Quantitative analysis of methanogenic community dynamics in three anaerobic batch digesters treating different wastewaters

Quantitative changes in methanogenic community structures, associated with performance data, were investigated in three anaerobic batch digesters treating synthetic glucose medium, whey permeate, and liquefied sewage sludge. All digesters were initially seeded with anaerobic sludge obtained from a local municipal wastewater treatment plant. Dynamics of methanogenic populations were monitored, at order and family levels, using real-time PCR based on the 16S rRNA gene. The molecular monitoring revealed that, in each digester, the quantitative structure of methanogenic community varied continuously over treatment time and the variation corresponded well to the changes in chemical profiles. Biphasic production of methane, associated with successive increases in aceticlastic (mainly Methanosarcinaceae) and hydrogenotrophic (mainly Methanomicrobiales) methanogenic groups, was observed in each digester. This corresponded to the diauxic utilization of acetate and longer-chain volatile fatty acids (C₃-C₆), mainly propionate. Additionally, the non-metric multidimensional scaling (NMDS) analysis of the quantification results demonstrated that the community shift patterns in three digesters were totally different from each other. Considering that the operating conditions in all trials were identical except substrates, the differences in quantitative shift profiles were suggested to be due to the different substrate compositions. This implied that the composition of wastewater could affect the evolution of quantitative methanogenic community structure in an anaerobic process. Overall, our results suggested that more attention to quantitative as well as qualitative approaches on microbial communities is needed for fundamental understanding of anaerobic processes, particularly under dynamic or transitional conditions.     

Development of high-rate anaerobic ammonium-oxidizing (anammox) biofilm reactors

To promptly establish anaerobic ammonium oxidation (anammox) reactors, appropriate seeding sludge with high abundance and activity of anammox bacteria was selected by quantifying 16S rRNA gene copy numbers of anammox bacteria by real-time quantitative PCR (RTQ-PCR) and batch culture experiments. The selected sludge was then inoculated into up-flow fixed-bed biofilm column reactors with nonwoven fabric sheets as biomass carrier and the reactor performances were monitored over 1 year. The anammox reaction was observed within 50 days and a total nitrogen removal rate of 26.0 kg-Nm(-3)day(-1) was obtained after 247 days. To our knowledge, such a high rate has never been reported before. Hydraulic retention time (HRT) and influent NH(4)(+) to NO(2)(-) molar ratio could be important determinant factors for efficient nitrogen removal in this study. The higher nitrogen removal rate was obtained at the shorter HRT and higher influent NH(4)(+)/NO(2)(-) molar ratio. After anammox reactors were fully developed, the community structure, spatial organization and in situ activity of the anammox biofilms were analyzed by the combined use of a full-cycle of 16S rRNA approach and microelectrodes. In situ hybridization results revealed that the probe Amx820-hybridized anaerobic anammox bacteria were distributed throughout the biofilm (accounting for more than 70% of total bacteria). They were associated with Nitrosomonas-like aerobic ammonia-oxidizing bacteria (AAOB) in the surface biofilm. The anammox bacteria present in this study were distantly related to the Candidatus Brocadia anammoxidans with the sequence similarity of 95%. Microelectrode measurements showed that a high in situ anammox activity (i.e., simultaneous consumption of NH(4)(+) and NO(2)(-)) of 4.45 g-N of (NH(4)(+)+NO(2)(-))m(-2)day(-1) was detected in the upper 800 microm of the biofilm, which was consistent with the spatial distribution of anammox bacteria.     

Functional bacterial and archaeal community structures of major trophic groups in a full-scale anaerobic sludge digester

