Predominance of Methanomicrobiales and diverse hydrocarbon-degrading taxa in the Appalachian coalbed biosphere revealed through metagenomics and genome-resolved metabolisms.

Coalbed deposits are a unique subsurface environment and represent an underutilized resource for methane generation. Microbial communities extant in coalbed deposits are responsible for key subsurface biogeochemical cycling and could be utilized to enhance methane production in areas where existing gas wells have depleted methane stores, or in coalbeds that are unmined, or conversely be utilized for mitigation of methane release. Here we utilize metagenomics and metagenome-assembled genomes (MAGs) to identify extant microbial lineages and genome-resolved microbial metabolisms of coalbed produced water, which has not yet been explored in the Appalachian Basin (AppB). Our analyses resulted in the recovery of over 40 MAGs from 8 coalbed methane wells. The most commonly identified taxa among samples were hydrogenotrophic methanogens from the order Methanomicrobiales and these dominant MAGs were highly similar to one another. Conversely, low-abundance coalbed bacterial populations were taxonomically and functionally diverse, mostly belonging to a variety of Proteobacteria classes, and encoding various hydrocarbon solubilization and degradation pathways. The data presented herein provides novel insights into AppB coalbed microbial ecology, and our findings provide new perspectives on underrepresented Methanocalculus species and low-relative abundance bacterial assemblages in coalbed environments, and their potential roles in stimulation or mitigation of methane release.

Formate-Dependent Heterodisulfide Reduction in a Methanomicrobiales Archaeon.

Hydrogenotrophic methanogens produce CH4 using H2 as an electron donor to reduce CO2 In the absence of H2, many are able to use formate or alcohols as alternate electron donors. Methanogens from the order Methanomicrobiales are capable of growth with H2, but many lack genes encoding hydrogenases that are typically found in other hydrogenotrophic methanogens. In an effort to better understand electron flow in methanogens from the Methanomicrobiales, we undertook a genetic and biochemical study of heterodisulfide reductase (Hdr) in Methanoculleus thermophilus Hdr catalyzes an essential reaction by coupling the first and last steps of methanogenesis through flavin-based electron bifurcation. Hdr from M. thermophilus copurified with formate dehydrogenase (Fdh) and only displayed activity when formate was supplied as an electron donor. We found no evidence of an Hdr-associated hydrogenase, and H2 could not function as an electron donor, even with Hdr purified from cells grown on H2 We found that cells catalyze a formate hydrogenlyase activity that is likely essential for generating the formate needed for the Hdr reaction. Together, these results highlight the importance of formate as an electron donor for methanogenesis and suggest the ability to use formate is closely integrated into the methanogenic pathway in organisms from the order MethanomicrobialesIMPORTANCE Methanogens from the order Methanomicrobiales are thought to prefer H2 as an electron donor for growth. They are ubiquitous in anaerobic environments, such as in wastewater treatment facilities, anaerobic digesters, and the rumen, where they catalyze the terminal steps in the breakdown of organic matter. However, despite their importance, the metabolism of these organisms remains understudied. Using a genetic and biochemical approach, we show that formate metabolism is closely integrated into methanogenesis in Methanoculleus thermophilus This is due to a requirement for formate as the electron donor to heterodisulfide reductase (Hdr), an enzyme responsible for catalyzing essential reactions in methanogenesis by linking the initial CO2 fixing step to the exergonic terminal reaction of the pathway. These results suggest that hydrogen is not necessarily the preferred electron donor for all hydrogenotrophic methanogens and provide insight into the metabolism of methanogens from the order Methanomicrobiales.

Effects of rumen cannulation on dissolved gases and methanogen community in dairy cows.

Rumen cannulation is a widely employed technique in ruminant nutrition research. However, the gap between skin and rumen cannula can cause leakage of fermentation gases and influx of atmospheric air, which may adversely affect the anaerobic environment in the rumen. The present study was designed to investigate the effects of rumen cannulation on headspace gases, dissolved gases, fermentation end products, and methanogen community in the rumen of dairy cows. Eight Holstein cows were used in the experiment. Four cows were surgically fitted with rumen cannulas, whereas the other 4 intact cows were used as control. Rumen cannulation decreased gaseous hydrogen and methane concentrations, dissolved carbon dioxide concentration, and relative abundances of Methanosphaera, and increased the saturation factor of dissolved hydrogen and dissolved methane, dissolved methane concentration, volatile fatty acid concentration, 16S ribosomal RNA gene copies of methanogens, and Simpson index of methanogen community. In summary, rumen cannulation causes a reduction in headspace gaseous hydrogen and gaseous methane, which may not decrease dissolved gas concentrations due to an increase in saturation factors. Furthermore, rumen cannulation alters methanogen community with increased methanogen population and decreased relative abundances of Methanosphaera.

