The root zone of graminoids: A niche for H2-consuming acetogens in a minerotrophic peatland.

The importance of acetogens for H2 turnover and overall anaerobic degradation in peatlands remains elusive. In the well-studied minerotrophic peatland fen Schlöppnerbrunnen, H2-consuming acetogens are conceptualized to be largely outcompeted by iron reducers, sulfate reducers, and hydrogenotrophic methanogens in bulk peat soil. However, in root zones of graminoids, fermenters thriving on rhizodeposits and root litter might temporarily provide sufficient H2 for acetogens. In the present study, root-free peat soils from around the roots of Molinia caerulea and Carex rostrata (i.e., two graminoids common in fen Schlöpnnerbrunnen) were anoxically incubated with or without supplemental H2 to simulate conditions of high and low H2 availability in the fen. In unsupplemented soil treatments, H2 concentrations were largely below the detection limit (∼10 ppmV) and possibly too low for acetogens and methanogens, an assumption supported by the finding that neither acetate nor methane substantially accumulated. In the presence of supplemental H2, acetate accumulation exceeded CH4 accumulation in Molinia soil whereas acetate and methane accumulated equally in Carex soil. However, reductant recoveries indicated that initially, additional unknown processes were involved either in H2 consumption or the consumption of acetate produced by H2-consuming acetogens. 16S rRNA and 16S rRNA gene analyses revealed that potential acetogens (Clostridium, Holophagaceae), methanogens (Methanocellales, Methanobacterium), iron reducers (Geobacter), and physiologically uncharacterized phylotypes (Acidobacteria, Actinobacteria, Bacteroidetes) were stimulated by supplemental H2 in soil treatments. Phylotypes closely related to clostridial acetogens were also active in soil-free Molinia and Carex root treatments with or without supplemental H2. Due to pronounced fermentation activities, H2 consumption was less obvious in root treatments, and acetogens likely thrived on root organic carbon and fermentation products (e.g., ethanol) in addition to H2. Collectively, the data highlighted that in fen Schlöppnerbrunnen, acetogens are associated to graminoid roots and inhabit the peat soil around the roots, where they have to compete for H2 with methanogens and iron reducers. Furthermore, the study underscored that the metabolically flexible acetogens do not rely on H2, potentially a key advantage over other H2 consumers under the highly dynamic conditions characteristic for the root-zones of graminoids in peatlands.

Organic carbon from graminoid roots as a driver of fermentation in a fen.

Fen Schlöppnerbrunnen is a moderately acidic methane-emitting peatland overgrown by Molinia caerulea and other wetland graminoids (e.g. Carex rostrata). Recently, the accumulation of H2, an indicator for fermentation, was observed with anoxically incubated C. rostrata roots but not with root-free fen soil. Based on this finding, we hypothesized that root-derived organic carbon has a higher capacity to promote fermentation processes than peat organic carbon from root-free fen soil. To address this hypothesis, C. rostrata and M. caerulea roots were anoxically incubated with or without fen soil and the product profiles of root treatments were compared with those of root-free soil treatments. Ethanol, acetate, propionate, butyrate, H2 and CO2 accumulated in root treatments and collective amounts of carbon in accumulating products were 20-200 times higher than those in root-free soil treatments, in which mainly CO2 accumulated. Analyses of 16S rRNA and 16S rRNA gene sequences revealed that Clostridium, Propionispira and Rahnella, representatives of butyrate, propionate and mixed acid fermenters, respectively, were relatively enriched in root treatments. In contrast, differences of the microbial community before and after incubation were marginal in root-free soil treatments. Collectively, these findings supported the assumed stimulatory effect of root-derived organic carbon on fen fermenters.

Impact of water content and dietary organic carbon richness on gut bacteria in the earthworm Lumbricus terrestris.

