Three manganese oxide-rich marine sediments harbor similar communities of acetate-oxidizing manganese-reducing bacteria

Dissimilatory manganese reduction dominates anaerobic carbon oxidation in marine sediments with high manganese oxide concentrations, but the microorganisms responsible for this process are largely unknown. In this study, the acetate-utilizing manganese-reducing microbiota in geographically well-separated, manganese oxide-rich sediments from Gullmar Fjord (Sweden), Skagerrak (Norway) and Ulleung Basin (Korea) were analyzed by 16S rRNA-stable isotope probing (SIP). Manganese reduction was the prevailing terminal electron-accepting process in anoxic incubations of surface sediments, and even the addition of acetate stimulated neither iron nor sulfate reduction. The three geographically distinct sediments harbored surprisingly similar communities of acetate-utilizing manganese-reducing bacteria: 16S rRNA of members of the genera Colwellia and Arcobacter and of novel genera within the Oceanospirillaceae and Alteromonadales were detected in heavy RNA-SIP fractions from these three sediments. Most probable number (MPN) analysis yielded up to 10⁶ acetate-utilizing manganese-reducing cells cm⁻³ in Gullmar Fjord sediment. A 16S rRNA gene clone library that was established from the highest MPN dilutions was dominated by sequences of Colwellia and Arcobacter species and members of the Oceanospirillaceae, supporting the obtained RNA-SIP results. In conclusion, these findings strongly suggest that (i) acetate-dependent manganese reduction in manganese oxide-rich sediments is catalyzed by members of taxa (Arcobacter, Colwellia and Oceanospirillaceae) previously not known to possess this physiological function, (ii) similar acetate-utilizing manganese reducers thrive in geographically distinct regions and (iii) the identified manganese reducers differ greatly from the extensively explored iron reducers in marine sediments.     

Diversity and Structure of the Methanogenic Community in Anoxic Rice Paddy Soil Microcosms as Examined by Cultivation and Direct 16S rRNA Gene Sequence Retrieval

A dual approach consisting of cultivation and molecular retrieval of partial archaeal 16S rRNA genes was carried out to characterize the diversity and structure of the methanogenic community inhabiting the anoxic bulk soil of flooded rice microcosms. The molecular approach identified four groups of known methanogens. Three environmental sequences clustered with Methanobacterium bryantii and Methanobacterium formicicum, six were closely related but not identical to those of strains of Methanosaeta concilii, two grouped with members of the genus Methanosarcina, and two were related to the methanogenic endosymbiont of Plagiopyla nasuta. The cultivation approach via most-probable-number counts with a subsample of the same soil as an inoculum yielded cell numbers of up to 10⁷ per g of dry soil for the H₂-CO₂-utilizing methanogens and of up to 10⁶ for the acetate-utilizing methanogens. Strain VeH52, isolated from the terminal positive dilution on H₂-CO₂, grouped within the phylogenetic radiation characterized by M. bryantii and M. formicicum and the environmental sequences of the Methanobacterium-like group. A consortium of two distinct methanogens grew in the terminal positive culture on acetate. These two organisms showed absolute 16S rRNA gene identities with environmental sequences of the novel Methanosaeta-like group and the Methanobacterium-like group. Methanosarcina spp. were identified only in the less-dilute levels of the same dilution series on acetate. These data correlate well with acetate concentrations of about 11 μM in the pore water of this rice paddy soil. These concentrations are too low for the growth of known Methanosarcina spp. but are at the acetate utilization threshold of Methanosaeta spp. Thus, our data indicated Methanosaeta spp. and Methanobacterium spp. to be the dominant methanogenic groups in the anoxic rice soil, whereas Methanosarcina spp. appeared to be less abundant.     