Anaerobic oxidation of methane coupled to sulfate reduction: Consortium characteristics and application in co-removal of H2S and methane.

Anaerobic sludge from a sewage treatment plant was used to acclimatize microbial colonies capable of anaerobic oxidation of methane (AOM) coupled to sulfate reduction. Clone libraries and fluorescence in situ hybridization were used to investigate the microbial population. Sulfate-reducing bacteria (SRB) (e.g., Desulfotomaculum arcticum and Desulfobulbus propionicus) and anaerobic methanotrophic archaea (ANME) (e.g., Methanosaeta sp. and Methanolinea sp.) coexisted in the enrichment. The archaeal and bacterial cells were randomly or evenly distributed throughout the consortia. Accompanied by sulfate reduction, methane was oxidized anaerobically by the consortia of methane-oxidizing archaea and SRB. Moreover, CH4 and SO42- were consumed by methanotrophs and sulfate reducers with CO2 and H2S as products. The H3CSH produced by methanotrophy was an intermediate product during the process. The methanotrophic enrichment was inoculated in a down-flow biofilter for the treatment of methane and H2S from a landfill site. On average, 93.33% of H2S and 10.71% of methane was successfully reduced in the biofilter. This study tries to provide effective method for the synergistic treatment of waste gas containing sulfur compounds and CH4.

Shifts of methanogenic communities in response to permafrost thaw results in rising methane emissions and soil property changes.

Permafrost thaw can bring negative consequences in terms of ecosystems, resulting in permafrost collapse, waterlogging, thermokarst lake development, and species composition changes. Little is known about how permafrost thaw influences microbial community shifts and their activities. Here, we show that the dominant archaeal community shifts from Methanomicrobiales to Methanosarcinales in response to the permafrost thaw, and the increase in methane emission is found to be associated with the methanogenic archaea, which rapidly bloom with nearly tenfold increase in total number. The mcrA gene clone libraries analyses indicate that Methanocellales/Rice Cluster I was predominant both in the original permafrost and in the thawed permafrost. However, only species belonging to Methanosarcinales showed higher transcriptional activities in the thawed permafrost, indicating a shift of methanogens from hydrogenotrophic to partly acetoclastic methane-generating metabolic processes. In addition, data also show the soil texture and features change as a result of microbial reproduction and activity induced by this permafrost thaw. Those data indicate that microbial ecology under warming permafrost has potential impacts on ecosystem and methane emissions.

In Vitro Response of Rumen Microbiota to the Antimethanogenic Red Macroalga Asparagopsis taxiformis.

The red macroalga Asparagopsis taxiformis has been shown to significantly decrease methane production by rumen microbial communities. This has been attributed to the bioaccumulation of halogenated methane analogues produced as algal secondary metabolites. The objective of this study was to evaluate the impact of A. taxiformis supplementation on the relative abundance of methanogens and microbial community structure during in vitro batch fermentation. Addition of A. taxiformis (2% organic matter) or the halogenated methane analogue bromoform (5 µM) reduced methane production by over 99% compared to a basal substrate-only control. Quantitative PCR confirmed that the decrease in methane production was correlated with a decrease in the relative abundance of methanogens. High-throughput 16S ribosomal RNA gene amplicon sequencing showed that both treatments reduced the abundance of the three main orders of methanogens present in ruminants (Methanobacteriales, Methanomassiliicoccales and Methanomicrobiales). Shifts in bacterial community structure due to the addition of A. taxiformis and 5 µM bromoform were similar and concomitant with increases in hydrogen concentration in the headspace of the fermenters. With high potency and broad-spectrum activity against rumen methanogens, A. taxiformis represents a promising natural strategy for reducing enteric methane emissions from ruminant livestock.

Potential Activity of Subglacial Microbiota Transported to Anoxic River Delta Sediments.