Many higher and lower animal gut ecosystems have complex resident microbial communities. In contrast, ingested soil is the primary source of the gut microbial diversity of earthworms, invertebrates of fundamental importance to the terrestrial biosphere. Earthworms also harbor a few endemic bacteria including Tenericutes-affiliated Candidatus Lumbricincola of unknown function. Gut microbes are subject to nutrient fluctuations due to dilution effects during gut passage, the nutrient richness of the anoxic gut, and dietary organic carbon, factors that could alter their activity/detection. This study's objective was to assess the potential impact of these factors on the occurrence and activity of ingested and endemic bacteria in gut content of Lumbricus terrestris. Fermentation product profiles of anoxic undiluted and diluted gut content treatments were similar, suggesting that experimental increase in water content and nutrient dilution had marginal impact on fermentation. However, 16S ribosomal Ribonucleic Acid (16S rRNA) sequence abundances indicated that stimulated bacterial taxa were not identical in undiluted and diluted treatments, with dominate potentially functionally redundant phylotypes being affiliated to the Firmicutes, Fusobacteria and Proteobacteria. Although the earthworm-associated Tenericutes were not stimulated in these treatments, the occurrence of three Tenericutes-affiliated phylotypes varied with the organic carbon richness of the earthworm diet, with two phylotypes being associated with high organic carbon richness. 16S rRNA sequence abundances indicated that other dominant gut taxa also varied with dietary organic carbon richness. These findings illustrate that functionally redundant ingested bacteria and earthworm-associated Tenericutes might be influenced by nutrient fluctuations in the gut and organic carbon richness of the earthworm diet.

Amino Acids and Ribose: Drivers of Protein and RNA Fermentation by Ingested Bacteria of a Primitive Gut Ecosystem.

Earthworms are among the most primitive animals and are of fundamental importance to the turnover of organic matter in the terrestrial biosphere. These invertebrates ingest materials that are colonized by microbes, some of which are subject to disruption by the crop/gizzard or other lytic events during gut passage. Protein and RNA are dominant polymers of disrupted microbial cells, and these biopolymers facilitate robust fermentations by surviving ingested bacteria. To further resolve these fermentations, amino acids and ribose (as fermentable constituents of protein and RNA, respectively) were evaluated as potential drivers of fermentation in gut content of the model earthworm Lumbricus terrestris (taxa were examined with 16S rRNA-based analyses). Of eight amino acids tested, glutamate, aspartate, and threonine were most stimulatory and yielded dissimilar fermentations facilitated by contrasting taxa (e.g., glutamate stimulated the Fusobacteriaceae and yielded H2 and formate, whereas aspartate stimulated the Aeromonadaceae and yielded succinate and propionate). A marginal Stickland fermentation was associated with the Peptostreptococcaceae and Lachnospiraceae Ribose fermentation yielded a complex product profile facilitated primarily by the Aeromonadaceae The transient nature of succinate was linked to its decarboxylation to propionate and the Fusobacteriaceae, whereas the transient nature of formate was linked to formate-hydrogen lyase activity and the Peptostreptococcaceae These findings reinforce the likelihood that (i) the animal host and hosted fermentative bacteria compete for the constituents of protein and RNA in the alimentary canal and (ii) diverse gut fermenters engaged in the fermentation of these constituents produce products that can be utilized by earthworms.IMPORTANCE Animal health is linked to gut ecosystems whose primary function is normally the digestion of dietary matter. Earthworms are representative of one of the oldest known animal lineages and, despite their primitive nature, have unique environmental impact by virtue of their dietary consumption of their habitat, i.e., soil-associated matter. A resident gut community is a hallmark of many gut ecosystems of evolutionarily more advanced animals, but the alimentary canal of earthworms is dominated by ingested transient soil microbes. Protein and RNA are (i) the primary organic components of microbial cells that are subject to lysis during gut passage and (ii) fermentable dietary substrates in the alimentary canal. This study examined the gut-associated fermentation of constituents of these biopolymers to determine how their fermentation is integrated to the microbiological dynamics of the gut and might contribute to earthworm-linked transformations of organic matter in the terrestrial biosphere.

