque assay, target host strains must be isolated, although almost all environmental prokaryotes are recalcitrant to cultivation. Here, we introduce a novel phage host range evaluation method using fluorescently labeled phages (the FLP method), which consists of the following four steps: (i) Fluorescently labeled phages are added to a microbial consortium, and host cells are infected and fluorescently labeled. (ii) Fluorescent cells are sorted by fluorescence-activated cell sorting. (iii) 16S rRNA gene sequences retrieved from sorted cells are analyzed, and specific oligonucleotide probes for fluorescence in situ hybridization (FISH) are designed. (iv) Cells labeled with both fluorescently labeled phage and FISH probe are identified as host cells. To verify the feasibility of this method, we used T4 phage and Escherichia coli as a model. We first used nucleic acid stain reagents for phage labeling; however, the reagents also stained non-host cells. Next, we employed the Click-iT EdU (5-ethynyl-2'-deoxyuridine) assay kit from Invitrogen for phage labeling. Using EdU-labeled T4 phage, we could specifically detect E. coli cells in a complex microbial consortium from municipal sewage. We also confirmed that FISH could be applied to the infected E. coli cells. These results suggest that this FLP method using the EdU assay kit is a useful method for evaluating phage host range and may have a potential application for various types of phages, even if their prokaryotic hosts are currently unculturable.

Ecophysiological role and function of uncultured Chloroflexi in an anammox reactor.

The coexistence of uncultured heterotrophic bacteria belonging to the phylum Chloroflexi has often been observed in anaerobic ammonium oxidation (anammox) reactors fed with synthetic nutrient medium without organic carbon compounds. To determine if coexisting Chloroflexi in anammox reactors scavenge organic matter derived from anammox bacterial cells, the present study was conducted to investigate the substrate uptake pattern of the uncultured Chloroflexi present in an anammox reactor and to clarify if they take up microbial products derived from anammox bacterial cells. To accomplish this, combined microautoradiography and fluorescence in situ hybridization (MAR-FISH) was conducted. Phylogenetic analysis revealed that 36% of the clones analyzed in this study were affiliated with Chloroflexi. The sequence similarities to Anaerolinea thermophila and Caldilinea aerophila within the phylum Chloroflexi were only 81.0-88.7% and 80.3-83.8%, respectively. The uncultured Chloroflexi were found to incorporate sucrose, glucose, and N-acetyl-glucosamine. The (14)C-tracing experiment revealed that the uncultured Chloroflexi were clearly MAR-positive, indicating the utilization of decaying anammox bacterial cell materials. Taken together, these results indicate that coexisting uncultured Chloroflexi in anammox reactors scavenge organic compounds derived from anammox bacterial cells.

Influence of temperature and salinity on microbial structure of marine anammox bacteria.

Anaerobic ammonium oxidation (anammox) is a type of biological oxidation mediated by a group of Planctomycete-like bacteria. Members of the genus Candidatus Scalindua are mainly found in marine environments, but not exclusively. This group is cultured using different inoculums and conditions; however, its optimal growth conditions are not clear. Additionally, little information is known about the factors that influence the activity and the selection of a population of marine anammox bacteria. This study was conducted to investigate the influence of temperature and salinity on the marine anammox community. To accomplish this, an up-flow fixed-bed column reactor was operated, and quantitative fluorescence in situ hybridization (FISH) with probes specific to dominant marine anammox bacteria was conducted. Anammox activity was observed at 20 and 30 °C, but not at 10 °C. A nitrogen removal rate of 0.32 kg TN m(-3) day(-1) was obtained at 20 °C. These results suggest that temperature affects the activity (nitrogen removal rate) of anammox bacteria, while salinity does not affect the activity in the marine anammox biofilm.

Development of anammox reactor equipped with a degassing membrane to improve biomass retention.

In up-flow anammox reactors, one of the contributing factors to biomass wash-out is the adherence of nitrogen gas produced by the anammox reaction to biomass. In this study, we operated an up-flow anammox reactor equipped with a degassing membrane to minimize the biomass wash-out from the reactor by separating the produced gas from the biomass. In addition, both the effect of degassing on the anammox reactor performance and the durability of the membrane submerged in the anammox reactor were investigated. The results show that the use of the degassing membrane in the anammox reactor could (1) improve the biomass retention ability (by separating the produced gas from the biomass), and (2) increase the component ratio of anammox bacteria in the reactor. In addition, degassing could reduce the N(2)O emission produced in the reactor (for the gas selectivity of the degassing membrane). No membrane fouling was observed even after 2 months of operation without washing, indicating an advantage to the use of the degassing membrane.

Treatment of low-strength wastewater in an anaerobic down-flow hanging sponge (AnDHS) reactor at low temperature.