Phylogenetic diversity and activity of anaerobic microorganisms of high-temperature horizons of the Dagang oil field (P. R. China)

The number of microorganisms of major metabolic groups and the rates of sulfate reduction and methanogenesis processes in the formation waters of the high-temperature horizons of Dagang oil field have been determined. Using cultural methods, it was shown that the microbial community contained aerobic bacteria oxidizing crude oil, anaerobic fermentative bacteria, sulfate-reducing bacteria, and methanogens. Using cultural methods, the possibility of methane production from a mixture of hydrogen and carbon dioxide (H₂ + CO₂) and from acetate was established, and this result was confirmed by radioisotope methods involving NaH¹⁴CO₃ and ¹⁴CH₃COONa. Analysis of enrichment cultures 16S rDNA of methanogens demonstrated that these microorganisms belong to Methanothermobacter sp. (M. thermautotrophicus), which consumes hydrogen and carbon dioxide as basic substrates. The genes of acetate-utilizing bacteria were not revealed. Phylotypes of the representatives of Thermococcus spp. were found among archaeal 16S rDNA. 16S rRNA genes of bacterial clones belong to the orders Thermoanaerobacteriales (Thermoanaerobacter, Thermovenabulum, Thermacetogenium, and Coprothermobacter spp.), Thermotogales, Nitrospirales (Thermodesulfovibrio sp.) and Planctomycetales. 16S rDNA of a bacterium capable of oxidizing acetate in the course of syntrophic growth with H₂-utilizing methanogens was found in high-temperature petroleum reservoirs for the first time. These results provide further insight into the composition of microbial communities of high-temperature petroleum reservoirs, indicating that syntrophic processes play an important part in acetate degradation accompanied by methane production.     

Identification of acetate-utilizing Bacteria and Archaea in methanogenic profundal sediments of Lake Kinneret (Israel) by stable isotope probing of rRNA

Acetate is an important intermediate in the decomposition of organic matter in anoxic freshwater sediments. Here, we identified distinct microorganisms active in its oxidation and transformation to methane in the anoxic methanogenic layers of Lake Kinneret (Israel) profundal sediment by rRNA-based stable isotope probing (RNA-SIP). After 18 days of incubation with amended [U-(13)C]acetate we found that archaeal 16S rRNA was (13)C-labelled to a far greater extent than bacterial rRNA. We identified acetoclastic methanogens related to Methanosaeta concilii as being most active in the degradation and assimilation of acetate. Oxidation of the acetate-methyl group played only a minor role, but nevertheless 'heavy'(13)C-labelled bacterial rRNA templates were identified. 'Heavy' bacteria were mainly affiliated with the Betaproteobacteria (mostly Rhodocyclales and Nitrosomonadales), the Nitrospira phylum (related to 'Magnetobacterium bavaricum' and Thermodesulfovibrio yellowstonii), and also with the candidate phylum 'Endomicrobia'. However, the mode of energy gain that allowed for the assimilation of (13)C-acetate by these bacterial groups remains unknown. It may have involved syntrophic oxidation of acetate, reduction of chlorinated compounds, reduction of humic substances, fermentation of organic compounds, or even predation of (13)C-labelled Methanosaeta spp. In summary, this SIP experiment shows that acetate carbon was predominantly consumed by acetoclastic methanogens in profundal Lake Kinneret sediment, while it was also utilized by a small and heterogeneous community of bacteria.     

Microbial rRNA gene expression and co‐occurrence profiles associate with biokinetics and elemental composition in full‐scale anaerobic digesters