The Watson River drains a portion of the SW Greenland ice sheet, transporting microbial communities from subglacial environments to a delta at the head of Søndre Strømfjord. This study investigates the potential activity and community shifts of glacial microbiota deposited and buried under layers of sediments within the river delta. A long-term (12-month) incubation experiment was established using Watson River delta sediment under anaerobic conditions, with and without CO2/H2 enrichment. Within CO2/H2-amended incubations, sulphate depletion and a shift in the microbial community to a 52% predominance of Desulfosporosinus meridiei by day 371 provides evidence for sulphate reduction. We found evidence of methanogenesis in CO2/H2-amended incubations within the first 5 months, with production rates of ~4 pmol g-1 d-1, which was likely performed by methanogenic Methanomicrobiales- and Methanosarcinales-related organisms. Later, a reduction in methane was observed to be paired with the depletion of sulphate, and we hypothesise that sulphate reduction out competed hydrogenotrophic methanogenesis. The structure and diversity of the original CO2/H2-amended incubation communities changed dramatically with a major shift in predominant community members and a decline in diversity and cell abundance. These results highlight the need for further investigations into the fate of subglacial microbiota within downstream environments.

Use of microbial indicators combined with environmental factors coupled with metrology tools for discrimination and classification of Luzhou-flavoured pit muds.

AIMS: Pit mud is essential for the quality and yield of Chinese Luzhou-flavoured liquor. A reliable and fast method based on the use of microbial indicators combined with environmental factors coupled with metrology tools is needed to discriminate and classify different maturity levels of Luzhou-flavoured pit muds. METHODS AND RESULTS: Firmicutes, Bacteroidetes, Actinobacteria, Lactobacillus, Bacillus, Methanosarcina, Methanocorpusculum, Methanoculleus and Clostridium kluyveri were microbial indicators in Luzhou-flavoured pit muds. They were detected by real-time quantitative PCR. Environmental factors investigated included moisture content, pH, total acid and ammonia nitrogen. Principal component analysis (PCA) and partial least square-discriminant analysis were employed to explore the structure of the data and construct discrimination and classification models by reduction in the data dimensionality. Pit muds were distinguished and classified as new, trend to-be aged and aged. Moisture content and pH were significantly negatively correlated with new pit mud, while pH, total acid, amino nitrogen, Firmicutes, Bacteroidetes, Actinobacteria, Methanosarcina, Methanoculleus and C. kluyveri were significantly positively correlated with aged pit mud. CONCLUSIONS: Microbial indicators combined with environmental factors coupled to metrology tools can reliably and quickly discriminate and classify different maturity levels of Luzhou-flavoured pit muds. SIGNIFICANCE AND IMPACT OF THE STUDY: Modern techniques and metrology tools verified the correctness of the traditional sensory evaluation used in controlling the quality of pit mud, and will contribute to distinguishing different maturity levels of Chinese Luzhou-flavoured pit muds.

Methanogenic paraffin degradation proceeds via alkane addition to fumarate by 'Smithella' spp. mediated by a syntrophic coupling with hydrogenotrophic methanogens.

Anaerobic microbial biodegradation of recalcitrant, water-insoluble substrates, such as paraffins, presents unique metabolic challenges. To elucidate this process, a methanogenic consortium capable of mineralizing long-chain n-paraffins (C28 -C50 ) was enriched from San Diego Bay sediment. Analysis of 16S rRNA genes indicated the dominance of Syntrophobacterales (43%) and Methanomicrobiales (26%). Metagenomic sequencing allowed draft genome assembly of dominant uncultivated community members belonging to the bacterial genus Smithella and the archaeal genera Methanoculleus and Methanosaeta. Five contigs encoding homologs of the catalytic subunit of alkylsuccinate synthase (assA) were detected. Additionally, mRNA transcripts for these genes, including a homolog binned within the 'Smithella' sp. SDB genome scaffold, were detected via RT-PCR, implying that paraffins are activated via 'fumarate addition'. Metabolic reconstruction and comparison with genome scaffolds of uncultivated n-alkane degrading 'Smithella' spp. are consistent with the hypothesis that syntrophically growing 'Smithella' spp. may achieve reverse electron transfer by coupling the reoxidation of ETFred to a membrane-bound FeS oxidoreductase functioning as an ETF:menaquinone oxidoreductase. Subsequent electron transfer could proceed via a periplasmic formate dehydrogenase and/or hydrogenase, allowing energetic coupling to hydrogenotrophic methanogens such as Methanoculleus. Ultimately, these data provide fundamental insight into the energy conservation mechanisms that dictate interspecies interactions salient to methanogenic alkane mineralization.