Functional Bacteria and Archaea community structures of a full-scale anaerobic sludge digester were investigated by using a full-cycle 16S rRNA approach followed by microautoradiography (MAR)-fluorescent in situ hybridization (FISH) technique and micromanipulation. FISH analysis with a comprehensive set of 16S and 23S rRNA-targeted oligonucleotide probes based on 16S rRNA clone libraries revealed that the Gram-positive bacteria represented by probe HGC69A-hybridized Actinobacteria (8.5+/-1.4% of total 4', 6-diamidino-2-phenylindole (DAPI)-stained cells) and probe LGC354-hybridized Firmicutes (3.8+/-0.8%) were the major phylogenetic bacterial phyla, followed by Bacteroidetes (4.0+/-1.2%) and Chloroflexi (3.7+/-0.8%). The probe MX825-hybridized Methanosaeta (7.6+/-0.8%) was the most abundant archaeal group, followed by Methanomicrobiales (2.8+/-0.6%) and Methanobacteriaceae (2.7+/-0.4%). The functional community structures (diversity and relative abundance) of major trophic groups were quantitatively analyzed by MAR-FISH. The results revealed that glucose-degrading microbial community had higher abundance (ca. 10.6+/-4.9% of total DAPI-stained cells) and diversity (at least seven phylogenetic groups) as compared with fatty acid-utilizing microbial communities, which were more specialized to a few phylogenetic groups. Despite the dominance of Betaproteobacteria, members of Chloroflexi, Smithella, Syntrophomonas and Methanosaeta groups dominated the [(14)C]glucose-, [(14)C]propionate-, [(14)C]butyrate- and [(14)C]acetate-utilizing microorganism community, and accounted for 27.7+/-4.3%, 29.6+/-7.0%, 34.5+/-7.6% and 18.2+/-9.5%, respectively. In spite of low abundance (ca. 1%), the hitherto unknown metabolic functions of Spirochaeta and candidate phylum of TM7 as well as Synergistes were found to be glucose and acetate utilization, respectively.     

Impact of inocula and growth mode on the molecular microbial ecology of anaerobic ammonia oxidation (anammox) bioreactor communities

The composition of distinctly inoculated granular anammox and biofilm-based completely autotrophic nitrogen removal over nitrite (CANON) bioreactors was investigated from start-up through continuous long-term operation via denaturing gradient gel electrophoresis (DGGE) and sequencing. The granular anammox reactor was seeded with sludge from an operational anammox reactor in Strass, Austria. The CANON reactor was seeded with activated sludge from a local wastewater treatment plant in New York City. The principal anammox bacteria (AMX) shifted from members related to Kuenenia stuttgartiensis present in the initial inoculum to members related to Candidatus Brocadia fulgida during pre-enrichment (before this study) and to members related to Candidatus Brocadia sp. 40 (during this study) in the granular reactor. AMX related to C. Brocadia sp. 40 were also enriched from activated sludge in the CANON reactor. The estimated doubling times of AMX in the granular and CANON reactors were 5.3 and 8.9 days, respectively, which are lower than the value of 11 days, reported previously. Both the granular anammox and CANON reactors also fostered significant amounts of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB). The fractions of AMX and two groups of NOB were generally similar in the granular anammox and CANON reactors. However, the diversity and fractions of AOB in the two reactors was markedly different. Therefore, it is suggested that the composition of the feed and extant substrate concentrations in the reactor likely select for the microbial community composition more than the inocula and reactor configuration. Further, such selection is not equivalent for all resident communities.     

Quantitative and qualitative analyses of methanogenic community development in high-rate anaerobic bioreactors

Methanogenic community structure and population dynamics were investigated in two anaerobic reactors treating a dairy wastewater, an Inverted Fluidized Bed (IFB) and Expanded Granular Sludge Bed (EGSB). A combination of real-time PCR, denaturing gradient gel electrophoresis and statistical techniques was employed. Distinct methanogenic communities developed in the IFB and EGSB reactors reflecting step-wise reductions in the applied hydraulic retention time from 72 to 12 h during the 200-day trial. The aceticlastic family Methanosarcinaceae was only detected in the IFB and the order Methanomicrobiales was also much more abundant in this reactor, while the aceticlastic family Methanosaetaceae was more abundant in the EGSB. The hydrogenotrophic order, Methanobacteriales, predominated in both reactors under all applied operational conditions. Non-metric multidimensional scaling (NMS) and moving-window analyses, based on absolute and relative abundance quantification data, demonstrated that the methanogenic communities developed in a different manner in the IFB, compared to the EGSB reactor. In our study, relative abundance-based quantification by NMS and moving-window analysis appeared to be a valuable molecular approach that was more applicable to reflect the changes in the anaerobic digestion process than approaches based either on qualitative analysis, or solely on absolute quantification of the various methanogenic groups. The overall results and findings provided a comparative, quantitative and qualitative insight into anaerobic digestion processes, which could be helpful for better future reactor design and process control.     