Dietary polysaccharides: fermentation potentials of a primitive gut ecosystem.

The alimentary canal of the earthworm is representative of primitive gut ecosystems, and gut fermenters capable of degrading ingested biomass-derived polysaccharides might contribute to the environmental impact and survival of this terrestrial invertebrate. Thus, this study evaluated the postulation that gut microbiota of the model earthworm Lumbricus terrestris ferment diverse biomass-derived polysaccharides. Structural polysaccharides (e.g. cellulose, chitin) had marginal impact on fermentation in anoxic gut content treatments. In contrast, nonstructural polysaccharides (e.g. starch, glycogen) greatly stimulated (a) the formation of diverse fermentation products (e.g. H2 , ethanol, fatty acids) and (b) the facultatively fermentative families Aeromonadaceae and Enterobacteriaceae. Despite these contrasting results with different polysaccharides, most saccharides derived from these biopolymers (e.g. glucose, N-acetylglucosamine) greatly stimulated fermentation, yielding 16S rRNA gene-based signatures of Aeromonadaceae-, Enterobacteriaceae- and Fusobacteriaceae-affiliated phylotypes. Roots and litter are dietary substrates of the earthworm, and as proof-of-principle, gut-associated fermenters responded rapidly to root- and litter-derived nutrients including saccharides. These findings suggest that (a) hydrolysis of certain ingested structural polysaccharides may be a limiting factor in the ability of gut fermenters to utilize them and (b) nonstructural polysaccharides of disrupted biomass are subject to rapid fermentation by gut microbes and yield fatty acids that can be utilized by the earthworm.

Fermenters in the earthworm gut: do transients matter?

Earthworms have profound impact on soil-based ecosystems. Although theoretical considerations suggest that most microbes in the earthworm gut are likely ingested and transient, the non-responsiveness of soil microbes to a specific high value gut nutrient and anoxia has made it difficult to demonstrate that responsive gut fermenters are derived from soil. Therefore, soil and gut content of the model earthworm Lumbricus terrestris were examined for their fermentative capabilities. In unsupplemented anoxic microcosms, fermentation was negligible with soil but rapid with gut content. However, both soil and gut content facilitated robust fermentations when challenged with complex nutrients indicative of those released from gizzard-disrupted cells. Based on the relative abundances of 16S rRNA and 16S rRNA gene sequences, the responsive fermentative taxa in unsupplemented gut content treatments were negligible in unsupplemented soil treatments. In contrast, the responsive fermentative taxa in soil and gut content treatments supplemented with complex nutrients displayed marked similarities, with numerous Proteobacteria- and Firmicutes-affiliated phylotypes being dominant. These findings indicated that detectable differences between the fermentative taxa in soil and gut contents are due in part to the nutrient-dependent metabolic status of community members and reinforce the likelihood that ingested transient microbes contribute to fermentation in the alimentary canal.

Protein- and RNA-Enhanced Fermentation by Gut Microbiota of the Earthworm Lumbricus terrestris.