The nomenclatural type of the genus Methanocorpusculum Zellner et al. 1988 and the selection of the correct name.

A recent Request for an Opinion has raised the issue of the inter-relationship between Methanocorpusculum parvum Zellner et al. 1988, the type species of the genus Methanocorpusculum Zellner et al. 1988 as defined at the time of valid publication of the genus name and the subsequent recognition of Methanocorpusculum aggregans (Ollivier et al., 1985) Xun et al.1989 as an earlier heterotypic synonym. Examination of the relevant literature indicates that there are a number of misunderstandings that have arisen. In particular misinterpretation of Rule 15 of the International Code of Nomenclature of Prokaryotes continues to be a source of confusion. Additional problems centre on whether the nomenclatural type of a taxon continues to be the nomenclatural type even if that name is not treated as the correct name and would not appear in a list of names in a given classification. It would be appropriate to clarify these issues.

Changes in microbial community structures due to varying operational conditions in the anaerobic digestion of oxytetracycline-medicated cow manure.

Management of manure containing veterinary antibiotics is a major concern in anaerobic treatment systems because of their possible adverse effects on microbial communities. Therefore, the aim of study was to investigate how oxytetracycline (OTC) influences bacteria and acetoclastic and hydrogenotrophic methanogens under varying operational conditions in OTC-medicated and non-medicated anaerobic cow manure digesters. Concentrations of OTC and its metabolites throughout the anaerobic digestion were determined using ultraviolet-high-performance liquid chromatography (UV-HPLC) and tandem liquid chromatography-mass spectrometry (LC/MS/MS), respectively. Fluorescent in situ hybridization, denaturing gradient gel electrophoresis, cloning, and sequencing analyses were used to monitor changes in microbial community structures. According to the results of analytical and molecular approaches, operating conditions highly influence active microbial community dynamics and associate with biogas production and elimination of OTC and its metabolites during anaerobic digestion of cow manure in the presence of an average initial concentration of 2.2 mg OTC/L. The impact of operating conditions has a drastic effect on acetoclastic methanogens than hydrogenotrophic methanogens and bacteria.

Genome of Methanoregula boonei 6A8 reveals adaptations to oligotrophic peatland environments.

Analysis of the genome sequence of Methanoregula boonei strain 6A8, an acidophilic methanogen isolated from an ombrotrophic (rain-fed) peat bog, has revealed unique features that likely allow it to survive in acidic, nutrient-poor conditions. First, M. boonei is predicted to generate ATP using protons that are abundant in peat, rather than sodium ions that are scarce, and the sequence of a membrane-bound methyltransferase, believed to pump Na+ in all methanogens, shows differences in key amino acid residues. Further, perhaps reflecting the hypokalemic status of many peat bogs, M. boonei demonstrates redundancy in the predicted potassium uptake genes trk, kdp and kup, some of which may have been horizontally transferred to methanogens from bacteria, possibly Geobacter spp. Overall, the putative functions of the potassium uptake, ATPase and methyltransferase genes may, at least in part, explain the cosmopolitan success of group E1/E2 and related methanogenic archaea in acidic peat bogs.

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.

The correct name of the type species of the genus Methanocorpusculum. Request for an Opinion.

We propose that Methanocorpusculum parvum with type strain XII must remain the nomenclatural type of the genus Methanocorpusculum, in spite of the fact that the description of Methanogenium aggregans with type strain MSt, later transferred to the genus Methanocorpusculum as comb. nov. and then proposed as a heterotypic synonym of Methanocorpusculum parvum, was published before Methanocorpusculum parvum strain XII.

Methanosalsum natronophilum sp. nov., and Methanocalculus alkaliphilus sp. nov., haloalkaliphilic methanogens from hypersaline soda lakes.