This study examined whether the abundance and expression of microbial 16S rRNA genes were associated with elemental concentrations and substrate conversion biokinetics in 20 full‐scale anaerobic digesters, including seven municipal sewage sludge (SS) digesters and 13 industrial codigesters. SS digester contents had higher methane production rates from acetate, propionate and phenyl acetate compared to industrial codigesters. SS digesters and industrial codigesters were distinctly clustered based on their elemental concentrations, with higher concentrations of NH₃‐N, Cl, K and Na observed in codigesters. Amplicon sequencing of 16S rRNA genes and reverse‐transcribed 16S rRNA revealed divergent grouping of microbial communities between mesophilic SS digesters, mesophilic codigesters and thermophilic digesters. Higher intradigester distances between Archaea 16S rRNA and rRNA gene profiles were observed in mesophilic codigesters, which also had the lowest acetate utilization biokinetics. Constrained ordination showed that microbial rRNA and rRNA gene profiles were significantly associated with maximum methane production rates from acetate, propionate, oleate and phenyl acetate, as well as concentrations of NH₃‐N, Fe, S, Mo and Ni. A co‐occurrence network of rRNA gene expression confirmed the three main clusters of anaerobic digester communities based on active populations. Syntrophic and methanogenic taxa were highly represented within the subnetworks, indicating that obligate energy‐sharing partnerships play critical roles in stabilizing the digester microbiome. Overall, these results provide new evidence showing that different feed substrates associate with different micronutrient compositions in anaerobic digesters, which in turn may influence microbial abundance, activity and function.     

Phylogenetic Identification and Substrate Uptake Patterns of Sulfate-Reducing Bacteria Inhabiting an Oxic-Anoxic Sewer Biofilm Determined by Combining Microautoradiography and Fluorescent In Situ Hybridization

We simultaneously determined the phylogenetic identification and substrate uptake patterns of sulfate-reducing bacteria (SRB) inhabiting a sewer biofilm with oxygen, nitrate, or sulfate as an electron acceptor by combining microautoradiography and fluorescent in situ hybridization (MAR-FISH) with family- and genus-specific 16S rRNA probes. The MAR-FISH analysis revealed that Desulfobulbus hybridized with probe 660 was a dominant SRB subgroup in this sewer biofilm, accounting for 23% of the total SRB. Approximately 9 and 27% of Desulfobulbus cells detected with probe 660 could take up [¹⁴C]propionate with oxygen and nitrate, respectively, as an electron acceptor, which might explain the high abundance of this species in various oxic environments. Furthermore, more than 40% of Desulfobulbus cells incorporated acetate under anoxic conditions. SRB were also numerically important members of H₂-utilizing and ¹⁴CO₂-fixing microbial populations in this sewer biofilm, accounting for roughly 42% of total H₂-utilizing bacteria hybridized with probe EUB338. A comparative 16S ribosomal DNA analysis revealed that two SRB populations, related to the Desulfomicrobium hypogeium and the Desulfovibrio desulfuricans MB lineages, were found to be important H₂ utilizers in this biofilm. The substrate uptake characteristics of different phylogenetic SRB subgroups were compared with the characteristics described to date. These results provide further insight into the correlation between the 16S rRNA phylogenetic diversity and the physiological diversity of SRB populations inhabiting sewer biofilms.     

Chemolithotrophic acetogenic H2/CO2 utilization in Italian rice field soil

Acetate oxidation in Italian rice field at 50 °C is achieved by uncultured syntrophic acetate oxidizers. As these bacteria are closely related to acetogens, they may potentially also be able to synthesize acetate chemolithoautotrophically. Labeling studies using exogenous H₂ (80%) and ¹³CO₂ (20%), indeed demonstrated production of acetate as almost exclusive primary product not only at 50 °C but also at 15 °C. Small amounts of formate, propionate and butyrate were also produced from ¹³CO₂. At 50 °C, acetate was first produced but later on consumed with formation of CH₄. Acetate was also produced in the absence of exogenous H₂ albeit to lower concentrations. The acetogenic bacteria and methanogenic archaea were targeted by stable isotope probing of ribosomal RNA (rRNA). Using quantitative PCR, ¹³C-labeled bacterial rRNA was detected after 20 days of incubation with ¹³CO₂. In the heavy fractions at 15 °C, terminal restriction fragment length polymorphism, cloning and sequencing of 16S rRNA showed that Clostridium cluster I and uncultured Peptococcaceae assimilated ¹³CO₂ in the presence and absence of exogenous H₂, respectively. A similar experiment showed that Thermoanaerobacteriaceae and Acidobacteriaceae were dominant in the ¹³C treatment at 50 °C. Assimilation of ¹³CO₂ into archaeal rRNA was detected at 15 °C and 50 °C, mostly into Methanocellales, Methanobacteriales and rice cluster III. Acetoclastic methanogenic archaea were not detected. The above results showed the potential for acetogenesis in the presence and absence of exogenous H₂ at both 15 °C and 50 °C. However, syntrophic acetate oxidizers seemed to be only active at 50 °C, while other bacterial groups were active at 15 °C.     