Earthworms are a dominant macrofauna in soil ecosystems and have determinative effects on soil fertility and plant growth. These invertebrates feed on ingested material, and gizzard-linked disruption of ingested fungal and bacterial cells is conceived to provide diverse biopolymers in the anoxic alimentary canals of earthworms. Fermentation in the gut is likely important to the utilization of ingested biopolymer-derived compounds by the earthworm. This study therefore examined the fermentative responses of gut content-associated microbes of the model earthworm Lumbricus terrestris to (i) microbial cell lysate (to simulate gizzard-disrupted cells) and (ii) dominant biopolymers of such biomass, protein, and RNA. The microbial cell lysate augmented the production of H2, CO2, and diverse fatty acids (e.g., formate, acetate, propionate, succinate, and butyrate) in anoxic gut content microcosms, indicating that the cell lysate triggered diverse fermentations. Protein and RNA also augmented diverse fermentations in anoxic microcosms of gut contents, each yielding a distinct product profile (e.g., RNA yielded H2 and succinate, whereas protein did not). The combined product profile of protein and RNA treatments was similar to that of cell lysate treatments, and 16S rRNA-based analyses indicated that many taxa that responded to cell lysate were similar to taxa that responded to protein or RNA. In particular, protein stimulated Peptostreptococcaceae, Clostridiaceae, and Fusobacteriaceae, whereas RNA stimulated Aeromonadaceae These findings demonstrate the capacity of gut-associated obligate anaerobes and facultative aerobes to catalyze biopolymer-driven fermentations and highlight the potential importance of protein and RNA as substrates linked to the overall turnover dynamics of organic carbon in the alimentary canal of the earthworm.IMPORTANCE The subsurface lifestyle of earthworms makes them an unnoticed component of the terrestrial biosphere. However, the propensity of these invertebrates to consume their home, i.e., soil and litter, has long-term impacts on soil fertility, plant growth, and the cycling of elements. The alimentary canals of earthworms can contain up to 500 ml anoxic gut content per square meter of soil, and ingested soil may contain 109 or more microbial cells per gram dry weight, considerations that illustrate that enormous numbers of soil microbes are subject to anoxia during gut passage. Feeding introduces diverse sources of biopolymers to the gut, and the gut fermentation of biopolymers could be important to the transformation of matter by the earthworm and its capacity to utilize fermentation-derived fatty acids. Thus, this study examined the capacity of microbes in earthworm gut contents to ferment protein and RNA, dominant biopolymers of cells that become disrupted during gut passage.

Differential Engagement of Fermentative Taxa in Gut Contents of the Earthworm Lumbricus terrestris.

The earthworm gut is an anoxic, saccharide-rich microzone in aerated soils. The apparent degradation of diverse saccharides in the alimentary canal of the model earthworm Lumbricusterrestris is concomitant with the production of diverse organic acids, indicating that fermentation is an ongoing process in the earthworm gut. However, little is known about how different gut-associated saccharides are fermented. The hypothesis of this investigation was that different gut-associated saccharides differentially stimulate fermentative microorganisms in gut contents of L. terrestris This hypothesis was addressed by (i) assessing the fermentation profiles of anoxic gut content microcosms that were supplemented with gut-associated saccharides and (ii) the concomitant phylogenic analysis of 16S rRNA sequences. Galactose, glucose, maltose, mannose, arabinose, fucose, rhamnose, and xylose stimulated the production of fermentation products, including H2, CO2, acetate, lactate, propionate, formate, succinate, and ethanol. Fermentation profiles were dependent on the supplemental saccharide (e.g., glucose yielded large amounts of H2 and ethanol, whereas fucose did not, and maltose yielded large amounts of lactate, whereas mannose did not). Approximately 1,750,000 16S rRNA sequences were affiliated with 37 families, and phylogenic analyses indicated that a respective saccharide stimulated a subset of the diverse phylotypes. An Aeromonas-related phylotype displayed a high relative abundance in all treatments, whereas key Enterobacteriaceae-affiliated phylotypes were stimulated by some but not all saccharides. Collectively, these results reinforce the likelihood that (i) different saccharides stimulate different fermentations in gut contents of the earthworm and (ii) facultative aerobes related to Aeromonadaceae and Enterobacteriaceae can be important drivers of these fermentations.IMPORTANCE The feeding habits of earthworms influence the turnover of elements in the terrestrial biosphere. The alimentary tract of the earthworm constitutes an anoxic saccharide-rich microzone in aerated soils that offers ingested microbes a unique opportunity for anaerobic growth. The fermentative activity of microbes in the alimentary tract are responsible for the in situ production of (i) organic compounds that can be assimilated by the earthworm and (ii) H2 that is subject to in vivo emission by the earthworm and can be trophically linked to secondary microbial events in soils. To gain insight on how fermentative members of the gut microbiome might respond to the saccharide-rich alimentary canal, this study examines the impact of diverse gut-associated saccharides on the differential activation of fermentative microbes in gut contents of the model earthworm L. terrestris.