Two groups of haloalkaliphilic methanogenic archaea were dominating in enrichments from hypersaline soda lake sediments at pH 10. At moderate salt concentrations with formate or H2 as electron donor, methanogens belonging to the genus Methanocalculus were enriched, while at high salt concentrations with methylated substrates, a group related to Methanosalsum zhilinae was dominating. For both groups, several pure cultures were obtained including the type strains AMF2T for the Methanocalculus group and AME2T for the Methanosalsum group. The Methanocalculus group is characterized by lithoheterotrophic growth with either formate (preferable substrate) or H2 at moderate salinity up to 1.5-2 M total Na+ and obligate alkaliphilic growth with an optimum at pH 9.5. According to phylogenetic analysis, the group also includes closely related strains isolated previously from the low-salt alkaline Lonar Lake. The novel Methanosalsum group is characterized by high salt tolerance (up to 3.5 M total Na+) and obligate alkaliphilic growth with an optimum at pH 9.5. It has a typical methylotrophic substrate profile, utilizing methanol, methylamines and dimethyl sulfide (at low concentrations) as methanogenic substrates. On the basis of physiological and phylogenetic data, it is proposed that the two groups of soda lake methanogenic isolates are assigned into two novel species, Methanocalculus alkaliphilus sp. nov. (type strain AMF2T = DSM 24457T = UNIQEM U859T) and Methanosalsum natronophilum sp. nov. (type strain AME2T = DSM 24634T = NBRC 110091T).

Electrochemical nutrient recovery enables ammonia toxicity control and biogas desulfurization in anaerobic digestion.

Organic waste streams can be valorized and reduced in volume with anaerobic digestion (AD). An often-encountered key issue however is the high ammonium (NH4(+)) content of certain waste streams. Ammonia (NH3), in equilibrium with NH4(+), is a toxic compound to the methanogenic community, which limits the organic loading rate and endangers process stability. An electrochemical system (ES) linked to a digester could, besides recovering this nutrient, decrease NH3 toxicity through electrochemical extraction. Therefore, two digesters with and without ES attached in the recirculation loop were operated to test whether the ES could control NH3 toxicity. During periods of high ammonium loading rates, the methane (CH4) production of the ES-coupled reactor was up to 4.5 times higher compared to the control, which could be explained through simultaneous NH4(+) extraction and electrochemical pH control. A nitrogen flux of 47 g N m(­2) membrane d(­1) could be obtained in the ES-coupled reactor, resulting in a current and removal efficiency of 38 ± 5% and 28 ± 2%, respectively, at an electrochemical power input of 17 ± 2 kWh kg(­1) N. The anode also oxidized sulfide, resulting in a significantly lower H2S emission via the biogas. Lastly, limited methanogenic community dynamics pointed to a nonselective influence of the different operational conditions.

Substrate sources regulate spatial variation of metabolically active methanogens from two contrasting freshwater wetlands.

There is ample evidence that methane (CH4) emissions from natural wetlands exhibit large spatial variations at a field scale. However, little is known about the metabolically active methanogens mediating these differences. We explored the spatial patterns in active methanogens of summer inundated Calamagrostis angustifolia marsh with low CH4 emissions and permanently inundated Carex lasiocarpa marsh with high CH4 emissions in Sanjiang Plain, China. In C. angustifolia marsh, the addition of (13)C-acetate significantly increased the CH4 production rate, and Methanosarcinaceae methanogens were found to participate in the consumption of acetate. In C. lasiocarpa marsh, there was no apparent increase in the CH4 production rate and no methanogen species were labeled with (13)C. When (13)CO2-H2 was added, however, CH4 production was found to be due to Fen Cluster (Methanomicrobiales) in C. angustifolia marsh and Methanobacterium Cluster B (Methanobacteriaceae) together with Fen Cluster in C. lasiocarpa marsh. These results suggested that CH4 was produced primarily by hydrogenotrophic methanogens using substrates mainly derived from plant litter in C. lasiocarpa marsh and by both hydrogenotrophic and acetoclastic methanogens using substrates mainly derived from root exudate in C. angustifolia marsh. The significantly lower CH4 emissions measured in situ in C. angustifolia marsh was primarily due to a deficiency of substrates compared to C. lasiocarpa marsh. Therefore, we speculate that the substrate source regulates both the type of active methanogens and the CH4 production pathway and consequently contributes to the spatial variations in CH4 productions observed in these freshwater marshes.

Performance of and methanogenic communities involved in an innovative anaerobic process for the treatment of food wastewater in a pilot plant.

