Assessing microbial stability and predicting biogas production in full-scale thermophilic dry methane fermentation of municipal solid waste.

Compared to typical anaerobic digestion processes, little is known about both sludge microbial compositions and biogas production models for full-scale dry methane fermentation treating municipal solid waste (MSW). The anaerobic sludge composed of one major hydrogenotrophic methanogen (Methanoculleus) and syntrophic acetate oxidizing bacteria (e.g., Caldicoprobacter), besides enrichment of MSW degraders such as Clostridia. The core population remained phylogenetically unchanged during the fermentation process, regardless of amounts of MSW supplied (∼35 ton/d) or biogas produced (∼12000 Nm3/d). Based on the correlations observed between feed amounts of MSW from 6 days in advance to the current day and biogas output (the strongest correlation: r = 0.77), the best multiple linear regression (MLR) model incorporating the temperature factor was developed with a good prediction for validation data (R2 = 0.975). The proposed simple MLR method with only data on the feedstock amounts will help decision-making processes to prevent low-efficient biogas production.

Startup performance and microbial composition of a pilot-scale rapid sand filter for the treatment of manganese-containing mine water.

The inexpensive removal of soluble manganese [Mn(II)] from mine water that contains large quantities of Mn(II) should be prioritized given that large quantities of alkaline reagents are typically used in the chemical treatment of Mn-rich water from abandoned mines. Rapid sand filter (RSF) systems are widely used as a cost-effective technology in drinking water treatment processes to remove iron and Mn from groundwater. Here, we applied a pilot-scale RSF to treat mine water with a neutral pH and containing approximately 22 mg/L of Mn(II). Following a lag phase from its startup (day 1-day 26), Mn removal rates increased to approximately 40% for around 1 month (day 27-day 55) without the use of alkaline reagents but did not increase during further operation. Quantitative elemental analysis revealed Mn oxides on the sand filters during the Mn removal period. The bacterial communities on the RSFs, recorded on day 42 and day 85, were characterized and compared using 16S rRNA gene amplicon sequencing. Although the well-known Mn-oxidizing bacteria (MOB) were not listed among the ten most dominant operational taxonomic units (OTUs) on the sand filters (relative abundances: >0.68%), a significant increase in the OTUs related to well-known alphaproteobacterial MOB, such as Pedomicrobium spp., were observed during the period.

Metabolic implications for predatory and parasitic bacterial lineages in activated sludge wastewater treatment systems.

Deciphering unclear microbial interactions is key to improving biological wastewater treatment processes. Microbial predation and parasitism in wastewater treatment ecosystems are unexplored survival strategies that have long been known and have recently attracted attention because these interspecies interactions may contribute to the reduction of excess sludge. Here, microbial community profiling of 600 activated sludge samples taken from six industrial and one municipal wastewater treatment processes (WWTPs) was conducted. To identify the shared lineages in the WWTPs, the shared microbial constituents were defined as the family level taxa that had ≥ 0.1% average relative abundance and detected in all processes. The microbial community analysis assigned 106 families as the shared microbial constituents in the WWTPs. Correlation analysis showed that 98 of the 106 shared families were significantly correlated with total carbon (TC) and/or total nitrogen (TN) concentrations, suggesting that they may contribute to wastewater remediation. Most possible predatory or parasitic bacteria belonging to the phyla Bdellovibrionota, Myxococcota, and Candidatus Patescibacteria were found to be the shared families and negatively correlated with TC/TN; thus, they were frequently present in the WWTPs and could be involved in the removal of carbon/nitrogen derived from cell components. Shotgun metagenome-resolved metabolic reconstructions indicated that gene homologs associated with predation or parasitism are conserved in the Bdellovibrionota, Myxococcota, and Ca. Patescibacteria genomes (e.g., host interaction (hit) locus, Tad-like secretion complexes, and type IV pilus assembly proteins). This study provides insights into the complex microbial interactions potentially linked to the reduction of excess sludge biomass in these processes.

Microbially produced fertilizer provides rhizobacteria to hydroponic tomato roots by forming beneficial biofilms.