Monitoring bacterial indicators and pathogens in cattle feedlot waste by real-time PCR

Quantitative microbial health risk assessment requires accurate enumeration of pathogens in hazard-containing matrices as part of the risk characterization process. As part of a risk management-oriented study of cattle feedlot waste contaminants, we investigated the utility of quantitative real-time PCR (qPCR) for surveying the microbial constituents of different faecal wastes. The abundance of Escherichia coli and enterococci were first estimated in five cattle feedlot waste types from five localities. Bacteria were quantified using two culture methods and compared to the number of genome copies detected by qPCR targeted at E. coli and Enterococcus faecalis. Bacterial numbers detected in the different wastes (fresh faeces, pen manure, aged manure, composted manure, carcass manure compost) ranged from 10−⁷ to 10² g⁻¹ (dry weight). Both indicator groups were detected by qPCR with a comparable sensitivity to culture methods across this range. qPCR measurements of E. coli and E. faecalis correlated well with MPN and spread plate data. As a second comparison, we inoculated green fluorescent protein (GFP) labeled reference bacteria into manure samples. GFP labeled E. coli and Listeria monocytogenes were detected by qPCR in concentrations corresponding to between 18% and 71% of the initial bacterial numbers, compared to only 2.5-16% by plating. Our results supported our selection of qPCR as a fast, accurate and reliable system for surveying the presence and abundance of pathogens in cattle waste.     

应用SBR实现有机废水厌氧氨氧化生物脱氮的研究

采用SBR反应器,以硝化污泥和厌氧氨氧化(ANAMMOX)颗粒污泥的混合污泥为接种污泥,以有机模拟废水为研究对象,进行了厌氧氨氧化生物脱氮工艺研究。结果表明,在控制温度为25℃,水力停留时间为12 d,pH值为7.2～8.5,进水NH4+-N为220 mg/L左右、NO2--N为138 mg/L左右、COD为294 mg/L的条件下成功启动了SBR反应器。在高氨氮、低有机物浓度的条件下,ANAMMOX菌和异养反硝化菌能够实现共存,且ANAMMOX菌仍能成为优势菌属,AN-AMMOX反应是反应器中的主导反应。镜检发现,优势菌尺寸约为1μm,呈圆形或椭圆形,成簇聚生,表面可观察到明显的漏斗状缺口,具有典型的厌氧氨氧化菌特征。污泥中形成了以厌氧氨氧化球状菌为主、其他杆状菌和丝状菌共存的微生物混培体。     

Optimization of anaerobic acidogenesis of an aquatic plant, Canna indica L., by rumen cultures

Anaerobic acidogenesis of Canna indica L. (canna) by rumen cultures was investigated in this study. Fractional factorial design (FFD) was used to explore the roles of the growth factors such as substrate concentration and pH in such a bioconversion, whereas response surface methodology (RSM) was employed for optimizing this acidogenic process. The optimum substrate concentration and pH for the acidogenesis of canna were found to be 8.2 g VSl(-1) and 6.6, respectively, and the corresponding degradation efficiency of canna was 52.3%. Volatile fatty acid yield peaked at 0.362 g g(-1)VS degraded at a substrate concentration of 6.9 g VSl(-1)and pH 6.7. These results were confirmed by the experimental results.     

ANAMMOX与反硝化协同脱氮反应器的启动特性研究

向成功启动并已稳定运行2年的ANAMMOX反应器中连续添加有机物(葡萄糖),研究ANAMMOX与反硝化协同脱氮反应器的启动特性.结果表明,在短期内(35 d)可成功启动ANAMMOX与反硝化协同脱氮反应器.启动过程可分为迟滞、适应和稳定运行三个阶段,在稳定运行阶段反应器对NH_4~+-N、NO_2~-—N、TN和COD的去除率分别高达95%、99%、94%和93%,NH_4~+-N去除量、NO_2~--N去除量与NO_3~--N生成量的比值为1:1.32:0.03,出水碱度和pH均略高于进水.     

Long-term effects of operating temperature and sulphate addition on the methanogenic community structure of anaerobic hybrid reactors

