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.

Cultivation of previously uncultured microorganisms with a continuous-flow down-flow hanging sponge (DHS) bioreactor, using a syntrophic archaeon culture obtained from deep marine sediment as a case study.

In microbiology, cultivation is a central approach for uncovering novel physiology, ecology, and evolution of microorganisms, but conventional methods have left many microorganisms found in nature uncultured. To overcome the limitations of traditional methods and culture indigenous microorganisms, we applied a two-stage approach: enrichment/activation of indigenous organisms by using a continuous-flow down-flow hanging sponge bioreactor and subsequent selective batch cultivation. Here, we provide a protocol for this bioreactor-mediated technique using activation of deep marine sediment microorganisms and downstream isolation of a syntrophic co-culture containing an archaeon closely related to the eukaryote ancestor (Candidatus Promethearchaeum syntrophicum strain MK-D1) as an example. Both stages can easily be tailored to target other environments and organisms by modifying the inoculum, feed solution/gases, attachment material and/or cultivation media. We anaerobically incubate polyurethane sponges inoculated with deep-sea methane seep sediment in a reactor at 10 °C and feed anaerobic artificial seawater medium and methane. Once phylogenetically diverse and metabolically active microorganisms are adapted to synthetic conditions in the reactor, we transition to growing community samples in glass tubes with the above medium, simple substrates and selective compounds (e.g., antibiotics). To accommodate for the slow growth anticipated for target organisms, primary cultures can be incubated for ≥6-12 months and analyzed for community composition even when no cell turbidity is observed. One casamino acid- and antibiotic-amended culture prepared in this way led to the enrichment of uncultured archaea. Through successive transfer in vitro combined with molecular growth monitoring, we successfully obtained the target archaeon with its partner methanogen as a pure syntrophic co-culture.

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.

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.

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.

Bioelectrical Methane Production with an Ammonium Oxidative Reaction under the No Organic Substance Condition.

The present study investigated bioelectrical methane production from CO2 without organic substances. Even though microbial methane production has been reported at relatively high electric voltages, the amount of voltage required and the organisms contributing to the process currently remain unknown. Methane production using a biocathode was investigated in a microbial electrolysis cell coupled with an NH4+ oxidative reaction at an anode coated with platinum powder under a wide range of applied voltages and anaerobic conditions. A microbial community analysis revealed that methane production simultaneously occurred with biological denitrification at the biocathode. During denitrification, NO3- was produced by chemical NH4+ oxidation at the anode and was provided to the biocathode chamber. H2 was produced at the biocathode by the hydrogen-producing bacteria Petrimonas through the acceptance of electrons and protons. The H2 produced was biologically consumed by hydrogenotrophic methanogens of Methanobacterium and Methanobrevibacter with CO2 uptake and by hydrogenotrophic denitrifiers of Azonexus. This microbial community suggests that methane is indirectly produced without the use of electrons by methanogens. Furthermore, bioelectrical methane production occurred under experimental conditions even at a very low voltage of 0.05| |V coupled with NH4+ oxidation, which was thermodynamically feasible.

Triggering Growth via Growth Initiation Factors in Nature: A Putative Mechanism for in situ Cultivation of Previously Uncultivated Microorganisms.

Most microorganisms resist cultivation under standard laboratory conditions. On the other hand, cultivating microbes in a membrane-bound device incubated in nature (in situ cultivation) can be an effective approach to overcome this limitation. In the present study, we applied in situ cultivation to isolate diverse previously uncultivated marine sponge-associated microbes and comparatively analyzed this method's efficiencies with those of the conventional method. Then, we attempted to investigate the key and previously unidentified mechanism of growing uncultivated microorganisms by in situ cultivation focusing on growth triggering via growth initiation factor. Significantly more novel and diverse microbial types were isolated via in situ cultivation than by standard direct plating (SDP). We hypothesized that some of environmental microorganisms which resist cultivation are in a non-growing state and require growth initiation factors for the recovery and that these can be provided from the environment (in this study from marine sponge). According to the hypothesis, the effect of the sponge extract on recovery on agar medium was compared between strains derived from in situ and SDP cultivation. Adding small amounts of the sponge extracts to the medium elevated the colony-formation efficiencies of the in situ strains at the starvation recovery step, while it showed no positive effect on that of SDP strains. Conversely, specific growth rates or saturated cell densities of all tested strains were not positively affected. These results indicate that, (1) the sponge extract contains chemical compounds that facilitate recovery of non-growing microbes, (2) these substances worked on the in situ strains, and (3) growth initiation factor in the sponge extract did not continuously promote growth activity but worked as triggers for regrowth (resuscitation from non-growing state).