Co-occurrence patterns for abundant marine archaeal and bacterial lineages in the deep chlorophyll maximum of coastal California

Microorganisms remineralize and respire half of marine primary production, yet the niches occupied by specific microbial groups, and how these different groups may interact, are poorly understood. In this study, we identify co-occurrence patterns for marine Archaea and specific bacterial groups in the chlorophyll maximum of the Southern California Bight. Quantitative PCR time series of marine group 1 (MG1) Crenarchaeota 16S rRNA genes varied substantially over time but were well-correlated (r²=0.94, P<0.001) with ammonia monooxygenase subunit A (amoA) genes, and were more weakly related to 16S rRNA genes for all Archaea (r²=0.39), indicating that other archaeal groups (for example, Euryarchaeota) were numerically important. These data sets were compared with variability in bacterial community composition based on automated ribosomal intergenic spacer analysis (ARISA). We found that archaeal amoA gene copies and a SAR11 (or Pelagibacter) group Ib operational taxonomic unit (OTU) displayed strong co-variation through time (r²=0.55, P<0.05), and archaeal amoA and MG1 16S rRNA genes also co-occurred with two SAR86 and two Bacteroidetes OTUs. The relative abundance of these groups increased and decreased in synchrony over the course of the time series, and peaked during periods of seasonal transition. By using a combination of quantitative and relative abundance estimates, our findings show that abundant microbial OTUs—including the marine Crenarchaeota, SAR11, SAR86 and the Bacteroidetes—co-occur non-randomly; they consequently have important implications for our understanding of microbial community ecology in the sea.     

Microbial Diversity in High Arsenic Groundwater in Hetao Basin of Inner Mongolia,China

The microbial diversity and community structure in twenty-one groundwater samples from high arsenic shallow aquifers of Hetao Basin, Inner Mongolia, China was investigated with an integrated approach including polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) and 16S rRNA gene phylogenetic analyses. A total of 25 bacterial and 32 archaeal DGGE bands were exercised for sequencing. Phylogenetic analyses showed that the bacterial DGGE bands were dominated by Proteobacteria, and the archaeal bands were dominated by Thaumarchaeota and Euryarchaeota. Based on arsenic concentrations, three samples (corresponding to low, medium, and high level of arsenic, respectively) were selected for construction of 16S rRNA gene clone libraries. A total of 912 (468 and 444 for bacteria and archaea, respectively) 16S rRNA gene clone sequences were obtained and subjected to phylogenetic analyses. The results showed that bacterial communities of these samples were dominated by Acinetobacter, Pseudomonas, Massilia, Dietzia, Planococcus, Brevundimonas, Aquabacterium and Geobacter, and archaeal communities by Nitrosophaera, Thermoprotei and Methanosaeta. The relative abundance of major groups varied as a function of changes in groundwater geochemistry. Acinetobacter, Brevundimonas, Geobacter, Thermoprotei and Methanosaeta dominated in high arsenic samples with high concentrations of methane and Fe(II), and low concentrations of SO^2? ₄ and NO^? ₃, while Pseudomonas and Nitrosophaera were abundant in low arsenic groundwater. These results imply that microbes play an important role in arsenic mobilization in the shallow aquifers of Hetao Basin, Inner Mongolia.     

