Presence of diverse nitrate-dependent anaerobic methane oxidizing archaea in sewage sludge.

AIM: The aim of this study was to explore the community diversity and abundance of nitrate-dependent anaerobic methane oxidizing archaea, Candidatus Methanoperedens nitroreducens, in sewage sludge from wastewater treatment plants. METHODS AND RESULTS: Seasonal sampling of the sewage sludge was carried out from two wastewater treatment plants (WWTPs) located in the northern and southern parts of China. Through amplicon sequencing using our newly designed primers, a large number of Candidatus Methanoperedens nitroreducens-like (M. nitroreducens) archaeal sequences (638 743) were generated. These sequences were assigned into 742 operational protein units (OPUs) at 90% cut-off level and classified as Group B member of M. nitroreducens archaea in the phylogenetic tree. More than 80% of the OPUs were not shared between these two WWTPs, showing the M. nitroreducens-like archaeal community in each WWTP was unique. Quantitative PCR assays also confirmed the presence of M. nitroreducens-like archaea and revealed a higher abundance in autumn and winter than other seasons, indicating that the environmental attributes in these seasons might favour the growth of this archaea. Further redundancy analysis revealed that volatile solid and pH were the significant environmental attributes (P < 0·05) in shaping the M. nitroreducens-like archaeal community based on variance inflation factor selection and Monte Carlo permutation test. CONCLUSIONS: The results confirmed the presence of diverse M. nitroreducens-like archaea in sewage sludge using Illumina-based mcrA gene sequencing and quantitative PCR assays. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study revealed the ecological characteristics of M. nitroreducens-like archaea in sewage sludge that improved our understanding of nitrate-dependent anaerobic methane oxidation process and may be the basis for future application of M. nitroreducens-like archaea for new nitrogen removal in WWTPs.

Metabolic capability and in situ activity of microorganisms in an oil reservoir.

BACKGROUND: Microorganisms have long been associated with oxic and anoxic degradation of hydrocarbons in oil reservoirs and oil production facilities. While we can readily determine the abundance of microorganisms in the reservoir and study their activity in the laboratory, it has been challenging to resolve what microbes are actively participating in crude oil degradation in situ and to gain insight into what metabolic pathways they deploy. RESULTS: Here, we describe the metabolic potential and in situ activity of microbial communities obtained from the Jiangsu Oil Reservoir (China) by an integrated metagenomics and metatranscriptomics approach. Almost complete genome sequences obtained by differential binning highlight the distinct capability of different community members to degrade hydrocarbons under oxic or anoxic condition. Transcriptomic data delineate active members of the community and give insights that Acinetobacter species completely oxidize alkanes into carbon dioxide with the involvement of oxygen, and Archaeoglobus species mainly ferment alkanes to generate acetate which could be consumed by Methanosaeta species. Furthermore, nutritional requirements based on amino acid and vitamin auxotrophies suggest a complex network of interactions and dependencies among active community members that go beyond classical syntrophic exchanges; this network defines community composition and microbial ecology in oil reservoirs undergoing secondary recovery. CONCLUSION: Our data expand current knowledge of the metabolic potential and role in hydrocarbon metabolism of individual members of thermophilic microbial communities from an oil reservoir. The study also reveals potential metabolic exchanges based on vitamin and amino acid auxotrophies indicating the presence of complex network of interactions between microbial taxa within the community.

Ecological and genomic profiling of anaerobic methane-oxidizing archaea in a deep granitic environment.