The diversity, population dynamics, and activity profiles of methanogens in anaerobic granular sludges from two anaerobic hybrid reactors treating a molasses wastewater both mesophilically (37 degrees C) and thermophilically (55 degrees C) during a 1081 day trial were determined. The influent to one of the reactors was supplemented with sulphate, after an acclimation period of 112 days, to determine the effect of competition with sulphate-reducing bacteria on the methanogenic community structure. Sludge samples were removed from the reactors at intervals throughout the operational period and examined by amplified ribosomal DNA (rDNA) restriction analysis (ARDRA) and partial sequencing of 16S rRNA genes. In total, 18 operational taxonomic units (OTUs) were identified, 12 of which were sequenced. The methanogenic communities in both reactors changed during the operational period. The seed sludge and the reactor biomass sampled during mesophilic operation, both in the presence and absence of sulphate, was characterised by a predominance of Methanosaeta spp. Following temperature elevation, the dominant methanogenic sequences detected in the non-sulphate supplemented reactor were closely related to Methanocorpusculum parvum. By contrast, the dominant OTUs detected in the sulphate-supplemented reactor upon temperature increase were related to the hydrogen-utilising methanogen, Methanobacterium thermoautotrophicum. The observed methanogenic community structure in the reactors correlated with the operational performance of the reactors during the trial and with physiological measurements of the reactor biomass. Both reactors achieved chemical oxygen demand (COD) removal efficiencies of over 90% during mesophilic operation, with or without sulphate supplementation. During thermophilic operation, the presence of sulphate resulted in decreased reactor performance (effluent acetate concentrations of >3000 mg/l and biogas methane content of <25%). It was demonstrated that methanogenic conversion of acetate at 55 degrees C was extremely sensitive to inhibition by sulphide (50% inhibition at 8-17 mg/l unionised sulphide at pH 7.6-8.0), while the conversion of H(2)/CO(2) methanogenically was favoured. The combination of experiments carried out demonstrated the presence of specific methanogenic populations during periods of successful operational performance.     

Effect of the presence of the antimicrobial tylosin in swine waste on anaerobic treatment

An anaerobic sequencing batch reactor (ASBR), seeded with a biomass inoculum that previously had not been exposed to the macrolide antimicrobial tylosin (mixture of Tylosin A, B, C, and D), was operated for 3 months with swine waste without Tylosin A and for 9 months with swine waste containing Tylosin A at an average concentration of 1.6 mg/L. When swine waste with tylosin was fed to the ASBR, methane production and volatile solids removal did not appear to be inhibited and a methane yield of 0.47 L methane per gram volatile solids fed to the ASBR was observed. Throughout the operating period, Tylosin A levels in ASBR biomass and effluent were below the detection limit of 0.01 mg/L. However, during the first 3 months of operation, the levels of macrolide-lincosamide-streptogramin B (MLSB)-resistant bacteria in the ASBR biomass increased substantially as determined by hybridizations with oligonucleotide probes designed to target MLSB-resistant bacteria. Since no Tylosin A was present in the swine waste during the initial 3 months, the presence of MLSB-resistant bacteria in the swine waste was likely the reason for the increase in resistance. Subsequently, the levels of MLSB-resistant bacteria in ASBR biomass stabilized with an average of 44.9% for the 9 months of operation with swine waste containing Tylosin A. The level of MLSB-resistant bacteria in the swine waste fed to the ASBR during this period averaged 18.0%. The results indicate that anaerobic treatment of a waste stream containing tylosin was effective (based on reactor performance) and that the level of resistant bacteria in the ASBR was substantially higher than in the waste stream fed to this system.     

Development of microbial community structure and actvity in a high-rate anaerobic bioreactor at 18 degrees C

Anaerobic digestion in the psychrophilic (< 20 degrees C) or sub-mesophilic temperature range has recently been proven as an effective treatment option for the mineralization of a wide variety of problematic wastewaters. In this study, an expanded granular sludge bed-anaerobic filter (EGSB-AF) bioreactor was seeded with a full-scale, mesophilic sludge and employed to evaluate the long-term operational potential, and underlying microbial ecology, of this approach for the treatment of a medium-strength (5 g chemical oxygen demand [COD] l(-1)), synthetic, volatile fatty acid-based wastewater. Throughout the trial period of 625 days, extended intervals of consistently stable and efficient wastewater treatment were sustained. These results were highlighted by a short start-up period (21 d), low hydraulic retention times (4.88h), high organic (up to 24.64kg CODm(-3)d(-1)), and volumetric loading rates (up to 4.92 m3 m(-3) d(-1)). A stable, well-settling granular sludge bed was maintained in the bioreactor for the majority of the trial; however, reduced treatment efficiency and biomass washout were observed at an imposed OLR of 36.96 kg COD m(-3) d(-1). The microbial biomass in the bioreactor was investigated using maximum specific methanogenic activity assays and polymerase chain reaction-denaturing gradient gel electrophoresis. A temporal succession of both the bacterial and archaeal populations was noted during the trial, compared to the seed sludge, in response to bioreactor operation at lower temperatures, loading rate increases and to VFA accumulation in the bioreactor. During the trial, an increased contribution of hydrogenotrophic methanogenesis as a pathway of methane production was observed, along with the overall emergence of a highly active psychrotolerent-though still mesophilic biomass.     