Hydroponic cultivation of Solanum lycopersicum (tomato) is important, and high tomato production depends on the use of nitrogen and phosphate fertilizers. We had developed a microbial fertilizer (MF), which is mainly composed of nitrate. To investigate the effect of MF on plant growth, hydroponic tomato was grown with MF or commercial inorganic fertilizer (IF), and the microbiomes of the rhizosphere and the liquid phase were analyzed by confocal microscopy and high-throughput sequencing. Plant biomass and biofilm formation were increased by growth in MF compared to IF. The microbial community structures of tomato roots and hydroponic water differed between the two conditions, and three operational taxonomic units (OTUs) dominated in plants grown with MF. The three OTUs were related to Rudaea spp., Chitinophaga spp., and Stenotrophobacter terrae, which are reported to be disease-suppressive epiphytic or endophytic microbes of plant roots. Because these three OTUs also predominated in the MF itself, they were likely provided to the rhizosphere or endophytic environments of tomato roots via hydroponic water. KEY POINTS: • Microbial fertilizer for hydroponic growth enhanced biofilm formation on tomato root. • Microbial fertilizer contains tomato-root epiphytic or endophytic microbes. • Microbial fertilizer provided beneficial microbes to the rhizosphere and endophytic environments of tomato roots via hydroponic water.

Evaluating the Optimal Oil Concentrations in the Startup Performance of a Membrane Bioreactor Treating Oily Noodle-soup Wastewater.

The discharge of high-strength oily wastewater adversely affects the environment; therefore, the treatment of wastewater containing fats, oils, and grease from the food industry is of importance. In this study, we used a membrane bioreactor (MBR) to treat Ramen noodle-soup wastewater, and we evaluated the optimal oil concentration in the wastewater for the startup of the MBR treatment in winter and summer. The MBR system had a sufficient startup in both seasons when fed with a 20-fold dilution of the original oily wastewater, containing approximately 950 to 1,200 mg/L oil and approximately 3,000 to 4,400 mg/L biological oxygen demand (BOD; BOD-SS load of 0.1 to 0.2 kg/kg/d). The reactor performance in winter were relatively stable during the operation. While, activated sludge microbes in summer were not highly active with a 40-fold dilution of wastewater, because of the decreased mixed liquor suspended solid concentration during the operation period. Population shifts in the sludge microbiome with increasing oil concentrations were analyzed using high-throughput sequencing, and the relative abundance of operational taxonomic units belonging to the phylum Bacteroidetes were highest in both winter and summer when fed with 20-fold dilution of the wastewater. In particular, the family Chitinophagaceae was dominant, with relative abundances of 13.5% in winter and 5.1% in summer, suggesting that this family may play important roles in the startup of a MBR treating the wastewater.

Discovery of diphenyl ether-degrading Streptomyces strains by direct screening based on ether bond-cleaving activity.

Diphenyl ethers (DEs), which are widely used in the agricultural and chemical industries, have become hazardous contaminants in the environment. Although several DE-degrading bacteria have been reported, discovering new types of such microorganisms could enhance understanding of the degradation mechanism in the environment. In this study, we used a direct screening method based on detection of ether bond-cleaving activity to screen for microorganisms that degrade 4,4'-dihydroxydiphenyl ether (DHDE) as a model DE. Microorganisms isolated from soil samples were incubated with DHDE, and strains producing hydroquinone via ether bond cleavage were selected using hydroquinone-sensitive Rhodanine reagent. This screening procedure resulted in the isolation of 3 bacteria and 2 fungi that transform DHDE. Interestingly, all of the isolated bacteria belonged to one genus, Streptomyces. To our knowledge, these are the first microorganisms of the genus Streptomyces shown to degrade a DE. Streptomyces sp. TUS-ST3 exhibited high and stable DHDE-degrading activity. HPLC, LC-MS, and GC-MS analyses revealed that strain TUS-ST3 converts DHDE to its hydroxylated analogue and generates hydroquinone as an ether bond-cleavage product. Strain TUS-ST3 also transformed DEs other than DHDE. In addition, glucose-grown TUS-ST3 cells began to transform DHDE after incubation with this compound for 12 h, and produced 75 µM hydroquinone in 72 h. These activities of streptomycetes may play an important role in DE degradation in the environment. We also report the whole genome sequence of strain TUS-ST3.

Evaluation of dye decolorization using anaerobic granular sludge from an expanded granular sludge bed based on spectrometric and microbiome analyses.

