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This study aimed to analyze the effect of temperature on performance and microbial community structure of an anaerobic packed bed reactor (APBR). The temperature was increased step-wise from room temperature (22 degrees C ± 1 degrees C) to psychrophilic (15 degrees C ± 1 degrees C), mesophilic (37 degrees C ± 1 degrees C) and thermophilic (55 degrees C ± 1 degrees C). The results showed that, in the temperature changing process, the higher the temperature of APBR was, the higher COD removal rate and daily gas production were. After temperature changed to psychrophilic, mesophilic and thermophilic, COD removal rate and daily gas production were 25%, 45%, 60% and 2.3 L x d(-1), 4.0 L x d(-1), 8.5 L x d(-1) respectively. However, there was no significant change in biogas composition (-60%). A sudden temperature change caused a simultaneous increase in the concentration of volatile fatty acids (VFA), which had been fluctuating. Using 16S rRNA gene clone library screening, Euryarchaeota was commonly found, including important methanogens: MBT (Methanobacteriales), Mst (Methanosaetaceae) , Msc (Methanosarcinaceae) and MMB (Methanomicrobiales), as well as thermophilic bacteria and few spring Archaea. However, the diversity of methanogenic groups was reduced, especially at mesophilic. The results of quantitative PCR showed that the 16S rRNA gene concentrations of Mst, MMB and Msc were reduced by temperature changes. Although the relative proportion of every kind of methanogen was significantly affected, Mst was the dominant methanogen.

Organic loading conditions are an important factor influencing reactor performances in methanogenic bioreactors. Yet the underlying microbiological basis of the process stability, deterioration, and recovery remains to be understood. Here, structural responses of the bacterial and archaeal populations to the change of organic loading conditions in a thermophilic anaerobic digester were investigated by process analyses and 16S rRNA gene-based molecular approaches. The biogas was produced stably without the accumulation of volatile fatty acids (VFAs) at low organic loading rates (OLRs) in the beginning of reactor operation. Increasing OLR in stages disrupted the stable reactor performance, and high OLR conditions continued the deteriorated performance with slight biogas production and high accumulation of VFAs. Thereafter, the gradual decrease of OLR resulted in the recovery from the deterioration, giving rise to the stable performance again. The stable performances before and after the high OLR conditions conducted were associated with compositionally similar but not identical methanogenic consortia. The bacterial and archaeal populations were synchronously changed at both the transient phases toward the deteriorated performance and in recovery process, during which the dynamic shift of aceticlastic and hydrogenotrophic methanogens including the recently identified Methanomassiliicoccus might contribute to the maintenance of the methanogenic activity. The distinctive bacterial population with a high predominance of Methanobacterium formicicum as archaeal member was found for the deteriorated performance. The results in this study indicate the coordinated reorganization of the bacterial and archaeal populations in response to functional states induced by the change of organic loading conditions in the anaerobic digester., (Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

The membraneless bioelectrochemical reactor (Ml-BER) is useful for dark hydrogen fermentation. The effect of the electrochemical reaction on microorganisms in the Ml-BER was investigated using glucose as the substrate and compared with organisms in a membraneless non-bioelectrochemical reactor (Ml-NBER) and bioelectrochemical reactor (BER) with a proton exchange membrane. The potentials on the working electrode of the Ml-BER and BER with membrane were regulated to -0.9 V (versus Ag/AgCl) to avoid water electrolysis with a carbon electrode. The Ml-BER showed suppressed methane production (19.8 ± 9.1 mg-C·L(-1)·day(-1)) and increased hydrogen production (12.6 ± 3.1 mg-H·L(-1)·day(-1)) at pHout 6.2 ± 0.1, and the major intermediate was butyrate (24.9 ± 2.4 mM), suggesting efficient hydrogen fermentation. In contrast, the Ml-NBER showed high methane production (239.3 ± 17.9 mg-C·L(-1)·day(-1)) and low hydrogen production (0.2 ± 0.0 mg-H·L(-1)·day(-1)) at pHout 6.3 ± 0.1. In the cathodic chamber of the BER with membrane, methane production was high (276.3 ± 20.4 mg-C·L(-1)·day(-1)) (pHout, 7.2 ± 0.1). In the anodic chamber of the BER with membrane (anode-BER), gas production was low because of high lactate production (43.6 ± 1.7 mM) at pHout 5.0 ± 0.1. Methanogenic archaea were not detected in the Ml-BER and anode-BER. However, Methanosarcina sp. and Methanobacterium sp. were found in Ml-NBER. Prokaryotic copy numbers in the Ml-BER and Ml-NBER were similar, as were the bacterial community structures. Thus, the electrochemical reaction in the Ml-BER affected hydrogenotrophic and acetoclastic methanogens, but not the bacterial community.