Recent single-gene-based surveys of deep continental aquifers demonstrated the widespread occurrence of archaea related to Candidatus Methanoperedens nitroreducens (ANME-2d) known to mediate anaerobic oxidation of methane (AOM). However, it is unclear whether ANME-2d mediates AOM in the deep continental biosphere. In this study, we found the dominance of ANME-2d in groundwater enriched in sulfate and methane from a 300-m deep underground borehole in granitic rock. A near-complete genome of one representative species of the ANME-2d obtained from the underground borehole has most of functional genes required for AOM and assimilatory sulfate reduction. The genome of the subsurface ANME-2d is different from those of other members of ANME-2d by lacking functional genes encoding nitrate and nitrite reductases and multiheme cytochromes. In addition, the subsurface ANME-2d genome contains a membrane-bound NiFe hydrogenase gene putatively involved in respiratory H2 oxidation, which is different from those of other methanotrophic archaea. Short-term incubation of microbial cells collected from the granitic groundwater with 13C-labeled methane also demonstrates that AOM is linked to microbial sulfate reduction. Given the prominence of granitic continental crust and sulfate and methane in terrestrial subsurface fluids, we conclude that AOM may be widespread in the deep continental biosphere.

Identification of Pelomyxa palustris Endosymbionts.

Pelomyxa palustris is a giant anaerobic/microaerobic amoeba, characterized by a number of exceptional cytological and physiological features, among them the presumed absence of energy producing organelles and the presence of endosymbiotic bacteria. These endosymbionts have been previously distinguished as: a large rectangular-shaped Gram-variable rod with a central cleft; a slender Gram-negative rod; and a slender Gram-positive rod. Using DNA extracted from P. palustris cysts, we have obtained three SSU rRNA gene sequences. We have determined that these sequences are affiliated to three different prokaryotic genera: Methanosaeta (a methanogenic archaea), Syntrophorhabdus (a syntrophic Gram-negative bacteria) and Rhodococcus (an aerobic chemoorganotrophic Gram-positive bacteria). To our knowledge, it is the first time that Syntrophorhabdus has been described as an endosymbiont in association with a methanogen. Strikingly, no traces of Methanobacterium formicicum could be detected, despite this methanogen had allegedly been isolated from trophozoites of P. palustris. It seems that the host and the endosymbionts have established a multipartite syntrophic consortium resembling to some extent those found in sewage treatment plants.

Metagenomic analysis reveals the contribution of anaerobic methanotroph-1b in the oxidation of methane at the Ulleung Basin, East Sea of Korea.

We have previously identified a sulfate methane transition zone (SMTZ) within the methane hydrate-bearing sediment in the Ulleung Basin, East Sea of Korea, and the presence of ANME-1b group in the sediment has been shown by phylogenetic analysis of a 16S rRNA gene. Herein, we describe taxonomic and functional profiling in the SMTZ sample by metagenomic analysis, comparing with that of surface sediment. Metagenomic sequences of 115 Mbp and 252 Mbp were obtained from SMTZ and surface sediments, respectively. The taxonomic profiling using BLASTX against the SEED within MG-RAST showed the prevalence of methanogens (19.1%), such as Methanosarcinales (12.0%) and Methanomicrobiales (4.1%) predominated within the SMTZ metagenome. A number of 185,200 SMTZ reads (38.9%) and 438,484 surface reads (62.5%) were assigned to functional categories, and methanogenesis-related reads were statistically significantly overrepresented in the SMTZ metagenome. However, the mapping analysis of metagenome reads to the reference genomes, most of the sequences of the SMTZ metagenome were mapped to ANME-1 draft genomes, rather than those of methanogens. Furthermore, the two copies of the methyl-coenzyme M reductase gene (mcrA) segments of the SMTZ metagenome were clustered with ANME-1b in the phylogenetic cluster. These results indicate that ANME-1b reads were miss-annotated to methanogens due to limitation of database. Many of key genes necessary for reverse methanogenesis were present in the SMTZ metagenome, except for N 5,N 10-methenyl-H4MPT reductase (mer) and CoB-CoM heterodisulfide reductase subunits D and E (hdrDE). These data suggest that the ANME-1b represents the primary player the anaerobic methane oxidation in the SMTZ, of the methane hydrate-bearing sediment at the Ulleung Basin, East Sea of Korea.

Microbial community dynamics during the early stages of plant polymer breakdown in paddy soil.