The decolorization of 11 dyes by granular sludge from an anaerobic expanded granular sludge bed (EGSB) reactor was evaluated. Biological decolorization of Reactive Red 21, 23, and 180, and Reactive Yellow 15, 17, and 23 in model textile wastewater was observed for the first time after a 7-day incubation (over 94% decolorization). According to the sequencing analysis of 16S rRNA gene amplicons from EGSB granular sludge, the operational taxonomic unit related to Paludibacter propionicigenes showed the highest increase in relative abundance ratios in the presence of dyes (7.12 times on average over 11 dyes) compared to those without dyes.

Isolation and characterization of microorganisms capable of cleaving the ether bond of 2-phenoxyacetophenone.

Lignin is a heterogeneous aromatic polymer and major component of plant cell walls. The ß-O-4 alkyl aryl ether is the most abundant linkage within lignin. Given that lignin is effectively degraded on earth, as yet unknown ether bond-cleaving microorganisms could still exist in nature. In this study, we searched for microorganisms that transform 2-phenoxyacetophenone (2-PAP), a model compound for the ß-O-4 linkage in lignin, by monitoring ether bond cleavage. We first isolated microorganisms that grew on medium including humic acid (soil-derived organic compound) as a carbon source. The isolated microorganisms were subsequently subjected to colorimetric assay for 2-PAP ether bond-cleaving activity; cells of the isolated strains were incubated with 2-PAP, and strains producing phenol via ether bond cleavage were selected using phenol-sensitive Gibbs reagent. This screening procedure enabled the isolation of various 2-PAP-transforming microorganisms, including 7 bacteria (genera: Acinetobacter, Cupriavidus, Nocardioides, or Streptomyces) and 1 fungus (genus: Penicillium). To our knowledge, these are the first microorganisms demonstrated to cleave the ether bond of 2-PAP. One Gram-negative bacterium, Acinetobacter sp. TUS-SO1, was characterized in detail. HPLC and GC-MS analyses revealed that strain TUS-SO1 oxidatively and selectively cleaves the ether bond of 2-PAP to produce phenol and benzoate. These results indicate that the transformation mechanism differs from that involved in reductive ß-etherase, which has been well studied. Furthermore, strain TUS-SO1 efficiently transformed 2-PAP; glucose-grown TUS-SO1 cells converted 1 mM 2-PAP within only 12 h. These microorganisms might play important roles in the degradation of lignin-related compounds in nature.

Optimal start-up conditions for the efficient treatment of acid mine drainage using sulfate-reducing bioreactors based on physicochemical and microbiome analyses.

Typically, sulfate-reducing bioreactors used to treat acid mine drainage (AMD) undergo an initial incubation period of a few weeks to acclimatize sulfate-reducing bacteria (SRB), although necessity of this preincubation has rarely been evaluated. To reduce time and economic cost, we developed an SRB acclimatization method using the continuous flow of AMD into bioreactors fed with rice bran, and compared with the conventional acclimatization method. We found that the SRB sufficiently acclimatized without the preincubation phase. Furthermore, we examined the performance and SRB communities in bioreactors operated for >200 days under seven different conditions, in which the amount of rice bran added and hydraulic retention times (HRTs) were varied. A comparison of the various bioreactor conditions revealed that the lowest rice bran amount (50 g) and the shortest HRT (6 h) caused a deterioration in reactor performance after day 144 and 229, respectively. In both cases, relatively aerobic environments developed due to the lack of organic matter seemed to inhibit sulfate reduction. Of the conditions tested, operation of the bioreactors with 200 g of rice bran and an HRT of 12.5 h was the most effective in treating AMD, showing a sulfate reduction rate of 20.7-77.9% during days 54-242. DATA AND MATERIALS AVAILABILITY: All data needed to evaluate the conclusions of this study are presented in the paper and/or the appendix.

Heterologous expression of membrane-bound alcohol dehydrogenase-encoding genes for glyceric acid production using Gluconobacter sp. CHM43 and its derivatives.