Competing formate- and carbon dioxide-utilizing prokaryotes in an anoxic methane-emitting fen soil

Methanogenesis in wetlands is dependent on intermediary substrates derived from the degradation of biopolymers. Formate is one such substrate and is stimulatory to methanogenesis and acetogenesis in anoxic microcosms of soil from the fen Schlöppnerbrunnen. Formate dissimilation also yields CO₂ as a potential secondary substrate. The objective of this study was to resolve potential differences between anaerobic formate- and CO₂-utilizing prokaryotes of this fen by stable isotope probing. Anoxic soil microcosms were pulsed daily with low concentrations of [¹³C]formate or ¹³CO₂ (i.e., [¹³C]bicarbonate). Taxa were evaluated by assessment of 16S rRNA genes, mcrA (encoding the alpha-subunit of methyl-coenzyme M reductase), and fhs (encoding formyltetrahydrofolate synthetase). Methanogens, acetogens, and formate-hydrogen lyase-containing taxa appeared to compete for formate. Genes affiliated with Methanocellaceae, Methanobacteriaceae, Acetobacteraceae, and Rhodospirillaceae were ¹³C enriched (i.e., labeled) in [¹³C]formate treatments, whereas genes affiliated with Methanosarcinaceae, Conexibacteraceae, and Solirubrobacteraceae were labeled in ¹³CO₂ treatments. [¹³C]acetate was enriched in [¹³C]formate treatments, but labeling of known acetogenic taxa was not detected. However, several phylotypes were affiliated with acetogen-containing taxa (e.g., Sporomusa). Methanosaetaceae-affiliated methanogens appeared to participate in the consumption of acetate. Twelve and 58 family-level archaeal and bacterial 16S rRNA phylotypes, respectively, were detected, approximately half of which had no isolated representatives. Crenarchaeota constituted half of the detected archaeal 16S rRNA phylotypes. The results highlight the unresolved microbial diversity of the fen Schlöppnerbrunnen, suggest that differing taxa competed for the same substrate, and indicate that Methanocellaceae, Methanobacteriaceae, Methanosarcinaceae, and Methanosaetaceae were linked to the production of methane, but they do not clearly resolve the taxa responsible for the apparent conversion of formate to acetate.     

Microbial community structure of a pilot-scale thermophilic anaerobic digester treating poultry litter

The microbial community structure of a stable pilot-scale thermophilic continuous stirred tank reactor digester stabilized on poultry litter was investigated. This 40-m³ digester produced biogas with 57 % methane, and chemical oxygen demand removal of 54 %. Bacterial and archaeal diversity were examined using both cloning and pyrosequencing that targeted 16S rRNA genes. The bacterial community was dominated by phylum Firmicutes, constituting 93 % of the clones and 76 % of the pyrotags. Of the Firmicutes, class Clostridia (52 % pyrotags) was most abundant followed by class Bacilli (13 % pyrotags). The bacterial libraries identified 94 operational taxonomic units (OTUs) and pyrosequencing identified 577 OTUs at the 97 % minimum similarity level. Fifteen OTUs were dominant (≥2 % abundance), and nine of these were novel unclassified Firmicutes. Several of the dominant OTUs could not be classified more specifically than Clostridiales, but were most similar to plant biomass degraders, including Clostridium thermocellum. Of the rare pyrotag OTUs (<0.5 % abundance), 75 % were Firmicutes. The dominant methanogen was Methanothermobacter which has hydrogenotrophic metabolism, and accounted for >99 % of the archaeal clones. Based on the primary methanogen, as well as digester chemistry (high VA and ammonia levels), we propose that bacterial acetate oxidation is the primary pathway in this digester for the control of acetate levels.     