We used paddy soil slurries amended with rice straw to identify the microbial populations involved in the methanogenic breakdown of plant polymers. Rice straw greatly stimulated microbial activity over the 28-day incubation period. On day 7, the transient peak concentration of acetate (24 mM) coincided with the onset of increased methane production. Microbial 16S rRNA transcript numbers increased by one to two orders of magnitude, but not the 16S rRNA gene copy numbers. Using metatranscriptomic rRNA, Clostridiaceae, Lachnospiraceae, Ruminococcaceae, Veillonellaceae and Pseudomonadaceae were identified to be the most abundant and the most dynamic bacterial groups. Changes in methanogen rRNA and mRNA abundances corresponded well with methanogenic activity. Acetate determined the abundance ratio between Methanosarcinaceae and Methanosaetaceae. Methanocellaceae dominated hydrogenotrophic methanogenesis. Transcript levels of mRNA families involved in plant polymer breakdown increased slightly with time. Glycosyl hydrolase (GH) transcripts involved in cellulose and chitin breakdown were predominantly expressed by the Firmicutes, whereas those involved in hemicellulose breakdown exhibited more diverse taxonomic sources, including Acidobacteria, Bacteriodetes and Chloroflexi. Taken together, we observed strong population dynamics and the expression of taxonomically diverse GH families, suggesting that not only Firmicutes, but also less abundant groups play a major functional role in the decomposition of rice straw.

Metagenome from a Spirulina digesting biogas reactor: analysis via binning of contigs and classification of short reads.

BACKGROUND: Anaerobic digestion is a biological process in which a consortium of microorganisms transforms a complex substrate into methane and carbon dioxide. A good understanding of the interactions between the populations that form this consortium can contribute to a successful anaerobic digestion of the substrate. In this study we combine the analysis of the biogas production in a laboratory anaerobic digester fed with the microalgae Spirulina, a protein rich substrate, with the analysis of the metagenome of the consortium responsible for digestion, obtained by high-throughput DNA sequencing. The obtained metagenome was also compared with a metagenome from a full scale biogas plant fed with cellulose rich material. RESULTS: The optimal organic loading rate for the anaerobic digestion of Spirulina was determined to be 4.0 g Spirulina L(-1) day(-1) with a specific biogas production of 350 mL biogas g Spirulina (-1) with a methane content of 68 %. Firmicutes dominated the microbial consortium at 38 % abundance followed by Bacteroidetes, Chloroflexi and Thermotogae. Euryarchaeota represented 3.5 % of the total abundance. The most abundant organism (14.9 %) was related to Tissierella, a bacterium known to use proteinaceous substrates for growth. Methanomicrobiales and Methanosarcinales dominated the archaeal community. Compared to the full scale cellulose-fed digesters, Pfam domains related to protein degradation were more frequently detected and Pfam domains related to cellulose degradation were less frequent in our sample. CONCLUSIONS: The results presented in this study suggest that Spirulina is a suitable substrate for the production of biogas. The proteinaceous substrate appeared to have a selective impact on the bacterial community that performed anaerobic digestion. A direct influence of the substrate on the selection of specific methanogenic populations was not observed.

Metagenome approaches revealed a biological prospect for improvement on mesophilic cellulose degradation.

Improvement on the bioconversion of cellulosic biomass depends much on the expanded knowledge on the underlying microbial structure and the relevant genetic information. In this study, metagenomic analysis was applied to characterize an enriched mesophilic cellulose-converting consortium, to explore its cellulose-hydrolyzing genes, and to discern genes involved in methanogenesis. Cellulose conversion efficiency of the mesophilic consortium enriched in this study was around 70 %. Apart from methane, acetate was the major fermentation product in the liquid phase, while propionate and butyrate were also detected at relatively high concentrations. With the intention to uncover the biological factors that might shape the varying cellulose conversion efficiency at different temperatures, results of this mesophilic consortium were then compared with that of a previously reported thermophilic cellulose-converting consortium. It was found that the mesophilic consortium harbored a larger pool of putative carbohydrate-active genes, with 813 of them in 54 GH modules and 607 genes in 13 CBM modules. Methanobacteriaceae and Methanosaetaceae were the two methanogen families identified, with a preponderance of the hydrogenotrophic Methanobacteriaceae. In contrast to its relatively high diversity and high abundance of carbohydrate-active genes, the abundance of genes involved in the methane metabolism was comparatively lower in the mesophilic consortium. A biological enhancement on the methanogenic process might serve as an effective option for the improvement of the cellulose bioconversion at mesophilic temperature.