In contrast to D-glyceric acid (D-GA) production with 99% enantiomeric excess (ee) by Acetobacter tropicalis NBRC 16470, Gluconobacter sp. CHM43 produced 19.6 g L-1 of D-GA with 73.7% ee over 4 days of incubation in flask culture. To investigate the reason for this enantiomeric composition of GA, the genes encoding membrane-bound alcohol dehydrogenase (mADH) of A. tropicalis NBRC 16470, composed of three subunits (adhA, adhB, and adhS), were cloned using the broad-host-range vector pBBR1MCS-2 and heterologously expressed in Gluconobacter sp. CHM43 and its ΔadhAB ΔsldBA derivative TORI4. Reverse-transcription quantitative real-time polymerase chain reaction demonstrated that adhABS genes from A. tropicalis were expressed in TORI4 transformants, and their membrane fraction exhibited mADH activities of 0.13 and 0.31 U/mg with or without AdhS, respectively. Compared with the GA production of TORI4-harboring pBBR1MCS-2 (1.23 g L-1), TORI4 transformants expressing adhABS and adhAB showed elevated GA production of 2.46 and 3.67 g L-1, respectively, suggesting a negative effect of adhS gene expression on GA production as well as mADH activity in TORI4. Although TORI4 was found to produce primarily L-GA with 42.5% ee, TORI4 transformants expressing adhABS and adhAB produced D-GA with 27.6% and 49.0% ee, respectively, demonstrating that mADH of A. tropicalis causes a sharp increase in the enantiomeric composition of D-GA. These results suggest that one reason for D-GA production with 73.7% ee in Gluconobacter spp. might be a property of the host, which possibly produces L-GA intracellularly. KEY POINTS: • Membrane-bound ADH from Acetobacter tropicalis showed activity in Gluconobacter sp. • D-GA production from glycerol was performed using recombinant Gluconobacter sp. • Enantiomeric excess of D-GA was affected by both membrane and intracellular ADHs.

Bacterial Community Coexisting with White-Rot Fungi in Decayed Wood in Nature.

Lignin-decomposing ability of several bacteria and the degradation mechanism have been revealed in vitro. However, the abundance of such bacteria in decayed wood in nature remains unknown at genus and species levels. This study was aimed at identifying bacterial communities in the decayed wood coexisting with white-rot fungi, which play a potential role in lignin degradation, and predicting the functional profile of bacterial lignin degradation in wood via bacterial community analyses. The bacterial flora of forest soil and four decayed wood samples showed marked differences; particularly, in addition to Methylobacterium and Acidibrevibacterium, sphingomonads, which degrade the major skeleton of lignin in vitro, were more abundant in the decayed wood than in forest soil, suggesting that multiple bacteria were involved in lignin degradation. The bacterial community in the decayed wood was more influenced by wood type and lignin structure than the fungal species observed in the decayed wood.

Efficient conversion of organic nitrogenous wastewater to nitrate solution driven by comammox Nitrospira.

A bacterium capable of complete ammonia oxidation (comammox) has been widely found in various environments, whereas its industrial application is limited due to the difficulty of cultivation and/or enrichment. We developed a biological system to produce a high-quality nitrate solution for use in hydroponic fertilizer. The system was composed of two separate reactors for ammonification and nitrification and was found to have a stable and efficient performance in the conversion of organic nitrogen to nitrate. To determine the key microbes involved and better understand the system, the microbial communities in the reactors were analyzed by 16S rRNA gene sequencing in combination with a shotgun metagenomic analysis. Canonical ammonia-oxidizing bacteria, which can only catalyze the oxidation of ammonia to nitrite, were detected with negligible relative abundances, while a comammox Nitrospira-related operational taxonomic unit (OTU) dominated the nitrification reactor. Furthermore, the comammox-type ammonia monooxygenase was found to be 500 times more highly expressed than the canonical one by quantitative PCR, indicating that comammox was the main driver of the stable and efficient ammonia oxidation in the system. A microbial co-occurrence analysis revealed a strong positive correlation between Nitrospira and several OTUs, some of which, such as Anaerolinea OTU, have been found to co-exist with comammox Nitrospira in the biofilms of water treatment systems. Given that these OTUs were abundant only on microbe-attached carriers in the system, their co-existence within the biofilm could be beneficial to stabilize the Nitrospira abundance, possibly by physically preventing oxygen exposure as well as cell spillage.

Enrichment and Isolation of Surfactin-degrading Bacteria.

A total of 100 environmental samples were investigated for their ability to degrade 1 g/L surfactin as a substrate. Among them, two enrichment cultures, which exhibited microbial growth as well as surfactin degradation, were selected and further investigated. After several successive cultivations, nanopore sequencing of full-length 16S rRNA genes with MinIONTM was used to analyze the bacterial species in the enrichment cultures. Variovorax spp., Caulobacter spp., Sphingopyxis spp., and Pseudomonas spp. were found to be dominant in these surfactin-degrading mixed cultures. Finally, one strain of Pseudomonas putida was isolated as a surfactin-degrading bacterium. This strain degraded 1 g/L surfactin below a detectable level within 14 days, and C13 surfactin was degraded faster than C15 surfactin.