Anti-bacterial Effects of MnO2 on the Enrichment of Manganese-oxidizing Bacteria in Downflow Hanging Sponge Reactors.

We focused on the use of abiotic MnO2 to develop reactors for enriching manganese-oxidizing bacteria (MnOB), which may then be used to treat harmful heavy metal-containing wastewater and in the recovery of useful minor metals. Downflow hanging sponge (DHS) reactors were used under aerobic and open conditions to investigate the potential for MnOB enrichment. The results of an experiment that required a continuous supply of organic feed solution containing Mn(II) demonstrated that MnOB enrichment and Mn(II) removal were unsuccessful in the DHS reactor when plain sponge cubes were used. However, MnOB enrichment was successful within a very short operational period when sponge cubes initially containing abiotic MnO2 were installed. The results of a microbial community analysis and MnOB isolation revealed that MnOB belonging to Comamonadaceae or Pseudomonas played a major role in Mn(II) oxidation. Successful MnOB enrichment was attributed to several unidentified species of Chitinophagaceae and Gemmataceae, which were estimated to be intolerant of MnO2, being unable to grow on sponge cubes containing MnO2. The present results show that MnO2 exerted anti-bacterial effects and inhibited the growth of certain non-MnOB groups that were intolerant of MnO2, thereby enabling enriched MnOB to competitively consume more substrate than MnO2-intolerant bacteria.

Pesticides in aquatic environments and their removal by adsorption methods.

Although pesticides are widely used in agriculture, industry and households, they pose a risk to human health and ecosystems. Based on target organisms, the main types of pesticides are herbicides, insecticides and fungicides, of which herbicides accounted for 46% of the total pesticide usage worldwide. The movement of pesticides into water bodies occurs through run-off, spray drift, leaching, and sub-surface drainage, all of which have negative impacts on aquatic environments and humans. We sought to define the critical factors affecting the fluxes of contaminants into receiving waters. We also aimed to specify the feasibility of using sorbents to remove pesticides from waterways. In Karun River in Iran (1.21 × 105 ng/L), pesticide concentrations are above regulatory limits. The concentration of pesticides in fish can reach 26.1 × 103 µg/kg, specifically methoxychlor herbicide in Perca fluviatilis in Lithuania. During the last years, research has focused on elimination of organic pollutants, such as pesticides, from aqueous solution. Pesticide adsorption onto low-cost materials can effectively remediate contaminated waters. In particular, nanoparticle adsorbents and carbon-based adsorbents exhibit high performance (nearly 100%) in removing pesticides from water bodies.

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.

PAHs emission source analysis for air and water environments by isomer ratios - Comparison by modified Cohen's d.

Possible emission sources of PAHs in air and water environments were discussed by a comparison between the data sets of emission sources and environmental fields using five isomer ratios. The similarity of a pair of the datasets of different sources or environment fields for each isomer ratio was evaluated by a newly developed modified effect size d, and the mean of those for the five isomer ratios was applied as an index of similarity. From the analysis, diesel emission and/or biomass burning residues were considered to be major emission sources for almost all the datasets of environments. The pollution loading and path to the PAHs loading of coastal sediments in Hiroshima bay area were examined and it was inferred emission sources was consistently assigned by these newly developed indicators of isomer ratios. Diesel and/or biomass burning were considered to be major sources for the west side area of the bay and the biomass burning was considered to be for the east side area. Further, it was evaluated the west side area, which confronts the Hiroshima city downtown area more directly, was more similar to diesel, and the east side area, which is a bit remoted to the urban central was more similar to the biomass burning. This newly developed method would be a promising alternative application of isomer ratio analysis.

Comprehensive review of polycyclic aromatic hydrocarbons in water sources, their effects and treatments.

Polycyclic aromatic hydrocarbons (PAHs) are principally derived from the incomplete combustion of fossil fuels. This study investigated the occurrence of PAHs in aquatic environments around the world, their effects on the environment and humans, and methods for their removal. Polycyclic aromatic hydrocarbons have a great negative impact on the humans and environment, and can even cause cancer in humans. Use of good methods and equipment are essential to monitoring PAHs, and GC/MS and HPLC are usually used for their analysis in aqueous solutions. In aquatic environments, the PAHs concentrations range widely from 0.03 ng/L (seawater; Southeastern Japan Sea, Japan) to 8,310,000 ng/L (Domestic Wastewater Treatment Plant, Siloam, South Africa). Moreover, bioaccumulation of ∑16PAHs in fish has been reported to range from 11.2 ng/L (Cynoscion guatucupa, South Africa) to 4207.5 ng/L (Saurida undosquamis, Egypt). Several biological, physical and chemical and biological techniques have been reported to treat water contaminated by PAHs, but adsorption and combined treatment methods have shown better removal performance, with some methods removing up to 99.99% of PAHs.