Genotyping of single Cryptosporidium oocysts in sewage by semi-nested PCR and direct sequencing

This study describes an approach for genotyping individual Cryptosporidium oocysts obtained from sewage. We isolated single immunofluorescent assay (IFA)-stained Cryptosporidium oocysts from sewage concentrate using glass capillary pipettes and inverted epifluorescence microscopy. Each isolated Cryptosporidium oocyst was analyzed by semi-nested PCR for the 18S rRNA gene and direct sequencing of the PCR products. A total of 74 of 107 oocysts isolated from sewage were genotyped successfully. Of the 74 genotyped isolates, 51% (38 oocysts) were identified as C. parvum genotype 1, 4% (3 oocysts) of C. parvum VF383 human isolates, 20% (15 oocysts) of C. parvum genotype 2, 14% (10 oocysts) of C. meleagridis, 7% (5 oocysts) of C. sp. Pig 1, 3% (2 oocysts) of C. sp PG1-26 pig isolates and 1% (1 oocyst) of C. parvum CPM1 isolated from mouse. The results of this study demonstrate that 18S rRNA-based semi-nested PCR and direct sequencing can be used to characterize individual Cryptosporidium oocysts and also to reveal the distribution of Cryptosporidium genotypes in environmental waters.     

Use of molecular techniques for the analysis of foam-causing bacteria in Al Bireh oxidation ditch,Palestine

Activated sludge foaming, a worldwide problem, usually consists of filamentous bacteria occurring predominantly in the mixed liquor. Because of a lack of pure cultures of most filamentous bacteria and the limited amount of characterisation data, molecular approaches were used to investigate dominant filamentous bacterial strains associated with foaming in Al Bireh Wastewater Treatment Plant in Palestine. Applying denaturing gradient gel electrophoresis (DGGE), 16S rRNA cloning and sequencing showed the dominance of several filamentous bacteria including Microthrix parvicella, Nocardia sp., Hyphomicrobium facilis, Chloroflexi, Candidates TM7 and Nocardioides oleivorans.     

Degradation of plasmid and plant DNA in water microcosms monitored by natural transformation and real-time polymerase chain reaction (PCR)

Extracellular DNA exists in the environment and can be taken up by competent bacterial cells, leading to horizontal gene transfer. The persistence of extracellular plasmid and plant DNA in water microcosms was monitored in this study. Water samples were two groundwater (GW1 and GW2) and one river water (RW) samples. Three treatments included: (1) intact, (2) 0.22 microm filter-sterilized, and (3) autoclaved water. DNA from a plasmid (pNS1) and a transgenic Bt (Bacillus thuringiensis) corn line, both carrying a neomycin phosphotransferase gene (nptII gene) conferring kanamycin and neomycin resistance, was inoculated into the microcosms at 0.4 and 0.8 microg/ml, respectively. By monitoring its ability to transform a competent Pseudomonas stutzeri strain harboring plasmid pMR7 (P. stutzeri pMR7), plasmid DNA was degraded to undetectable levels in the intact and/or filter-sterilized water treatments within 48-96 h in GW1, GW2, and RW. Meanwhile, plasmid DNA persisted in the autoclaved treatment throughout the entire incubation period. For plant DNA, a highly sensitive real-time PCR method using SYBR Green I was developed to monitor the degradation dynamics of the nptII gene carried by the transgenic corn line in the microcosms. The results showed that the concentration of plant DNA was reduced by two orders of magnitude (from 0.8-0.008 microg/ml) within 96 h in the intact and filter-sterilized treatments of GW1, GW2, and RW, in contrast to its persistence in the autoclaved treatment. In addition, no kanamycin resistant (Km R) transformants were detected from in situ transformation of P. stutzeri pMR7 with plasmid pNS1 DNA.     