Disentangling the influence of earthworms in sugarcane rhizosphere.

For the last 150 years many studies have shown the importance of earthworms for plant growth, but the exact mechanisms involved in the process are still poorly understood. Many important functions required for plant growth can be performed by soil microbes in the rhizosphere. To investigate earthworm influence on the rhizosphere microbial community, we performed a macrocosm experiment with and without Pontoscolex corethrurus (EW+ and EW-, respectively) and followed various soil and rhizosphere processes for 217 days with sugarcane. In EW+ treatments, N2O concentrations belowground (15 cm depth) and relative abundances of nitrous oxide genes (nosZ) were higher in bulk soil and rhizosphere, suggesting that soil microbes were able to consume earthworm-induced N2O. Shotgun sequencing (total DNA) revealed that around 70 microbial functions in bulk soil and rhizosphere differed between EW+ and EW- treatments. Overall, genes indicative of biosynthetic pathways and cell proliferation processes were enriched in EW+ treatments, suggesting a positive influence of worms. In EW+ rhizosphere, functions associated with plant-microbe symbiosis were enriched relative to EW- rhizosphere. Ecological networks inferred from the datasets revealed decreased niche diversification and increased keystone functions as an earthworm-derived effect. Plant biomass was improved in EW+ and worm population proliferated.

Formate-derived H2 , a driver of hydrogenotrophic processes in the root-zone of a methane-emitting fen.

Wetlands are important sources of globally emitted methane. Plants mediate much of that emission by releasing root-derived organic carbon, including formate, a direct precursor of methane. Thus, the objective of this study was to resolve formate-driven processes potentially linked to methanogenesis in the fen root-zone. Although, formate was anticipated to directly trigger methanogenesis, the rapid anaerobic consumption of formate by Carex roots unexpectedly yielded H2 and CO2 via enzymes such as formate-H2 -lyase (FHL), and likewise appeared to enhance the utilization of organic carbon. Collectively, 57 [FeFe]- and [NiFe]-hydrogenase-containing family level phylotypes potentially linked to FHL activity were detected. Under anoxic conditions, root-derived fermentative Citrobacter and Hafnia isolates produced H2 from formate via FHL. Formate-derived H2 fueled methanogenesis and acetogenesis, and methanogenic (Methanoregula, Methanobacterium, Methanocella) and acetogenic (Acetonema, Clostridum, Sporomusa) genera potentially linked to these hydrogenotrophic activities were identified. The findings (i) provide novel insights on highly diverse root-associated FHL-containing taxa that can augment secondary hydrogenotrophic processes via the production of formate-derived H2 , (ii) demonstrate that formate can have a 'priming' effect on the utilization of organic carbon, and (iii) raise questions regarding the fate of formate-derived H2 when it diffuses away from the root-zone.

Peat: home to novel syntrophic species that feed acetate- and hydrogen-scavenging methanogens.