The process performance of a novel anaerobic down-flow hanging sponge (AnDHS) reactor for the treatment of low strength wastewater was investigated. A lab-scale experiment was conducted in which 300-400 mgCOD L(-1) of artificial wastewater was fed in over 600 days. The reactor exhibited sufficient performance: 70-90% of total COD removal, and 60-90% of methane recovery were maintained at 20°C, with a hydraulic retention time (HRT) of 2 h. It was possible to maintain COD removal by extending the HRT to 4 h at 15°C and 10 h at 10°C. With regard to the wastewater feed, one-pass mode (without effluent recirculation) gave better performance in COD removal as compared with recirculation mode. The results of batch feeding experiments using single substrates (such as acetate, propionate or sucrose) indicated that acetate degradation was more strongly affected by decreasing operational temperature. In addition, the AnDHS reactor system had no significant problems related to sludge retention such as massive loss of sludge throughout the experiment. Microbial structure analysis of the retained sludge with respect to the domain Archaeal 16S rRNA gene showed the proliferation of relatives of both the acetate-utilizing genus Methanosaeta and the hydrogen-utilizing genus Methanolinea.

Metabolic Potential of the Superphylum Patescibacteria Reconstructed from Activated Sludge Samples from a Municipal Wastewater Treatment Plant.

Patescibacteria are widely distributed in various environments and often detected in activated sludge. However, limited information is currently available on their phylogeny, morphology, and ecophysiological role in activated sludge or interactions with other microorganisms. In the present study, we identified microorganisms that interacted with Patescibacteria in activated sludge via a correlation ana-lysis using the 16S rRNA gene, and predicted the metabolic potential of Patescibacteria using a metagenomic ana-lysis. The metagenome-assembled genomes of Patescibacteria consisted of three Saccharimonadia, three Parcubacteria, and one Gracilibacteria, and showed a strong positive correlation of relative abundance with Chitinophagales. Metabolic predictions from ten recovered patescibacterial and five Chitinophagales metagenome-assembled genomes supported mutualistic interactions between a member of Saccharimonadia and Chitinophagales via N-acetylglucosamine, between a member of Parcubacteria and Chitinophagales via nitrogen compounds related to denitrification, and between Gracilibacteria and Chitinophagales via phospholipids in activated sludge. The present results indicate that various interactions between Patescibacteria and Chitinophagales are important for the survival of Patescibacteria in activated sludge ecosystems.

Environmental Factors Affecting the Community of Methane-oxidizing Bacteria.

Methane-oxidizing bacteria (MOB) are ubiquitous and play an important role in the mitigation of global warming by reducing methane. MOB are commonly classified into Type I and Type II, belonging to Gammaproteobacteria and Alphaproteobacteria, respectively, and the diversity of MOB has been examined. However, limited information is currently available on favorable environments for the respective MOB. To investigate the environmental factors affecting the dominant type in the MOB community, we performed MOB enrichment using down-flow hanging sponge reactors under 38 different environmental conditions with a wide range of methane (0.01-80%) and ammonium concentrations (0.001-2,000| |mg N L-1) and pH 4-7. Enrichment results revealed that pH was a crucial factor influencing the MOB type enriched. Type II was dominantly enriched at low pH (4-5), whereas Type I was dominant around neutral pH (6-7). However, there were some unusual cultivated biomass samples. Even though high methane oxidation activity was observed, very few or zero conventional MOB were detected using common FISH probes and primer sets for the 16S rRNA gene and pmoA gene amplification. Mycobacterium mostly dominated the microbial community in the biomass cultivated at very high NH4+ concentrations, strongly implying that it exhibits methane oxidation activity. Collectively, the present results revealed the presence of many unknown phylogenetic groups with the capacity for methane oxidation other than the reported MOB.

Mutualistic relationship between Nitrospira and concomitant heterotrophs.

Nitrifying chemoautotrophs support the growth of diverse concomitant heterotrophs in natural or engineered environments by supplying organic compounds. In this study, we aimed to investigate this microbial association, especially (i) to distinguish whether the relationship between nitrifying chemoautotrophs and heterotrophs is commensal or mutualistic, and (ii) to clarify how heterotrophs promote the growth of autotrophic nitrite-oxidizing bacteria (Nitrospira). Pure cultured Nitrospira (Nitrospira sp. ND1) was employed in this study. Heterotrophs growing with metabolic by-products of Nitrospira as a sole carbon source were isolated from several environmental samples and used to test the growth-promoting activity of Nitrospira. Furthermore, liquid chromatography-mass spectrometry analysis was conducted to evaluate how heterotrophs consumed chemical compounds produced by Nitrospira and newly produced during co-cultivation. Notably, Nitrospira growth was stimulated by co-cultivation with some heterotrophs and the addition of spent media of some strains, suggesting that not only heterotrophs but also Nitrospira received benefits from their mutual co-existence. Furthermore, the data suggested that some of the growth-promoting heterotrophs provided as-yet-unidentified growth-promoting factors to Nitrospira. Overall, Nitrospira and heterotrophs thus appear to exhibit a mutualistic relationship. Such mutualistic relationships between autotrophs and heterotrophs would contribute to the stability and diversity of microbial ecosystems.