Low-temperature anaerobic digestion is associated with differential methanogenic protein expression.

Anaerobic digestion (AD) is an attractive wastewater treatment technology, leading to the generation of recoverable biofuel (methane). Most industrial AD applications, carry excessive heating costs, however, as AD reactors are commonly operated at mesophilic temperatures while handling waste streams discharged at ambient or cold temperatures. Consequently, low-temperature AD represents a cost-effective strategy for wastewater treatment. The comparative investigation of key microbial groups underpinning laboratory-scale AD bioreactors operated at 37, 15 and 7°C was carried out. Community structure was monitored using 16S rRNA clone libraries, while abundance of the most prominent methanogens was investigated using qPCR. In addition, metaproteomics was employed to access the microbial functions carried out in situ. While δ-Proteobacteria were prevalent at 37°C, their abundance decreased dramatically at lower temperatures with inverse trends observed for Bacteroidetes and Firmicutes. Methanobacteriales and Methanosaeta were predominant at all temperatures investigated while Methanomicrobiales abundance increased at 15°C compared to 37 and 7°C. Changes in operating temperature resulted in the differential expression of proteins involved in methanogenesis, which was found to occur in all bioreactors, as corroborated by bioreactors' performance. This study demonstrated the value of employing a polyphasic approach to address microbial community dynamics and highlighted the functional redundancy of AD microbiomes.

In situ DNA-hybridization chain reaction (HCR): a facilitated in situ HCR system for the detection of environmental microorganisms.

In situ detection of microorganisms by fluorescence in situ hybridization (FISH) is a powerful tool for environmental microbiology, but analyses can be hampered by low rRNA content in target organisms, especially in oligotrophic environments. Here, we present a non-enzymatic, hybridization chain reaction (HCR)-based signal amplified in situ whole-cell detection technique (in situ DNA-HCR). The components of the amplification buffer were optimized to polymerize DNA amplifier probes for in situ DNA-HCR. In situ hybridization of initiator probes followed by signal amplification via HCR produced bright signals with high specificity and probe permeation into cells. The detection rates for Bacteria in a seawater sample and Archaea in anaerobic sludge samples were comparable with or greater than those obtained by catalyzed reporter deposition (CARD)-FISH or standard FISH. Detection of multiple organisms (Bacteria, Archaea and Methanosaetaceae) in an anaerobic sludge sample was achieved by simultaneous in situ DNA-HCR. In summary, in situ DNA-HCR is a simple and easy technique for detecting single microbial cells and enhancing understanding of the ecology and behaviour of environmental microorganisms in situ.

Dominance of Methanosarcinales phylotypes and depth-wise distribution of methanogenic community in fresh water sediments of Sitka stream from Czech Republic.

The variation in the diversity of methanogens in sediment depths from Sitka stream was studied by constructing a 16S rRNA gene library using methanogen-specific primers and a denaturing gradient gel electrophoresis (DGGE)-based approach. A total of nine different phylotypes from the 16S rRNA library were obtained, and all of them were clustered within the order Methanosarcinales. These nine phylotypes likely represent nine new species and at least 5-6 new genera. Similarly, DGGE analysis revealed an increase in the diversity of methanogens with an increase in sediment depth. These results suggest that Methanosarcinales phylotypes might be the dominant methanogens in the sediment from Sitka stream, and the diversity of methanogens increases as the depth increases. Results of the present study will help in making effective strategies to monitor the dominant methanogen phylotypes and methane emissions in the environment.