A bioelectrochemical reactor (BER) containing carbon fiber fabric (CFF) (BER+CFF) enabled efficient methane fermentation from thickened sewage sludge. A cylindrical BER+CFF was proposed and scaled-up to a volume of 4.0-L. Thickened sewage sludge was treated using three types of methanogenic reactors. The working electrode potential in the BER+CFF was regulated at -0.8 V (vs. Ag/AgCl). BER+CFF showed gas production of 3.57 L L(-1) day(-1) at a hydraulic retention time (HRT) of 4.0 days; however, non-BER+CFF showed a lower gas production rate (0.83 L L(-1) day(-1)) at this HRT, suggesting positive effects of electrochemical regulation. A stirred tank reactor (without CFF) deteriorated at an HRT of 10 days, suggesting positive effects of CFF. 16S rRNA gene analysis showed that the BER+CFF included 3 kinds of hydrogenotrophic methanogens and 1 aceticlastic methanogen. These results demonstrate the effectiveness of the BER+CFF for scale-up and flexibility of this technology., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The aim of this study was to show the effectiveness of the membrane free bioelectrochemical system (BES) using three electrodes on inhibition of methanogenesis and construction of hydrogen fermentation from the artificial garbage slurry. The electrical redox-potential on the working electrode was adjusted to -1.0V (vs. Ag/AgCl) that has positive effect on methanogenesis. The redox-potential on the counter electrode was measured to be 1.6V. The pH in the effluents was 5.5-6.4. Hydrogen production rate at the cathode side was similar to that at the anode side and much higher than that calculated from current, and reached a maximum of 2445±815 (average±standard deviation) mL L(-1) d(-1) at an organic loading rate of 58.7g dichromate chemical oxygen demand per L d(-1). Methane production was negligible throughout the experiment. Acetate and butyrate were the main products of the fermentation using a BES; these offered favorable conditions for hydrogen production. The bacterial community in the bioelectrochemical hydrogen fermentor differed from that in the methanogenic seed sludge and included hitherto unknown species. These results show that high redox-potential on the anodic electrode and acidic pH in the membrane free BES can be utilized for hydrogen fermentation from the artificial garbage slurry by avoiding methanogenesis., (Copyright © 2012 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

A packed-bed system includes supporting materials to retain microorganisms and a bioelectrochemical system influences the microbial metabolism. In our study, carbon fiber textiles (CFT) as a supporting material was attached onto a carbon working electrode in a bioelectrochemical reactor (BER) that degrades garbage slurry to methane, in order to investigate the effect of combining electrochemical regulation and packing CFT. The potential on the working electrode in the BER containing CFT was set to -1.0 V or -0.8 V (vs. Ag/AgCl). BERs containing CFT exhibited higher methane production, elimination of dichromate chemical oxygen demand, and the ratio of methanogens in the suspended fraction than reactors containing CFT without electrochemical regulation at an organic loading rate (OLR) of 27.8 gCODcr/L/day. In addition, BERs containing CFT exhibited higher reactor performances than BERs without CFT at this OLR. Our results revealed that the new design that combined electrochemical regulation and packing CFT was effective., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Ammonia accumulation is one of the main causes of the loss of methane production observed during fermentation. We investigated the effect of addition of carbon fiber textiles (CFT) to thermophilic methanogenic bioreactors with respect to ammonia tolerance during the process of degradation of artificial garbage slurry, by comparing the performance of the reactors containing CFT with the performance of reactors without CFT. Under total ammonia-N concentrations of 3,000 mg L(-1), the reactors containing CFT were found to mediate stable removal of organic compounds and methane production. Under these conditions, high levels of methanogenic archaea were retained at the CFT, as determined by 16S rRNA gene analysis for methanogenic archaea. In addition, Methanobacterium sp. was found to be dominant in the suspended fraction, and Methanosarcina sp. was dominant in the retained fraction of the reactors with CFT. However, the reactors without CFT had lower rates of removal of organic compounds and production of methane under total ammonia-N concentrations of 1,500 mg L(-1). Under this ammonia concentration, a significant accumulation of acetate was observed in the reactors without CFT (130.0 mM), relative to the reactors with CFT (4.2 mM). Only Methanobacterium sp. was identified in the reactors without CFT. These results suggest that CFT enables stable proliferation of aceticlastic methanogens by preventing ammonia inhibition. This improves the process of stable garbage degradation and production of methane in thermophilic bioreactors that include high levels of ammonia.