Archaeal and bacterial community structures of rural household biogas digesters with different raw materials in Qinghai Plateau

The present study aims to investigate microbial community structures household biogas digesters with different raw materials in Qinghai Plateau rural. High-throughput 16S rRNA gene sequencing analysis revealed that Firmicutes, Bacteroidetes, and Proteobacteria are the most abundant bacterial phyla (64.08%). Prevotella group 7 was the most abundant genus in digester YL9 and YL10 (69.72% and 26.96%, respectively) using vegetable waste raw materials. Trichococcus exhibited the highest abundance (14.55%) in YL1 digester using sheep and pig manure. Clostridium sensu stricto 1 (13.89%) and Synergistaceae_uncultured (15.52%) comprised the highest abundances in digester YL5 with mixed raw materials (i.e., dairy manure, sheep manure, and human feces). In addition, Proteiniphilum and Pseudomonas exhibited the highest abundances among bacterial genera in YL4 digester using pig manure. Methanomicrobiales was the most dominant archaeal communities, ranging from 13.35% to 81.34% in abundance. Methanocorpusculum exhibited dominant abundances in all digesters using various raw materials. Methanogenium was the most abundant archaeal genera in YL4 and YL6 digesters, which consume pig manure as primary raw material. In addition, Methanosarcina and Methanosaeta exhibited the highest abundances in digester YL1 (55.03%) and YL9 (51.40%), respectively. Moreover, fermentation temperatures and pH both contributed to the archaeal and bacterial community structures in all the investigated digesters. Specially, fermentation temperature showed positive correlation with the abundances of Synergistaceae_uncultured, Methanogenium, and Methanosaeta, and pH was positively correlated with the abundances of Prevotella group 7 and Methanosarcina abundances.

     

Dynamic Transition of a Methanogenic Population in Response to the Concentration of Volatile Fatty Acids in a Thermophilic Anaerobic Digester

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

Anaerobic oxidation of methane at different temperature regimes in Guaymas Basin hydrothermal sediments

Anaerobic oxidation of methane (AOM) was investigated in hydrothermal sediments of Guaymas Basin based on δ¹³C signatures of CH₄, dissolved inorganic carbon and porewater concentration profiles of CH₄ and sulfate. Cool, warm and hot in-situ temperature regimes (15–20 °C, 30–35 °C and 70–95 °C) were selected from hydrothermal locations in Guaymas Basin to compare AOM geochemistry and 16S ribosomal RNA (rRNA), mcrA and dsrAB genes of the microbial communities. 16S rRNA gene clone libraries from the cool and hot AOM cores yielded similar archaeal types such as Miscellaneous Crenarchaeotal Group, Thermoproteales and anaerobic methane-oxidizing archaea (ANME)-1; some of the ANME-1 archaea formed a separate 16S rRNA lineage that at present seems to be limited to Guaymas Basin. Congruent results were obtained by mcrA gene analysis. The warm AOM core, chemically distinct by lower porewater sulfide concentrations, hosted a different archaeal community dominated by the two deep subsurface archaeal lineages Marine Benthic Group D and Marine Benthic Group B, and by members of the Methanosarcinales including ANME-2 archaea. This distinct composition of the methane-cycling archaeal community in the warm AOM core was confirmed by mcrA gene analysis. Functional genes of sulfate-reducing bacteria and archaea, dsrAB, showed more overlap between all cores, regardless of the core temperature. 16S rRNA gene clone libraries with Euryarchaeota-specific primers detected members of the Archaeoglobus clade in the cool and hot cores. A V6-tag high-throughput sequencing survey generally supported the clone library results while providing high-resolution detail on archaeal and bacterial community structure. These results indicate that AOM and the responsible archaeal communities persist over a wide temperature range.     