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.

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.

Effects of Salts on the Activity and Growth of "Candidatus Scalindua sp.", a Marine Anammox Bacterium.

Four salts, SEALIFE (a synthetic sea salt), NaCl, Na2SO4, and NaCl+KCl, were applied to monitor the effects of salinity on "Candidatus Scalindua sp.", a marine anaerobic ammonium oxidation (anammox) bacterium. The highest ammonium consumption of 10 µmol mg protein-1 d-1 was observed at 88 mmol L-1 of Na in the presence of NaCl. The highest inorganic carbon uptake of 0.6 µmol mg protein-1 d-1 was observed at 117 mmol L-1 of Na and at 16 mmol L-1 of K in the presence of NaCl+KCl. Thus, Na and K are both important for maintaining a high growth rate of "Candidatus Scalindua sp."

Pollutant Removal from Synthetic Aqueous Solutions with a Combined Electrochemical Oxidation and Adsorption Method.

Eliminating organic and inorganic pollutants from water is a worldwide concern. In this study, we applied electrochemical oxidation (EO) and adsorption techniques to eliminate ammonia, phenols, and Mo(VI) from aqueous solutions. We analyzed the first stage (EO) with response surface methodology, where the reaction time (1⁻3 h), initial contaminant concentration (10⁻50 mg/L), and pH (3⁻6) were the three independent factors. Sodium sulfate (as an electrolyte) and Ti/RuO2⁻IrO2 (as an electrode) were used in the EO system. Based on preliminary experiments, the current and voltage were set to 50 mA and 7 V, respectively. The optimum EO conditions included a reaction time, initial contaminant concentration, and pH of 2.4 h, 27.4 mg/L, and 4.9, respectively. The ammonia, phenols, and Mo elimination efficiencies were 79.4%, 48.0%, and 55.9%, respectively. After treating water under the optimum EO conditions, the solution was transferred to a granular composite adsorbent column containing bentonite, limestone, zeolite, cockleshell, activated carbon, and Portland cement (i.e., BAZLSC), which improved the elimination efficiencies of ammonia, phenols, and molybdenum(VI) to 99.9%. The energy consumption value (8.0 kWh kg−1 N) was detected at the optimum operating conditions.

Specificities and Efficiencies of Primers Targeting Candidatus Phylum Saccharibacteria in Activated Sludge.

Candidatus Saccharibacteria is a well-described candidate phylum that has not been successfully isolated. Nevertheless, its presence was suggested by 16S rRNA gene sequencing data, and it is frequently detected in natural environments and activated sludge. Because pure culture representatives of Candidatus Saccharibacteria are lacking, the specificity of primers for the determination of their abundance and diversity should be carefully evaluated. In this study, eight Candidatus Saccharibacteria-specific primers were selected from previous studies and evaluated for their coverage against a public database, annealing temperature of the combined primer sets, as well as their utilization to determine the detection frequencies and phylogenetic diversity by cloning analysis, and in quantification by quantitative polymerase chain reaction (PCR). Among the eight primers, four primers (TM7314F, TM7580F, TM7-910R, and TM7-1177R) showed high coverage. Cloning analysis showed that four primer sets (TM7314F and TM7-910R, TM7314F and TM7-1177R, TM7580F and TM7-910R, and TM7580F and TM7-1177R) yielded high detection frequencies for Candidatus Saccharibacteria in activated sludge from a wastewater treatment plant in Higashihiroshima City, Japan. Quantitative PCR results indicated that the primer set containing TM7314F and TM7-910R was superior for the specific detection of Candidatus Saccharibacteria in activated sludge.

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.

Draft Genome Sequence of Mn(II)-Oxidizing Pseudomonas resinovorans Strain MO-1.

Pseudomonas resinovorans strain MO-1, which possesses a high ability to oxidize Mn(II), has been isolated from oligotrophic pond sediment. The draft genome sequence consists of 6,252,942 bp and has a G+C content of 63.4%. Strain MO-1 has 5,694 coding sequences, including 13 putative Mn(II) oxidation genes.