In this study, dual-cylindrical anaerobic digesters were designed and built on the pilot plant scale for the improvement of anaerobic digestion efficiency. The removal efficiency of organics, biogas productivity, yield, and microbial communities was evaluated as performance parameters of the digester. During the stable operational period in the continuous mode, the removal efficiencies of chemical oxygen demand and total solids were 74.1 and 65.1%, respectively. Biogas productivities of 63.9 m(3)/m(3)-FWW and 1.3 m(3)/kg-VSremoved were measured. The hydrogenotrophic methanogen orders, Methanomicrobiales and Methanobacteriales, were predominant over the aceticlastic methanogen order, Methanosarcinaceae, probably due to the tolerance of the hydrogenotrophs to environmental perturbation in the field and their faster growth rate compared with that of the aceticlastics.

New PCR primers based on mcrA gene for retrieving more anaerobic methanotrophic archaea from coastal reedbed sediments.

Two pairs of PCR primes ANMEallF/R and ANME23F/R were designed by Codehop method based on sequences available to retrieve more anaerobic methanotrophic (ANME) archaea mcrA gene sequences and ANME 2 and 3 subtypes from reedbed in two seasons. Overall, the PCR primers showed slightly favor for ANME group mcrA gene sequences. Due to the predominance of methanogens mainly affiliated to Methanomicrobiales in the samples, a large portion of mcrA gene sequences amplified in the clone libraries belonged to methanogens. Differences in PCR primers and performance affected the mcrA gene-PCR-amplified community composition to a minor extent. PCR primers targeting ANME mcrA group g-h were designed to apply real-time PCR for quantifying more groups of mcrA gene-affiliated ANME archaea and tested with these same samples, and the most abundant group in the whole ANME mcrA community was ANME group g-h. In addition, a stable mcrA gene-harboring archaeal community pattern was detected in the reedbed sediment samples collected from two distinctively different seasons. The PCR and qPCR primers designed in this study can expand our knowledge on the distribution of ANME mcrA genes and community composition in the ecosystem to better understand the carbon cycle.

Sample prefractionation with liquid isoelectric focusing enables in depth microbial metaproteome analysis of mesophilic and thermophilic biogas plants.

Biogas production from energy crops and biodegradable waste is one of the major sources for renewable energies in Germany. Within a biogas plant (BGP) a complex microbial community converts biomass to biogas. Unfortunately, disturbances of the biogas process occur occasionally and cause economic losses of varying extent. Besides technical failures the microbial community itself is commonly assumed as a reason for process instability. To improve the performance and efficiency of BGP, a deeper knowledge of the composition and the metabolic state of the microbial community is required and biomarkers for monitoring of process deviations or even the prediction of process failures have to be identified. Previous work based on 2D-electrophoresis demonstrated that the analysis of the metaproteome is well suited to provide insights into the apparent metabolism of the microbial communities. Using SDS-PAGE with subsequent mass spectrometry, stable protein patterns were evaluated for a number of anaerobic digesters. Furthermore, it was shown that severe changes in process parameters such as acidification resulted in significant modifications of the metaproteome. Monitoring of changing protein patterns derived from anaerobic digesters, however, is still a challenge due to the high complexity of the metaproteome. In this study, different combinations of separation techniques to reduce the complexity of proteomic BGP samples were compared with respect to the subsequent identification of proteins by tandem mass spectrometry (MS/MS): (i) 1D: proteins were tryptically digested and the resulting peptides were separated by reversed phase chromatography prior to MS/MS. (ii) 2D: proteins were separated by GeLC-MS/MS according to proteins molecular weights before tryptic digestion, (iii) 3D: proteins were separated by gel-free fractionation using isoelectric focusing (IEF) conducted before GeLC-MS/MS. For this study, a comparison of two anaerobic digesters operated at mesophilic and at thermophilic conditions was conducted. The addition of further separation dimensions before protein identification increased the number of identified proteins. On the other hand additional fractionation steps increased the experimental work load and the time required for LC-MS/MS measurement. The high resolution of the 3D-approach enabled the detection of approximately 750 to 1650 proteins covering the main pathways of hydrolysis, acidogenesis, acetogenesis and methanogenesis. Methanosarcinales dominated in the mesophilic BGP, whereas Methanomicrobiales were highly abundant in the thermophilic BGP. Pathway analysis confirmed the taxonomic results and revealed that the acetoclastic methanogenesis occurred preferentially at mesophilic conditions, whereas exclusively hydrogenotrophic methanogenesis was detected in thermophilic BGP. However, for the identification of process biomarkers by comprehensive screening of BGP it will be indispensable to find a balance between the experimental efforts and analytical resolution.