Phylogenetic Characterization of Methanogenic Assemblages in Eutrophic and Oligotrophic Areas of the Florida Everglades

Agricultural activities have produced well-documented changes in the Florida Everglades, including establishment of a gradient in phosphorus concentrations in Water Conservation Area 2A (WCA-2A) of the northern Everglades. An effect of increased phosphorus concentrations is increased methanogenesis in the eutrophic regions compared to the oligotrophic regions of WCA-2A. The goal of this study was to identify relationships between eutrophication and composition and activity of methanogenic assemblages in WCA-2A soils. Distributions of two genes associated with methanogens were characterized in soils taken from WCA-2A: the archaeal 16S rRNA gene and the methyl coenzyme M reductase gene. The richness of methanogen phylotypes was greater in eutrophic than in oligotrophic sites, and sequences related to previously cultivated and uncultivated methanogens were found. A preferential selection for the order Methanomicrobiales was observed in mcrA clone libraries, suggesting primer bias for this group. A greater diversity within the Methanomicrobiales was observed in mcrA clone libraries than in 16S rRNA gene libraries. 16S rRNA phylogenetic analyses revealed a dominance of clones related to Methanosaeta spp., an acetoclastic methanogen dominant in environments with low acetate concentrations. A significant number of clones were related to Methanomicrobiales, an order characterized by species utilizing hydrogen and formate as methanogenic substrates. No representatives of the orders Methanobacteriales and Methanococcales were found in any 16S rRNA clone library, although some Methanobacteriales were found in mcrA libraries. Hydrogenotrophs are the dominant methanogens in WCA-2A, and acetoclastic methanogen genotypes that proliferate in low acetate concentrations outnumber those that typically dominate in higher acetate concentrations.     

Clostridiaceae and Enterobacteriaceae as active fermenters in earthworm gut content

The earthworm gut provides ideal in situ conditions for ingested heterotrophic soil bacteria capable of anaerobiosis. High amounts of mucus- and plant-derived saccharides such as glucose are abundant in the earthworm alimentary canal, and high concentrations of molecular hydrogen (H₂) and organic acids in the alimentary canal are indicative of ongoing fermentations. Thus, the central objective of this study was to resolve potential links between fermentations and active fermenters in gut content of the anecic earthworm Lumbricus terrestris by 16S ribosomal RNA (rRNA)-based stable isotope probing, with [¹³C]glucose as a model substrate. Glucose consumption in anoxic gut content microcosms was rapid and yielded soluble organic compounds (acetate, butyrate, formate, lactate, propionate, succinate and ethanol) and gases (carbon dioxide and H₂), products indicative of diverse fermentations in the alimentary canal. Clostridiaceae and Enterobacteriaceae were users of glucose-derived carbon. On the basis of the detection of 16S rRNA, active phyla in gut contents included Acidobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Cyanobacteria, Firmicutes, Gemmatimonadetes, Nitrospirae, Planctomycetes, Proteobacteria, Tenericutes and Verrucomicrobia, taxa common to soils. On the basis of a 16S rRNA gene similarity cutoff of 87.5%, 82 families were detected, 17 of which were novel family-level groups. These findings (a) show the large diversity of soil taxa that might be active during gut passage, (b) show that Clostridiaceae and Enterobacteriaceae (fermentative subsets of these taxa) are selectively stimulated by glucose and might therefore be capable of consuming mucus- and plant-derived saccharides during gut passage and (c) indicate that ingested obligate anaerobes and facultative aerobes from soil can concomitantly metabolize the same source of carbon.     

Investigation of an acetate-fed denitrifying microbial community by stable isotope probing, full-cycle rRNA analysis, and fluorescent in situ hybridization-microautoradiography