Spectroscopic Investigation of Increased Fluorescent Intensity of Fluorescent Dyes When Adsorbed onto Polystyrene Microparticles.

Microplastics as environmental pollutants are increasingly a source of alarm. The characterization of microplastics will be necessary to discriminate microplastics from other types of particles. To discriminate specific microplastics, plastic-adsorbable fluorescent dyes are used, the stained microplastics are separated from the dye-microplastic mixture by filtration, and the type of fluorescent staining of the microplastics is analyzed by fluorescent microscopy. In this study, to realize the in situ analysis of fluorescent staining, i.e., to discriminate microplastics without any separation or filtration processes, we studied the change in the fluorescent properties after adsorption of the fluorescent dyes to the microplastic particle surfaces using a 3D excitation emission matrix fluorescence spectroscopy (the excitation wavelength-dependent emission spectrum). We used three fluorescent dyes: Fluorescein, Rhodamine 6G, and Methylene Blue, and polystyrene microparticles as our model microplastic. Fluorescein and Methylene Blue showed increases in the fluorescent intensity, while Rhodamine 6G showed negligible intensity changes. This is likely due to the degree of affinity of the dyes to the polystyrene particle surface, the structural stability of the dyes on the surface, and the changes in the environment around the dyes after the adsorption of each dye to the surface. We conclude that we have demonstrated the potential to look for appropriate fluorescent dyes using the method studied here to identify and estimate individual plastic materials.

Naked-eye detection of specific DNA sequences amplified by the polymerase chain reaction with nanocomposite beads.

We developed a novel nanocomposite bead system for detection by the naked eye of specific DNA sequences amplified by the polymerase chain reaction (PCR). The DNA probes, which were complementary to the target DNA, are conjugated with the nanocomposite beads. If the amplified products contained sequences complementary to the probes, the beads aggregated through sandwich hybridization. The aggregation was detectable as precipitation of the nanocomposite beads. The results were determined visually and did not require instrumental detection. The assay was sensitive enough to detect PCR products with a detection limit of 10 copies/tube for DNA templates. This technique is that all needed components are included within the initial cap, so that the risk of carryover contamination is very low. The nanocomposite bead system has broad application prospects for the detection of specific DNA sequences in biological and biomedical research.

Effective Se reduction by lactate-stimulated indigenous microbial communities in excavated waste rocks.

Waste rocks generated from tunnel excavation contain the metalloid selenium (Se) and its concentration sometimes exceeds the environmental standards. The possibility and effectiveness of dissolved Se removal by the indigenous microorganisms are unknown. Chemical analyses and high-throughput 16S rRNA gene sequencing were implemented to investigate the functional and structural responses of the rock microbial communities to the Se and lactate amendment. During anaerobic incubation of the amended rock slurries from two distinct sites, dissolved Se concentrations decreased significantly, which coincided with lactate degradation to acetate and/or propionate. Sequencing indicated that relative abundances of Desulfosporosinus burensis increased drastically from 0.025 % and 0.022% to 67.584% and 63.716 %, respectively, in the sites. In addition, various Desulfosporosinus spp., Symbiobacterium-related species and Brevibacillus ginsengisoli, as well as the Se(VI)-reducing Desulfitobacterium hafniense, proliferated remarkably. They are capable of incomplete lactate oxidation to acetate as only organic metabolite, strongly suggesting their involvement in dissimilatory Se reduction. Furthermore, predominance of Pelosinus fermentans that ferments lactate to propionate and acetate implied that Se served as the electron sink for its fermentative lactate degradation. These results demonstrated that the indigenous microorganisms played vital roles in the lactate-stimulated Se reduction, leading to the biological Se immobilization treatment of waste rocks.

Activated sludge microbiome in a membrane bioreactor for treating Ramen noodle-soup wastewater.

Population shifts in the activated sludge microbiome of a membrane bioreactor (MBR) during the treatment of Ramen noodle-soup wastewater were analyzed by high-throughput sequencing. An MBR underwent stable treatment of wastewater containing increasing oil concentrations (from 135 to 1,350 mg/L) for 26 days; however, after feeding with wastewater containing 2,700 mg/L of oil, the mixed liquor suspended solids and transmembrane pressure exhibited gradual and rapid increases, respectively, leading to clogging of the membrane. Phylogenetic analysis revealed an oil supply-dependent increase in the abundance of Cupriavidus gilardii (relative abundance of 26.2% at Day 30) in the sludge together with Parasegetibacter terrae (9.9%) and Ferruginibacter yonginensis (9.4%). These dominant species may play important roles in noodle-soup wastewater treatment.