Biogas production and methanogenic archaeal community in mesophilic and thermophilic anaerobic co-digestion processes.

Over 258 Mt of solid waste are generated annually in Europe, a large fraction of which is biowaste. Sewage sludge is another major waste fraction. In this study, biowaste and sewage sludge were co-digested in an anaerobic digestion reactor (30% and 70% of total wet weight, respectively). The purpose was to investigate the biogas production and methanogenic archaeal community composition in the anaerobic digestion reactor under meso- (35-37 °C) and thermophilic (55-57 °C) processes and an increasing organic loading rate (OLR, 1-10 kg VS m(-3) d(-1)), and also to find a feasible compromise between waste treatment capacity and biogas production without causing process instability. In summary, more biogas was produced with all OLRs by the thermophilic process. Both processes showed a limited diversity of the methanogenic archaeal community which was dominated by Methanobacteriales and Methanosarcinales (e.g. Methanosarcina) in both meso- and thermophilic processes. Methanothermobacter was detected as an additional dominant genus in the thermophilic process. In addition to operating temperatures, the OLRs, the acetate concentration, and the presence of key substrates like propionate also affected the methanogenic archaeal community composition. A bacterial cell count 6.25 times higher than archaeal cell count was observed throughout the thermophilic process, while the cell count ratio varied between 0.2 and 8.5 in the mesophilic process. This suggests that the thermophilic process is more stable, but also that the relative abundance between bacteria and archaea can vary without seriously affecting biogas production.

Start-up of an anaerobic dynamic membrane digester for waste activated sludge digestion: temporal variations in microbial communities.

An anaerobic dynamic membrane digester (ADMD) was developed to digest waste sludge, and pyrosequencing was used to analyze the variations of the bacterial and archaeal communities during the start-up. Results showed that bacterial community richness decreased and then increased over time, while bacterial diversity remained almost the same during the start-up. Proteobacteria and Bacteroidetes were the major phyla. At the class level, Betaproteobacteria was the most abundant at the end of start-up, followed by Sphingobacteria. In the archaeal community, richness and diversity peaked at the end of the start-up stage. Principle component and cluster analyses demonstrated that archaeal consortia experienced a distinct shift and became stable after day 38. Methanomicrobiales and Methanosarcinales were the two predominant orders. Further investigations indicated that Methanolinea and Methanosaeta were responsible for methane production in the ADMD system. Hydrogenotrophic pathways might prevail over acetoclastic means for methanogenesis during the start-up, supported by specific methanogenic activity tests.

Is aceticlastic methanogen composition in full-scale anaerobic processes related to acetate utilization capacity?

In this study, biomass samples were obtained from six municipal and nine industrial full-scale anaerobic processes to investigate whether the aceticlastic methanogen population composition is related to acetate utilization capacity and the nature of the wastewater treated, i.e. municipal sludge or industrial wastewater. Batch serum bottle tests were used to determine the specific acetate utilization rate (AUR), and a quantitative real-time polymerase chain reaction protocol was used to enumerate the acetate-utilizing Methanosaeta and Methanosarcina populations in the biomass samples. Methanosaeta was the dominant aceticlastic methanogen in all samples, except for one industrial wastewater-treating anaerobic process. However, Methanosarcina density in industrial biomass samples was higher than the Methanosarcina density in the municipal samples. The average AUR values of municipal and industrial wastewater treatment plant biomass samples were 10.49 and 10.65 mg CH3COO(-)/log(aceticlastic methanogen gene copy).d, respectively. One-way ANOVA test and principle component analysis showed that the acetate utilization capacities and aceticlastic methanogen community composition did not show statistically significant correlation among the municipal digesters and industrial wastewater-treating processes investigated.

¹³C pulse-chase labeling comparative assessment of the active methanogenic archaeal community composition in the transgenic and nontransgenic parental rice rhizospheres.