Bacterial community dynamics in an anaerobic plug-flow type bioreactor treating swine manure

A plug-flow type anaerobic reactor consisting of eight sequential compartments was used to study shifts in a bacterial community adapted to degrade swine manure at 25 °C. The investigation was carried out during the first 6 months of reactor operation. The reactor successfully separated the hydrolysis/acidogenesis stage from the methanogenesis stage. Bacterial 16S rDNA- and rRNA-based fingerprints obtained through amplicon length heterogeneity PCR (LH-PCR) were analyzed with a view to characterizing the bacterial community structure and the metabolically active community, respectively. Multivariate statistical tools showed that the rDNA-based fingerprints described a more temporal than compartmentalized distribution of similar bacterial communities. By contrast, the rRNA-based multivariate analyses described a distribution that was linked more to reactor performance parameters, especially during short time periods. Diversity indices calculated from fingerprint data were used to assess overall diversity shifts. The decrease in rRNA-based diversity observed through the reactor compartments was greater than the decrease in rDNA-based diversity. This finding indicates that the analysis of metabolically active bacteria diversity was more discriminative than the analysis based on the mere presence of bacteria. The observed decrease in diversity suggests that the bacterial community became specialized in degrading less diversified substrates through the compartments. All these findings suggest that rRNA-based analyses are more appropriate for monitoring reactor performance.     

Quantitative real-time PCR analysis of total and propidium monoazide-resistant fecal indicator bacteria in wastewater

A real-time quantitative PCR (qPCR) method and a modification of this method incorporating pretreatment of samples with propidium monoazide (PMA) were evaluated for respective analyses of total and presumptively viable Enterococcus and Bacteroidales fecal indicator bacteria. These methods were used in the analyses of wastewater samples to investigate their feasibility as alternatives to current fecal indicator bacteria culture methods for predicting the efficiency of viral pathogen removal by standard treatment processes. PMA treatment was effective in preventing qPCR detection of target sequences from non-viable cells. Concentrates of small volume, secondary-treated wastewater samples, collected from a publicly owned treatment works (POTW) under normal operating conditions, had little influence on this effectiveness. Higher levels of total suspended solids, such as those associated with normal primary treatment and all treatment stages during storm flow events, appeared to interfere with PMA effectiveness under the sample preparation conditions employed. During normal operating conditions at three different POTWs, greater reductions were observed in PMA-qPCR detectable target sequences of both Enterococcus and Bacteroidales than in total qPCR detectable sequences. These reductions were not as great as those observed for cultivable fecal indicator bacteria in response to wastewater disinfection. Reductions of PMA-qPCR as well as total qPCR detectable target sequences from enterococci and, to a lesser extent, Bacteroidales correlated well with reductions in infectious viruses during both normal and storm flow operating conditions and therefore may have predictive value in determining the efficiency at which these pathogens are removed.     

Influence of the agitation rate on the treatment of partially soluble wastewater in anaerobic sequencing batch biofilm reactor

This work reports on the influence of the agitation rate on the organic matter degradation in an anaerobic sequencing batch reactor, containing biomass immobilized on 3 cm cubic polyurethane matrices, stirred mechanically and fed with partially soluble soymilk substrate with mean chemical oxygen demand (COD) of 974+/-70 mg l(-1). Hydrodynamic studies informed on the homogenization time under agitagion rates from 500 to 1100 rpm provided by three propeller impellers. It occurred very quickly compared to the total cycle time. The results showed that agitation provided good mixing and improved the overall organic matter consumption rates. A modified first-order kinetic model represented adequately the data in the entire range of agitation rate. The apparent first-order kinetic constant for suspended COD rose approximately 360% when the agitation rate was changed from 500 to 900 rpm, whereas the apparent first-order kinetic constant for soluble COD did not vary significantly.     

Quantitative detection of previously characterized syntrophic bacteria in anaerobic wastewater treatment systems by sequence-specific rRNA cleavage method

Quantitative monitoring method of two important trophic groups of bacteria in methanogenic communities was established and applied to six different anaerobic processes. The method we employed was based upon our previous sequence-specific rRNA cleavage method that allows quantification of rRNA of target groups so that the populations reflecting in situ activity could be determined. We constructed a set of scissor probes targeting the Chloroflexi group known as ‘semi-syntrophic’ heterotrophic bacteria and fatty acid-oxidizing syntrophs to determine their relative abundance in the processes. By using the method, we found that several reactors harbored a large amount of organisms belonging to the phylum Chloroflexi accounting for up to 20% of the total prokaryotic populations. Propionate-oxidizing syntrophs, Syntrophobacter, Smithella and Pelotomaculum were also found to be significant comprising up to 3.9% of the total populations, but their distribution is highly dependent on the process examined. This is the first clear, non-PCR based quantitative evidence that those organisms play active roles under in situ methanogenic conditions.