Microbial and enzymatic conversion of levulinic acid, an alternative building block to fermentable sugars from cellulosic biomass.

Levulinic acid (LA) is an important chemical building block listed among the top 12 value-added chemicals by the United States Department of Energy, and can be obtained through the hydrolysis of lignocellulosic biomass. Using the same approach as in the catalytic production of LA from biomass, catalytic methods to upgrade LA to higher value chemicals have been investigated. Since the discovery of the catabolic genes and enzymes in the LA metabolic pathway, bioconversion of LA into useful chemicals has attracted attention, and can potentially broaden the range of biochemical products derived from cellulosic biomass. With a brief introduction to the LA catabolic pathway in Pseudomonas spp., this review summarizes the current studies on the microbial conversion of LA into bioproducts, including the recent developments to achieve higher yields through genetic engineering of Escherichia coli cells. Three different types of reactions during the enzymatic conversion of LA are also discussed. KEY POINTS: • Levulinic acid is an alternative building block to sugars from cellulosic biomass. • Introduction of levulinic acid bioconversion with natural and engineered microbes. • Initial enzymatic conversion of levulinic acid proceeds via three different pathways. • 4-Hydroxyvalerate is one of the target chemicals for levulinic acid bioconversion.

Design, application, and microbiome of sulfate-reducing bioreactors for treatment of mining-influenced water.

Sulfate-reducing bioreactors, also called biochemical reactors, represent a promising option for passive treatment of mining-influenced water (MIW) based on similar technology to aerobic/anaerobic-constructed wetlands and vertical-flow wetlands. MIW from each mine site has a variety of site-specific properties related to its treatment; therefore, design factors, including the organic substrates and inorganic materials packed into the bioreactor, must be tested and evaluated before installation of full-scale sulfate-reducing bioreactors. Several full-scale sulfate-reducing bioreactors operating at mine sites provide examples, but holistic understanding of the complex treatment processes occurring inside the bioreactors is lacking. With the recent introduction of high-throughput DNA sequencing technologies, microbial processes within bioreactors may be clarified based on the relationships between operational parameters and key microorganisms identified using high-resolution microbiome data. In this review, the test design procedures and precedents of full-scale bioreactor application for MIW treatment are briefly summarized, and recent knowledge on the sulfate-reducing microbial communities of field-based bioreactors from fine-scale monitoring is presented.Key points• Sulfate-reducing bioreactors are promising for treatment of mining-influenced water.• Various design factors should be tested for application of full-scale bioreactors.• Introduction of several full-scale passive bioreactor systems at mine sites.• Desulfosporosinus spp. can be one of the key bacteria within field-based bioreactors.

Microbial community in an anaerobic membrane bioreactor and its performance in treating organic solid waste under controlled and deteriorated conditions.

The adoption of anaerobic membrane bioreactors (AnMBRs) for organic solid waste management is important for the recovery of energy and high-quality treated water. However, few studies have focused on AnMBR treatment of high-strength organic solid waste and the microorganisms involved under deteriorated operating conditions. In the present study, a 15-L bench-scale AnMBR was operated using a model slurry of high-strength organic solid waste with the organic loading rate (OLR) increasing from 2.3 g chemical oxygen demand (COD) L-1 day-1 (represented as a controlled condition) to 11.6 g COD L-1 day-1 (represented as a deteriorated condition), and microbial community dynamics over 120 days of operation were analyzed. The abundances of methanogens and bacteria that were dominant under the controlled condition decreased as a result of both high organic loading and sludge withdrawal under the deteriorated condition and did not recover thereafter. Instead, numbers of putative volatile fatty acid (VFA)-producing bacterial operational taxonomic units (OTUs) related to the genus Prevotella increased rapidly, reaching a relative abundance of 43.2%, leading to the deterioration of methanogenic AnMBR operation. Considering that the sequences of these OTUs exhibited relatively low sequence identity (91-95%) to those of identified Prevotella species, the results strongly suggest that the accumulation of VFAs by novel VFA-producing bacteria in the digestion sludge promotes the disruption of the methanogen community under deteriorated conditions.