Observation of Acoustically Induced Dressed States of Rare-Earth Ions.

Acoustically induced dressed states of long-lived erbium ions in a crystal are demonstrated. These states are formed by rapid modulation of two-level systems via strain induced by surface acoustic waves whose frequencies exceed the optical linewidth of the ion ensemble. Multiple sidebands and the reduction of their intensities appearing near the surface are evidence of a strong interaction between the acoustic waves and the ions. This development allows for on-chip control of long-lived ions and paves the way to highly coherent hybrid quantum systems with telecom photons, acoustic phonons, and electrons.

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.

Microscopic evidence of sandstone deterioration and damage by fungi isolated from the Angkor monuments in simulation experiments.

The Angkor monuments have been registered on the World Cultural Heritage List of UNESCO, while the buildings built mostly of sandstone are suffering from serious deterioration and damage. Microorganisms are one of the leading causes for the sandstone deterioration. Identification of the mechanisms underlying the biodeterioration is of significance because it reveals the biochemical reaction involved so that effective conservation and restoration of cultural properties can be achieved. In this study, the fungal colonization and biodeterioration of sandstone in simulation experiments were examined using confocal reflection microscopy (CRM) and scanning electron microscopy-energy dispersive X-ray spectroscopy (SEM-EDS). Aspergillus sp. strain AW1 and Paecilomyces sp. strain BY8 isolated from the deteriorated sandstone of Angkor Wat and Bayon of Angkor Thom, respectively, were inoculated and incubated with the sandstone used for construction of Angkor Wat. With CRM, we could visualize that strain AW1 tightly attached to and broke in the sandstone with extension of the hyphae. Quantitative imaging analyses showed that the sandstone surface roughness increased and the cavities formed under the fungal hyphae deepened during the incubation of strains AW1 and BY8. These highlighted that the massive growth of fungi even under the culture conditions was associated with the cavity formation of the sandstone and its expansion. Furthermore, SEM-EDS indicated the flat and Si-rich materials, presumably quartz and feldspar, were found frequently at the intact sandstone surface. But the flatness was lost during the incubation, possibly due to the detachment of the Si-rich mineral particles by the fungal deterioration. Consequently, this study proposed a biodeterioration model of the sandstone in that the hyphae of fungi elongated on the surface of the sandstone to penetrate into the soft and porous sandstone matrix, damaging the matrix and gradually destabilize the hard and Si-rich minerals, such as quartz and feldspar, to the collapse and cavities.

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.

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.

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.

Aqueous mechano-bactericidal action of acicular aragonite crystals.

Nanoneedle structures on dragonfly and cicada wing surfaces or black silicon nanoneedles demonstrate antibacterial phenomena, namely mechano-bactericidal action. These air-exposed, mechano-bactericidal surfaces serve to destroy adherent bacteria, but their bactericidal action in the water is no precedent to report. Calcium carbonate easily accumulates on solid surfaces during long-term exposure to hard water. We expect that aragonite nanoneedles, in particular, which grow on TiO2 during the photocatalytic treatment of calcium-rich groundwater, exhibit mechano-bactericidal action against bacteria in water. Here, we showed that acicular aragonite modified on TiO2 ceramics prepared from calcium bicarbonate in mineral water by photocatalysis exhibits mechanical bactericidal activity against E. coli in water. Unmodified, calcite-modified and aragonite-modified TiO2 ceramics were exposed to water containing E. coli (in a petri dish), and their bactericidal action over time was investigated under static and agitated conditions. The surfaces of the materials were observed by scanning electron microscopy, and the live/dead bacterial cells were observed by confocal laser scanning microscopy. As a result, the synergistic bactericidal performance achieved by mechano-bactericidal action and photocatalysis was demonstrated. Aragonite itself has a high biological affinity for the human body different from the other whisker-sharpen nanomaterials, therefore, the mechano-bactericidal action of acicular aragonite in water is expected to inform the development of safe water purification systems for use in developing countries.

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.

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.

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.

Epitaxial single-crystal rare-earth oxide in horizontal slot waveguide for silicon-based integrated active photonic devices.

We have epitaxially grown high-quality single-crystal rare-earth oxide thin films, including Gd2O3 and erbium-incorporated (ErGd)2O3, on silicon-on-insulator substrate, and investigated their optical properties when embedded in horizontal slot waveguides. (ErGd)2O3 with an erbium concentration in the mid-1021 cm-3 range shows well-resolved Stark-split photoluminescence emission peaks in the telecommunications band and a photoluminescence lifetime-concentration product as large as 2.67×1018 s·cm-3 at room-temperature. Using these materials, horizontal slot waveguides with strong optical confinement in low-refractive-index rare-earth oxide layers, have been fabricated for silicon-based integrated active photonic devices. Thanks to the strong light-matter interaction, a large waveguide modal absorption of 88 dB/cm related to erbium ions is achieved, leading to a large potential optical gain. Intense emissions from the waveguides are also observed, with a radiation efficiency on the order of 10-4. These results indicate that a combination of epitaxial rare-earth oxide thin films and horizontal slot waveguides provides a promising platform for light amplification and generation on silicon.

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.

Biofilm Formation by Streptococcus mutans is Enhanced by Indole via the Quorum Sensing Pathway.

Interspecies interactions among oral microorganisms in the pathogenic biofilms causing dental caries have not yet been elucidated in detail. We herein demonstrated that indole and its derivatives induced biofilm formation by Streptococcus mutans. Indole is an intercellular signaling molecule that is produced by oral bacteria other than S. mutans. The amounts of biofilm and extracellular DNA were significantly increased by the addition of indole and 4-hydroxyindole (4-HI). An examination with quorum sensing mutants showed that the induction of biofilm formation by indole and 4-HI required a quorum sensing system. These results suggest that this intercellular signaling molecule plays a role in pathogenic biofilm formation.