Syntrophic bacteria drive the anaerobic degradation of certain fermentation products (e.g., butyrate, ethanol, propionate) to intermediary substrates (e.g., H2, formate, acetate) that yield methane at the ecosystem level. However, little is known about the in situ activities and identities of these syntrophs in peatlands, ecosystems that produce significant quantities of methane. The consumption of butyrate, ethanol or propionate by anoxic peat slurries at 5 and 15 °C yielded methane and CO2 as the sole accumulating products, indicating that the intermediates H2, formate and acetate were scavenged effectively by syntrophic methanogenic consortia. 16S rRNA stable isotope probing identified novel species/strains of Pelobacter and Syntrophomonas that syntrophically oxidized ethanol and butyrate, respectively. Propionate was syntrophically oxidized by novel species of Syntrophobacter and Smithella, genera that use different propionate-oxidizing pathways. Taxa not known for a syntrophic metabolism may have been involved in the oxidation of butyrate (Telmatospirillum-related) and propionate (unclassified Bacteroidetes and unclassified Fibrobacteres). Gibbs free energies (ΔGs) for syntrophic oxidations of ethanol and butyrate were more favorable than ΔGs for syntrophic oxidation of propionate. As a result of the thermodynamic constraints, acetate transiently accumulated in ethanol and butyrate treatments but not in propionate treatments. Aceticlastic methanogens (Methanosarcina, Methanosaeta) appeared to outnumber hydrogenotrophic methanogens (Methanocella, Methanoregula), reinforcing the likely importance of aceticlastic methanogenesis to the overall production of methane. ΔGs for acetogenesis from H2 to CO2 approximated to -20 kJ mol(-1) when acetate concentrations were low, indicating that acetogens may have contributed to the flow of carbon and reductant towards methane.

Anaerobic trophic interactions of contrasting methane-emitting mire soils: processes versus taxa.

Natural wetlands such as mires contribute up to 33% to the global emission of methane. The emission of methane is driven by trophic interactions of anaerobes that collectively degrade biopolymers. The hypothesis of this study was that these interactions in contrasting methane-emitting mire soils are functionally similar but linked to dissimilar taxa. This hypothesis was addressed by evaluating anaerobic processes and microbial taxa of eutrophic, mesotrophic and oligotrophic mire soils. Glucose was fermented to various products (e.g. H2, CO2, butyrate, acetate). Acetoclastic methanogenesis occurred, and acetogenesis and methanogenesis transformed H2-CO2 to acetate and methane, respectively. Although product profiles, cultivable cell numbers and gene copy numbers [mcrA (encodes alpha-subunit of methyl-CoM reductase) and 16S rRNA genes] were similar for all mire soils, only approximately 15% of detected family-level bacteria and species-level methanogens were shared by all mire soils. Approximately, 40% of the detected family-level taxa of each mire soil have no cultured isolates. Acidic conditions appeared to restrict the number of dominant phylotypes. The results indicated (a) that microbial processes which drive methanogenesis are similar but facilitated by dissimilar microbial communities in contrasting mire soils and (b) that mire soils harbor a large number of taxa with no cultured isolates.

Methanogenic food web in the gut contents of methane-emitting earthworm Eudrilus eugeniae from Brazil.

The anoxic saccharide-rich conditions of the earthworm gut provide an ideal transient habitat for ingested microbes capable of anaerobiosis. It was recently discovered that the earthworm Eudrilus eugeniae from Brazil can emit methane (CH4) and that ingested methanogens might be associated with this emission. The objective of this study was to resolve trophic interactions of bacteria and methanogens in the methanogenic food web in the gut contents of E. eugeniae. RNA-based stable isotope probing of bacterial 16S rRNA as well as mcrA and mrtA (the alpha subunit of methyl-CoM reductase and its isoenzyme, respectively) of methanogens was performed with [(13)C]-glucose as a model saccharide in the gut contents. Concomitant fermentations were augmented by the rapid consumption of glucose, yielding numerous products, including molecular hydrogen (H2), carbon dioxide (CO2), formate, acetate, ethanol, lactate, succinate and propionate. Aeromonadaceae-affiliated facultative aerobes, and obligate anaerobes affiliated to Lachnospiraceae, Veillonellaceae and Ruminococcaceae were associated with the diverse fermentations. Methanogenesis was ongoing during incubations, and (13)C-labeling of CH4 verified that supplemental [(13)C]-glucose derived carbon was dissimilated to CH4. Hydrogenotrophic methanogens affiliated with Methanobacteriaceae and Methanoregulaceae were linked to methanogenesis, and acetogens related to Peptostreptoccocaceae were likewise found to be participants in the methanogenic food web. H2 rather than acetate stimulated methanogenesis in the methanogenic gut content enrichments, and acetogens appeared to dissimilate supplemental H2 to acetate in methanogenic enrichments. These findings provide insight on the processes and associated taxa potentially linked to methanogenesis and the turnover of organic carbon in the alimentary canal of methane-emitting E. eugeniae.