Acidification is one of the most common and serious problems inducing process failure in anaerobic digesters. The production of volatile fatty acids (VFAs) mainly triggers acidic shock. However, little is known about the bacteria involved in the processes of acidogenic metabolism, such as fermentation and reductive acetogenesis. Here, the metabolic responses of a methanogenic community to the acidification and resulting process deterioration were investigated using transcriptional profiling of both the 16S rRNA and formyltetrahydrofolate synthetase (FTHFS) genes. The 16S rRNA-based analyses demonstrated that the dynamic shift of bacterial populations was closely correlated with reactor performance, especially with VFA accumulation levels. The pH drop accompanied by an increase in VFAs stimulated the metabolic activation of an uncultured Chloroflexi subphylum I bacterium. The subphylum has been characterized as a fermentative carbohydrate degrader using culture- and molecular-based ecophysiological assays. At the beginning of VFA accumulation, FTHFS genes were expressed; the transcripts were derived from phylogenetically predicted homoacetogens, suggesting that reductive acetogenesis was operated by hitherto unidentified bacteria. When acetate concentrations were high, the FTHFS expression ceased and Thermoanaerobacterium aciditolerans proliferated selectively. This thermoacidophilic bacterium would play a decisive role in acetate production via fermentative metabolism. The results of this study reveal for the first time that an uncultured Chloroflexi, T. aciditolerans, and novel homoacetogens were metabolically associated with acidic shock and subsequent VFA accumulation in an anaerobic digester.

The methanogenic pathway and microbial community in a thermophilic anaerobic digestion process of organic solid waste were investigated in a continuous-flow stirred-tank reactor using artificial garbage slurry as a feedstock. The decomposition pathway of acetate, a significant precursor of CH(4) and a key intermediate metabolite in the anaerobic digestion process, was analyzed by using stable isotopes. A tracer experiment using (13)C-labeled acetate revealed that approximately 80% of the acetate was decomposed via a non-aceticlastic oxidative pathway, whereas the remainder was converted to methane via an aceticlastic pathway. Archaeal 16S rRNA analyses demonstrated that the hydrogenotrophic methanogens Methanoculleus spp. accounted for >90% of detected methanogens, and the aceticlastic methanogens Methanosarcina spp. were the minor constituents. The clone library targeting bacterial 16S rRNA indicated the predominance of the novel Thermotogales bacterium (relative abundance: ~53%), which is related to anaerobic acetate oxidizer Thermotoga lettingae TMO, although the sequence similarity was low. Uncultured bacteria that phylogenetically belong to municipal solid waste cluster I were also predominant in the microflora (~30%). These results imply that the microbial community in the thermophilic degrading process of organic solid waste consists exclusively of unidentified bacteria, which efficiently remove acetate through a non-aceticlastic oxidative pathway., (Copyright © 2010 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

We have reported for the first time that agricultural and cellulosic waste, i.e., rice straw was directly applied to methanogenic bioreactors containing carbon fiber textiles (CFT) as supporting material. Addition of CFT to the methanogenic bioreactors enhanced the conversion of dichromate chemical oxygen demand of the substrate to methane (41%) to a greater extent than bioreactors without CFT (9%). In addition, removal of rice straw as a suspended solid was increased from 31% (in bioreactors without CFT) to 57% (in those with CFT). Methanogenic 16S rRNA gene analysis showed that the abundance of acetoclastic methanogen, genus Methanosarcina, was about 11 times higher in bioreactors with CFT (suspended fraction plus retained fraction to CFT) than in bioreactors without CFT (suspended fraction), resulting in lower concentration of acetate in bioreactors with CFT (0.4 mM) than in those without CFT (29.7 mM). On the other hand, the abundance of hydrogenotrophic methanogen, genus Methanobacterium, in bioreactors with CFT was similar to those without CFT. Bacterial communities in bioreactors with CFT were different from those in bioreactors without CFT. Our results indicated that specific microbial community and cooperative relationships between microorganisms in reactors containing CFT facilitated efficient decomposition of rice straw and its conversion to methane.