Unexpected diversity of acetate degraders in anaerobic membrane bioreactor treating organic solid waste revealed by high-sensitivity stable isotope probing.

In anaerobic membrane bioreactor (AnMBR) treating organic solid waste, acetate is one of the most important precursors to CH4. However, the identity and diversity of anaerobic acetate degraders are largely unknown, possibly due to their slow growth rates and low abundances. Here, we identified acetate-degrading microorganisms in the AnMBR sludges by high-sensitivity stable isotope probing. Degradation of the amended 13C-acetate coincided with production of 13CH4 and 13CO2 during the sludge incubation. High-throughput sequencing of RNA density fractions indicated that the aceticlastic and hydrogenotrophic methanogens, i.e., Methanosaeta sp. (acetate dissimilator) and Methanolinea sp. (acetate assimilator), incorporated 13C-acetate significantly. Remarkably, 22 bacterial species incorporating 13C-acetate were identified, whereas their majority was distantly related to the cultured representatives. Only two of them were the class Deltaproteobacteria-affiliated lineages with syntrophic volatile fatty acid oxidation activities. Phylogenetic tree analysis and population dynamics tracing revealed that novel species of the hydrolyzing and/or fermenting taxa, such as the phyla Bacteroidetes, Chloroflexi and Lentisphaerae, exhibited low relative abundances comparable to that of Methanolinea sp. (0.00011%) during the AnMBR operation, suggesting that these bacteria were involved in anaerobic acetate assimilation. Meanwhile, novel species of the phyla Firmicutes, Synergistetes and Caldiserica, the candidate phyla Aminicenantes and Atribacteria and the candidate division GOUTA4-related clade, as well as the known Deltaproteobacteria members, existed at relatively high abundances (0.00031%-0.31121%) in the reactor, suggesting that these bacterial species participated in anaerobic dissimilation of acetate, e.g., syntrophic acetate oxidation. The results of this study demonstrated the unexpected diversity and ecophysiological features of the anaerobic acetate degraders in the AnMBR treating organic solid waste.

Clarifying prokaryotic and eukaryotic biofilm microbiomes in anaerobic membrane bioreactor by non-destructive microscopy and high-throughput sequencing.

Anaerobic membrane bioreactor (AnMBR) is used for the treatment of organic solid waste. Clogging of filtration membrane pores, called membrane fouling, is one of the most serious issues for the sustainable operation of AnMBR. Although the physical and chemical mechanisms of the membrane fouling have been widely studied, the biological mechanisms are still unclear. The biofilm formation and development on the membrane might cause the membrane fouling. In this study, the prokaryotic and eukaryotic microbiomes of the membrane-attached biofilms in an AnMBR treating a model slurry of organic solid waste were investigated by non-destructive microscopy and high-throughput sequencing of 16S and 18S rRNA genes. The non-destructive visualization indicated that the biofilm was layered with different structures. The lowermost residual fouling layer was mesh-like and composed of filamentous microorganisms, while the upper cake layer was mainly the non-dense and non-cell region. The principal coordinate and phylogenetic analyses of the sequence data showed that the biofilm microbiomes were different from the sludge. The lowermost layer consisted of operational taxonomic units that were related to Leptolinea tardivitalis and Methanosaeta concilii (9.53-10.07% and 1.14-1.64% of the total prokaryotes, respectively) and Geotrichum candidum (30.22-82.31% of the total eukaryotes), all of which exhibited the filamentous morphology. Moreover, the upper layer was inhabited by the presumably cake-degrading bacteria and predatory eukaryotes. The biofilm microbiome features were consistent with the microscope-visualized structure. These results demonstrated that the biofilm structure and microbiome were the layer specific, which provides better understanding of biological mechanisms of membrane fouling in the AnMBR.

Complete Genome Sequence of Desulfuromonas sp. Strain AOP6, an Iron(III) Reducer Isolated from Subseafloor Sediment.

Desulfuromonas sp. strain AOP6, with iron(III)-reducing activity, was isolated from subseafloor sediment in Nankai Trough. We report the complete genome of this strain determined by Illumina MiSeq sequencing and PCR/Sanger sequencing-based gap closing. The genome includes the genes encoding c-type cytochromes, type IV pili, and fatty acid degradation enzymes.

Transition of microbial community structures after development of membrane fouling in membrane bioreactors (MBRs).

Although membrane fouling is a major issue when operating membrane bioreactors (MBRs), information regarding MBR performance and the sludge microbiome after the development of fouling remains limited. For the present study, two MBRs were operated for approximately 1 month under conditions of membrane fouling to investigate the effects of highly stressed environments on the sludge microbiome. After the development of fouling, a Collimonas-related operational taxonomic unit (OTU) was highly dominant in both reactors (relative abundances were ⁓ 63%) and this predomination caused a precipitous decline in the diversity indices of the sludge microbiomes. Because the excessive predomination by limited numbers of OTUs can lead to reductions in the adaptability to environmental changes, monitoring microbial diversity may be a valuable indicator for maintaining the robustness of a sludge microbiome. While, the decrease in the abundance of the Collimonas-related OTU resulted in the predomination of distinct microorganisms in each of the reactors despite being operated under the same conditions; this finding indicates existence of strong pressure to perturb the microbiomes. Detailed analyses suggested that the availability of terminal electron acceptors and competitive interactions between microbes via the secretion of extracellular proteins appeared to differentiate the structures of the respective microbial communities. During the extracellular proteins were secreted in the sludge, considerable portion of microbes were dead and large amounts of biomolecules seemed to be released; resultantly facilitated the predomination of fermentative anaerobes in one reactor as they use organic substances but not inorganic terminal electron acceptors to generate ATP under anaerobic conditions.