Temperature impacts differentially on the methanogenic food web of cellulose-supplemented peatland soil.

The impact of temperature on the largely unresolved intermediary ecosystem metabolism and associated unknown microbiota that link cellulose degradation and methane production in soils of a moderately acidic (pH 4.5) fen was investigated. Supplemental [(13) C]cellulose stimulated the accumulation of propionate, acetate and carbon dioxide as well as initial methane production in anoxic peat soil slurries at 15°C and 5°C. Accumulation of organic acids at 15°C was twice as fast as that at 5°C. 16S rRNA [(13) C]cellulose stable isotope probing identified novel unclassified Bacteria (79% identity to the next cultured relative Fibrobacter succinogenes), unclassified Bacteroidetes (89% identity to Prolixibacter bellariivorans), Porphyromonadaceae, Acidobacteriaceae and Ruminococcaceae as main anaerobic degraders of cellulose-derived carbon at both 15°C and 5°C. Holophagaceae and Spirochaetaceae were more abundant at 15°C. Clostridiaceae dominated the degradation of cellulose-derived carbon only at 5°C. Methanosarcina was the dominant methanogenic taxa at both 15°C and 5°C. Relative abundance of Methanocella increased at 15°C whereas that of Methanoregula and Methanosaeta increased at 5°C. Thaumarchaeota closely related to Nitrosotalea (presently not known to grow anaerobically) were abundant at 5°C but absent at 15°C indicating that Nitrosotalea sp. might be capable of anaerobic growth at low temperatures in peat.

Consumers of 4-chloro-2-methylphenoxyacetic acid from agricultural soil and drilosphere harbor cadA, r/sdpA, and tfdA-like gene encoding oxygenases.

Microbial degradation of 2-methyl-4-chlorophenoxyacetic acid (MCPA) in soil is enhanced by earthworms and initiated by tfdA-like, cadA and r/sdpA gene encoding oxygenases. Copy numbers of such genes increased during MCPA degradation in soil, and MCPA stimulated transcription of tfdA-like and r/sdpA genes up to 4×. Transcription of cadA was detected in the presence of MCPA only. DNA stable isotope probing after consumption of 0.6-0.8 mg 13C-MCPA gdw -1 in oxic microcosms indicated diverse labeled oxygenase genes in bulk soil, burrow walls, and cast. 9, 6, and 3 operational taxonomic units of tfdA-like, cadA, and r/sdpA genes, respectively, were labeled and affiliated with group 2 Alphaproteobacteria including Bradyrhizobia and group 1 class III Betaproteobacteria. New genes encoding putative MCPA degrading oxygenases were identified. Diversity of labeled OTUs tended to be lower for cast than for bulk soil. The collective data indicate (1) hitherto unknown active MCPA degraders and/or oxygenase genes in soil; (2) that multiple oxygenases are associated with MCPA degradation in soil at the same time; (3) that earthworms impact the capability of MCPA degraders in soil to respond to MCPA; and (4) the collective data enable a more in-depth analysis of MCPA degrader communities in soil by future structural gene-based experimental strategies.

Emission of nitrous oxide and dinitrogen by diverse earthworm families from Brazil and resolution of associated denitrifying and nitrate-dissimilating taxa.