More and more investigations indicate that genetic modification has no significant or persistent effects on microbial community composition in the rice rhizosphere. Very few studies, however, have focused on its impact on functional microorganisms. This study completed a ¹³C-CO2 pulse-chase labeling experiment comparing the potential effects of cry1Ab gene transformation on ¹³C tissue distribution and rhizosphere methanogenic archaeal community composition with its parental rice variety (Ck) and a distant parental rice variety (Dp). Results showed that ¹³C partitioning in aboveground biomass (mainly in stems) and roots of Dp was significantly lower than that of Ck. However, there were no significant differences in ¹³C partitioning between the Bt transgenic rice line (Bt) and Ck. RNA-stable isotope probing combined with clone library analyses inferred that the group Methanosaetaceae was the predominant methanogenic Archaea in all three rice rhizospheres. The active methanogenic archaeal community in the Bt rhizosphere was dominated by Methanosarcinaceae, Methanosaetaceae, and Methanomicrobiaceae, while there were only two main methanogenic clusters (Methanosaetaceae and Methanomicrobiaceae) in the Ck and Dp rhizospheres. These results indicate that the insertion of cry1Ab gene into the rice genome has the potential to result in the modification of methanogenic community composition in its rhizosphere.

Carbon source--a strong determinant of microbial community structure and performance of an anaerobic reactor.

Industrial effluents differ in their organic composition thereby providing different carbon sources to the microbial communities involved in its treatment. This study aimed to investigate the correlation of microbial community structure with wastewater composition and reactor's performance. Self-immobilized granules were developed in simulated wastewater based on different carbon sources (glucose, sugarcane molasses, and milk) in three hybrid anaerobic reactors operated at 37°C. To study archaeal community structure, a polyphasic approach was used with both qualitative and quantitative analysis. While PCR-denaturing gradient gel electrophoresis of 16S rRNA gene did not reveal major shifts in diversity of archaea with change in substrate, quantification of different groups of methanogens and total bacteria by real-time PCR showed variations in relative abundances with the dominance of Methanosaetaceae and Methanobacteriales. These data were supported by differences in the ratio of total counts of archaea and bacteria analyzed by catalyzed reporter deposition - fluorescence in situ hybridization. During hydraulic and organic shocks, the molasses-based reactor showed the best performance followed by the milk- and the glucose-based reactor. The study indicates that carbon source shapes the microbial community structure more in terms of relative abundance with distinct metabolic capacities rather than its diversity itself.

The effect of enzymatic pre-hydrolysis of dairy wastewater on the granular and immobilized microbial community in anaerobic bioreactors.

The effect of a lipase-rich enzyme preparation produced by the fungus Penicillium sp. on solid-state fermentation was evaluated in two anaerobic bioreactors (up-flow anaerobic sludge blanket (UASB) and horizontal-flow anaerobic immobilized biomass (HAIB)) treating dairy wastewater with 1200 mg oil and grease/L. The oil and grease hydrolysis step was carried out with 0.1% (w/v) of the solid enzymatic preparation at 30 degrees C for 24 h. This resulted in a final concentration of free acids eight times higher than the initial value. The bioreactors operated at 30 degrees C with hydraulic retention times of 12 h (HAIB) and 20 h (UASB) for a period of 430 days, and had high chemical oxygen demand (COD) removal efficiencies (around 90%) when fed with pre-hydrolyzed wastewater. There was, however, an increase in the effluent oil and grease concentration (from values as low as 17 mg/L to values above 150 mg/L in the UASB bioreactor, and from 38-242 mg/L in the HAIB bioreactor), and oil and grease accumulation in the biomass throughout the operational period (the oil and grease content reached 1.7 times that found in the inoculum of the UASB bioreactor). The HAIB bioreactor gave better results because the support for biomass immobilization acted as a filter, retaining oil and grease at the entry of the bioreactor. The molecular analysis of the Bacteria and Archaea domains revealed significant differences in the microbial profiles in experiments conducted with and without the pre-hydrolysis step. The differences observed in the overall parameters could be related to the microbial diversity of the anaerobic sludge.