Adding a supporting material to a methanogenic bioreactor treating garbage slurry can improve efficiency of methane production. However, little is known on how characteristics (e.g., porosity and hydrophobicity) of the supporting material affect the bioreactor degrading garbage slurry. We describe the reactor performances and microbial communities in bioreactors containing hydrophilic or hydrophobic sheets, or fibrous hydrophilic or hydrophobic sponges. The porosity affected the efficiency of methane production and solid waste removal more than the hydrophilic or hydrophobic nature of the supporting material. When the terminal restriction fragment length polymorphism technique was used at a lower organic loading rate (OLR), microbial diversities in the suspended fraction were retained on the hydrophobic, but not the hydrophilic, sheets. Moreover, real-time quantitative polymerase chain reaction (PCR) performed at a higher OLR revealed that the excellent performance of reactors containing fibrous sponges with high porosity (98%) was supported by a clear increase in the numbers of methanogens on these sponges, resulting in larger total numbers of methanogens in the reactors. In addition, the bacterial communities in fractions retained on both the hydrophobic and hydrophilic fibrous sponges differed from those in the suspended fraction, thus increasing bacterial diversity in the reactor. Thus, higher porosity of the supporting material improves the bioreactor performance by increasing the amount of methanogens and bacterial diversity; surface hydrophobicity contributes to maintaining the suspended microbial community.

To compare the performances and microbial populations of methanogenic reactors with and without carbon fiber textiles (CFT), we operated small-scale (200 ml) reactors using a slurry of artificial garbage. For both types of reactors, the organic loading rate (OLR) was stepwisely and rapidly increased in the same manner. Start-up period was shortened by adding CFT. Reactors with CFT showed greater efficiency for removal of suspended solid and volatile suspended solid than reactors without CFT at a long hydraulic retention time (HRT) between 8 and 13 days. The reactors with CFT maintained stable methane production at an OLR of 15.3 g dichromate chemical oxygen demand (CODcr)/l/day and DNAs from microorganisms were highly concentrated in adhering fractions on CFT. As shown by quantitative PCR analysis, the proportions of methanogenic archaea were conserved more than 25% in adhering fractions on CFT in reactors with CFT. By contrast, reactors without CFT showed accumulation of volatile fatty acid and deteriorated at an OLR of 2.4 gCODcr/l/day. Methanogenic proportions dropped to 17.1% in suspended fractions of reactors without CFT. Denaturing gradient gel electrophoresis (DGGE) analysis revealed that all archaeal DGGE bands in both types of reactors were related to methanogens, but more bands were observed in reactors with CFT. Thus the higher performance of reactors with CFT likely reflects the greater abundance of microorganisms and methanogenic diversity.

An anaerobic packed-bed reactor using carbon fiber textiles (CFT) as the supporting material was continuously operated using an artificial garbage slurry. 16S rRNA gene analysis showed that many bacteria in the biomass adhering to CFT were closely related to those observed from other anaerobic environments, although a wide variety of unidentified bacteria were also found. Dot blot hybridization results clarified that 16S rRNA levels of methanogens in the adhering biomass were higher than those in the effluent. Based on microscopic observation, the adhering biomass consisted of microorganisms, organic material, and void areas. Bacteria and Archaea detected by fluorescence in situ hybridization were distributed from the surface to the inner regions of the adhering biomass. Methanosarcina sp. tended to be more abundant in the inner part of the adhering biomass than at the surface. This is the first report to elucidate the structure of the microbial community on CFT in a packed-bed reactor.

The population of methanogenic archaea in a packed-bed reactor was determined. A difference between populations on the supporting material and in effluent was observed at operation under a high organic loading rate and a short hydraulic retention time. This difference was characterized by the predominance of Methanosarcina sp. on the supporting material.