The anoxic earthworm gut augments the activity of ingested microorganisms capable of anaerobiosis. Small earthworms (Lumbricidae) emit denitrification-derived N(2)O, whereas the large Octochaetus multiporus (Megascolecidae) does not. To examine this paradox, differently sized species of the families Glossoscolecidae (Rhinodrilus, Glossoscolex, Pontoscolex), Megascolecidae (Amynthas, Perionyx), Acanthodrilidae (Dichogaster), and Eudrilidae (Eudrilus) from Brazil were analyzed. Small species and the large Rhinodrilus alatus emitted N(2)O, whereas the large Glossoscolex paulistus did not, even though its gut could denitrify. N(2) and N(2)O were emitted concomitantly, and R. alatus emitted the highest amount of N(2). Denitrifiers and dissimilatory nitrate reducers were analyzed by barcoded amplicon pyrosequencing of narG, nirK, and nosZ. Gene sequences in gut and soil of the large G. paulistus were similar, whereas sequences in gut and soil of the small Amynthas gracilis were different and were also different compared with those of the gut and soil of G. paulistus. However, the denitrifying gut microbiota for both earthworms appeared to be soil-derived and dominated by Rhizobiales. The results demonstrated that (1) the emission of denitrification-derived N(2)O is widespread in different earthworm families, (2) large earthworms can also emit nitrogenous gases, and (3) ingested members of Rhizobiales are associated with this emission.

Microbiology of Lonar Lake and other soda lakes.

Soda lakes are saline and alkaline ecosystems that are believed to have existed throughout the geological record of Earth. They are widely distributed across the globe, but are highly abundant in terrestrial biomes such as deserts and steppes and in geologically interesting regions such as the East African Rift valley. The unusual geochemistry of these lakes supports the growth of an impressive array of microorganisms that are of ecological and economic importance. Haloalkaliphilic Bacteria and Archaea belonging to all major trophic groups have been described from many soda lakes, including lakes with exceptionally high levels of heavy metals. Lonar Lake is a soda lake that is centered at an unusual meteorite impact structure in the Deccan basalts in India and its key physicochemical and microbiological characteristics are highlighted in this article. The occurrence of diverse functional groups of microbes, such as methanogens, methanotrophs, phototrophs, denitrifiers, sulfur oxidizers, sulfate reducers and syntrophs in soda lakes, suggests that these habitats harbor complex microbial food webs that (a) interconnect various biological cycles via redox coupling and (b) impact on the production and consumption of greenhouse gases. Soda lake microorganisms harbor several biotechnologically relevant enzymes and biomolecules (for example, cellulases, amylases, ectoine) and there is the need to augment bioprospecting efforts in soda lake environments with new integrated approaches. Importantly, some saline and alkaline lake ecosystems around the world need to be protected from anthropogenic pressures that threaten their long-term existence.

Methanol oxidation by temperate soils and environmental determinants of associated methylotrophs.

The role of soil methylotrophs in methanol exchange with the atmosphere has been widely overlooked. Methanol can be derived from plant polymers and be consumed by soil microbial communities. In the current study, methanol-utilizing methylotrophs of 14 aerated soils were examined to resolve their comparative diversities and capacities to utilize ambient concentrations of methanol. Abundances of cultivable methylotrophs ranged from 10(6)-10(8) gsoilDW(-1). Methanol dissimilation was measured based on conversion of supplemented (14)C-methanol, and occurred at concentrations down to 0.002 µmol methanol gsoilDW(-1). Tested soils exhibited specific affinities to methanol (a(0)s=0.01 d(-1)) that were similar to those of other environments suggesting that methylotrophs with similar affinities were present. Two deep-branching alphaproteobacterial genotypes of mch responded to the addition of ambient concentrations of methanol (0.6 µmol methanol gsoilDW(-1)) in one of these soils. Methylotroph community structures were assessed by amplicon pyrosequencing of genes of mono carbon metabolism (mxaF, mch and fae). Alphaproteobacteria-affiliated genotypes were predominant in all investigated soils, and the occurrence of novel genotypes indicated a hitherto unveiled diversity of methylotrophs. Correlations between vegetation type, soil pH and methylotroph community structure suggested that plant-methylotroph interactions were determinative for soil methylotrophs.