The acetate-utilizing microbial consortium in a full-scale activated sludge process was investigated without prior enrichment using stable isotope probing (SIP). [13C]acetate was used in SIP to label the DNA of the denitrifiers. The [13C]DNA fraction that was extracted was subjected to a full-cycle rRNA analysis. The dominant 16S rRNA gene phylotypes in the 13C library were closely related to the bacterial families Comamonadaceae and Rhodocyclaceae in the class Betaproteobacteria. Seven oligonucleotide probes for use in fluorescent in situ hybridization (FISH) were designed to specifically target these clones. Application of these probes to the sludge of a continuously fed denitrifying sequencing batch reactor (CFDSBR) operated for 16 days revealed that there was a significant positive correlation between the CFDSBR denitrification rate and the relative abundance of all probe-targeted bacteria in the CFDSBR community. FISH-microautoradiography demonstrated that the DEN581 and DEN124 probe-targeted cells that dominated the CFDSBR were capable of taking up [14C]acetate under anoxic conditions. Initially, DEN444 and DEN1454 probe-targeted bacteria also dominated the CFDSBR biomass, but eventually DEN581 and DEN124 probe-targeted bacteria were the dominant bacterial groups. All probe-targeted bacteria assessed in this study were denitrifiers capable of utilizing acetate as a source of carbon. The rapid increase in the number of organisms positively correlated with the immediate increase in denitrification rates observed by plant operators when acetate is used as an external source of carbon to enhance denitrification. We suggest that the impact of bacteria on activated sludge subjected to intermittent acetate supplementation should be assessed prior to the widespread use of acetate in the wastewater industry to enhance denitrification.     

Cluster Structure of Anaerobic Aggregates of an Expanded Granular Sludge Bed Reactor

The metabolic properties and ultrastructure of mesophilic aggregates from a full-scale expanded granular sludge bed reactor treating brewery wastewater are described. The aggregates had a very high methanogenic activity on acetate (17.19 mmol of CH₄/g of volatile suspended solids [VSS]·day or 1.1 g of CH₄ chemical oxygen demand/g of VSS·day). Fluorescent in situ hybridization using 16S rRNA probes of crushed granules showed that 70 and 30% of the cells belonged to the archaebacterial and eubacterial domains, respectively. The spherical aggregates were black but contained numerous whitish spots on their surfaces. Cross-sectioning these aggregates revealed that the white spots appeared to be white clusters embedded in a black matrix. The white clusters were found to develop simultaneously with the increase in diameter. Energy-dispersed X-ray analysis and back-scattered electron microscopy showed that the whitish clusters contained mainly organic matter and no inorganic calcium precipitates. The white clusters had a higher density than the black matrix, as evidenced by the denser cell arrangement observed by high-magnification electron microscopy and the significantly higher effective diffusion coefficient determined by nuclear magnetic resonance imaging. High-magnification electron microscopy indicated a segregation of acetate-utilizing methanogens (Methanosaeta spp.) in the white clusters from syntrophic species and hydrogenotrophic methanogens (Methanobacterium-like and Methanospirillum-like organisms) in the black matrix. A number of physical and microbial ecology reasons for the observed structure are proposed, including the advantage of segregation for high-rate degradation of syntrophic substrates.     

Microbial sulfate reduction at low (8 °C) temperature using waste sludge as a carbon and seed source

Biological treatment of sulfate and metal-containing wastewater (such as acid mine drainage) is a viable option due to lower cost and better sludge quality compared to conventional chemical treatment. Although several substrates can be used as carbon source, a low-cost substrate is required for large scale applications. This study was conducted to investigate the suitability of waste sludge as a carbon and seed source for sulfate reduction at 8 °C in batch bioassays. Around 7 mmol of sulfate was reduced when the waste sludge mixture (WS) (6700 mg SS l^?1) from primary and secondary settling tank was supplemented as a carbon and seed source. However, only 1.6 mmol of sulfate was reduced with anaerobic digester effluent (ADS) (5300 mg SS l^?1). The produced H₂S from 1 g VSS l^?1 WS and ADS oxidation can theoretically precipitate around 90 and 35 mg Fe²⁺, respectively. Both WS and ADS oxidized ethanol to acetate at similar rates. It appears that WS is a good candidate for carbon and start-up seed source of sulfate reduction at 8 °C, whereas sulfidogenic acetate oxidation was the limiting step. Denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA genes showed that both sludge sources contain Desulfomicrobium apsheronum strain.