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.