Characterization of microbial communities using single-strand conformation polymorphism (SSCP) was compared with that using denaturing gradient gel electrophoresis (DGGE). This comparison was based on the V3-4 region (Escherichia coli positions: 341-806) of 16S rRNA gene of bacterial or archaeal communities obtained from a methanogenic bioreactor. Significant differences in the bacterial banding profiles were observed while attempting to detect the diversity of the community and its succession during the reactor operation. The SSCP produced a number of sharp bands and differentiated the bacterial community structures to which the DGGE gave an identical pattern. On the other hand, the SSCP and DGGE provided similar succession patterns for archaeal community.

The microbial community in a thermophilic anaerobic packed-bed reactor, which had been successfully operated to convert acetic and butyric acids to methane at a short hydraulic retention time (from 24 h to 1.9 h), was investigated. Archaea closely related to known methanogens were detected by 16S rRNA gene analyses of the effluents, together with diverse types of unidentified bacteria.

In this study, the microbial community succession in a thermophilic methanogenic bioreactor under deteriorative and stable conditions that were induced by acidification and neutralization, respectively, was investigated using PCR-mediated single-strand conformation polymorphism (SSCP) based on the 16S rRNA gene, quantitative PCR, and fluorescence in situ hybridization (FISH). The SSCP analysis indicated that the archaeal community structure was closely correlated with the volatile fatty acid (VFA) concentration, while the bacterial population was impacted by pH. The archaeal community consisted mainly of two species of hydrogenotrophic methanogen (i.e., a Methanoculleus sp. and a Methanothermobacter sp.) and one species of aceticlastic methanogen (i.e., a Methanosarcina sp.). The quantitative PCR of the 16S rRNA gene from each methanogen revealed that the Methanoculleus sp. predominated among the methanogens during operation under stable conditions in the absence of VFAs. Accumulation of VFAs induced a dynamic transition of hydrogenotrophic methanogens, and in particular, a drastic change (i.e., an approximately 10,000-fold increase) in the amount of the 16S rRNA gene from the Methanothermobacter sp. The predominance of the one species of hydrogenotrophic methanogen was replaced by that of the other in response to the VFA concentration, suggesting that the dissolved hydrogen concentration played a decisive role in the predominance. The hydrogenotrophic methanogens existed close to bacteria in aggregates, and a transition of the associated bacteria was also observed by FISH analyses. The degradation of acetate accumulated during operation under deteriorative conditions was concomitant with the selective proliferation of the Methanosarcina sp., indicating effective acetate degradation by the aceticlastic methanogen. The simple methanogenic population in the thermophilic anaerobic digester significantly responded to the environmental conditions, especially to the concentration of VFAs.

With particular focus on the microbial diversity in garbage treatment, the current status of garbage treatment in Japan and microbial ecological studies on various bioprocesses for garbage treatment are described in detail. The future direction of research in this field is also discussed.

Abstract: Methanogenic bioreactors, which are packed with supporting material, have attracted attention as an efficient means of degrading garbage. We aimed to increase bioreactor performance by using an electrochemical system to regulate the electrical potential on supporting material. At an organic loading rate of 26.9g dichromate chemical oxygen demand (CODcr)/L/day, reactors with a potential of −0.6 or −0.8V, generated by a cathodic electrochemical reaction, showed greater removal of CODcr and methanogenesis than reactors with a potential of 0.0 or −0.3V, generated by anodic reaction, or control reactors without electrochemical regulation. 16S rRNA gene analysis revealed that the same methanogens were present in all our reactors, but quantitative real-time polymerase chain reaction showed that higher prokaryotic and methanogenic copy numbers were present on cathodic electrodes than on anodic or control electrodes. These results indicate that cathodic electrochemical regulation can support methane fermentation from garbage. [Copyright &y& Elsevier]

Abstract: Methane fermentation was successfully carried out in bioelectrochemical reactors without membranes under a working potential of −0.6 or −0.8V (vs. Ag/AgCl) and neutral pH conditions. The hydrogenotrophic methanogens that dominated on the anodic and cathodic electrodes differed from those found on the electrodes in the control reactors without electrochemical reactions. [ABSTRACT FROM AUTHOR]