Microbial community structure and dynamics in two-stage vs single-stage thermophilic anaerobic digestion of mixed swine slurry and market bio-waste.

The microbial community of a thermophilic two-stage process was monitored during two-months operation and compared to a conventional single-stage process. Qualitative and quantitative microbial dynamics were analysed by Denaturing Gradient Gel Electrophoresis (DGGE) and real-time PCR techniques, respectively. The bacterial community was dominated by heat-shock resistant, spore-forming clostridia in the two-stage process, whereas a more diverse and dynamic community (Firmicutes, Bacteroidetes, Synergistes) was observed in the single-stage process. A significant evolution of bacterial community occurred over time in the acidogenic phase of the two-phase process with the selection of few dominant species associated to stable hydrogen production. The archaeal community, dominated by the acetoclastic Methanosarcinales in both methanogen reactors, showed a significant diversity change in the single-stage process after a period of adaptation to the feeding conditions, compared to a constant stability in the methanogenic reactor of the two-stage process. The more diverse and dynamic bacterial and archaeal community of single-stage process compared to the two-stage process accounted for the best degradation activity, and consequently the best performance, in this reactor. The microbiological perspective proved a useful tool for a better understanding and comparison of anaerobic digestion processes.

Performance and dynamic characteristics of microbial communities in an internal circulation reactor for treating brewery wastewater.

A laboratory-scale internal circulation (IC) anaerobic reactor fed with brewery wastewater was operated at 35 degrees C + 1 degrees C. The influent was pumped into the bottom of the IC reactor by a pulse pump, whereas the effluent was drawn from the upper outlet and allowed to flow into the effluent tank. The biogas volume was recorded using a gas container connected to a biogas metre. The results indicated that the maximum organic loading rate (OLR) of the IC reactor was 19.5 kg chemical oxygen demand (COD)/m3/day; at which point, the dominant archaeal populations found in the sludge using the polymerase chain reaction with denaturing gradient gel electrophoresis were Methanosaeta species. The COD removal efficiencies of the reactor exceeded 85%, with a maximum specific methane production rate of 210 mL CH4/g volatile suspended solids (VSS)/day and a coenzyme F420 content of 0.16 micromol/g VSS, respectively. The main archaeal species in the sludge samples at different OLRs varied greatly, as compared with the organisms in the inoculated sludge. The dominant archaeal species in the treated sludge at low OLRs were Methanosarcina species, whereas those at high OLRs were Methanosaeta species.

Community and proteomic analysis of methanogenic consortia degrading terephthalate.

Degradation of terephthalate (TA) through microbial syntrophy under moderately thermophilic (46 to 50°C) methanogenic conditions was characterized by using a metagenomic approach (A. Lykidis et al., ISME J. 5:122-130, 2011). To further study the activities of key microorganisms responsible for the TA degradation, community analysis and shotgun proteomics were used. The results of hierarchical oligonucleotide primer extension analysis of PCR-amplified 16S rRNA genes indicated that Pelotomaculum, Methanosaeta, and Methanolinea were predominant in the TA-degrading biofilms. Metaproteomic analysis identified a total of 482 proteins and revealed a distinctive distribution pattern of microbial functions expressed in situ. The results confirmed that TA was degraded by Pelotomaculum spp. via the proposed decarboxylation and benzoyl-coenzyme A-dependent pathway. The intermediate by-products, including acetate, H(2)/CO(2), and butyrate, were produced to support the growth of methanogens, as well as other microbial populations that could further degrade butyrate. Proteins related to energy production and conservation, and signal transduction mechanisms (that is, chemotaxis, PAS/GGDEF regulators, and stress proteins) were highly expressed, and these mechanisms were important for growth in energy-limited syntrophic ecosystems.