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Microorganisms play an essential role in nitrogen cycling and greenhouse gas emissions in soils and sediments. The recently discovered oxygenic denitrifiers are proposed to reduce nitrate and nitrite via nitric oxide dismutation directly to N 2 and O 2 . So far, the ecological role of these microbes is not well understood. The only available tool for a targeted study of oxygenic denitrifiers is their respective maker gene, nitric oxide dismutase (nod). Here, we established the use of PacBio long-read sequencing of nod gene amplicons to study the diversity and community structure of oxygenic denitrifiers. Two distinct sets of environmental samples, agricultural soil and lake sediment, were investigated as examples. The circular consensus sequences (ca 1.0 kb) obtained covered most substitution characteristic of NO dismutase and allowed for reliable classification of oxygenic denitrifiers. Distinct nod gene pools and community structure were revealed for the different habitats, with most sequence types affiliated to yet unidentified environmental nod lineages. The abundance of nod genes ranged 2.2 × 10 6 -3.2 × 10 7 gene copies g -1 soil or sediment, accounting for up to 3% of total bacterial 16S rRNA gene counts. This study indicates that nod-gene-targeted long-read sequencing can be a powerful tool for studying the ecology of these novel microbes, and the results also suggest that oxygenic denitrifiers are prevalent and abundant in different terrestrial samples, where they could play an important, but yet overlooked role in nitrogen transformations.

Root exudates shape microbial communities at the plant-soil interface. Here we compared bacterial communities that utilize plant-derived carbon in the rhizosphere of wheat in different soil depths, including topsoil, as well as two subsoil layers up to 1 m depth. The experiment was performed in a greenhouse using soil monoliths with intact soil structure taken from an agricultural field. To identify bacteria utilizing plant-derived carbon, 13 C-CO 2 labelling of plants was performed for two weeks at the EC50 stage, followed by isopycnic density gradient centrifugation of extracted DNA from the rhizosphere combined with 16S rRNA gene-based amplicon sequencing. Our findings suggest substantially different bacterial key players and interaction mechanisms between plants and bacteria utilizing plant-derived carbon in the rhizosphere of subsoils and topsoil. Among the three soil depths, clear differences were found in 13 C enrichment pattern across abundant operational taxonomic units (OTUs). Whereas, OTUs linked to Proteobacteria were enriched in 13 C mainly in the topsoil, in both subsoil layers OTUs related to Cohnella, Paenibacillus, Flavobacterium showed a clear 13 C signal, indicating an important, so far overseen role of Firmicutes and Bacteriodetes in the subsoil rhizosphere., (© 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.)

In abandoned coal mines, methanogenic archaea are responsible for the production of substantial amounts of methane. The present study aimed to directly unravel the active methanogens mediating methane release as well as active bacteria potentially involved in the trophic network. Therefore, the stable-isotope-labeled precursors of methane, [(13)C]acetate and H(2)-(13)CO(2), were fed to liquid cultures from hard coal and mine timber from a coal mine in Germany. Guided by methane production rates, samples for DNA stable-isotope probing (SIP) with subsequent quantitative PCR and denaturing gradient gel electrophoretic (DGGE) analyses were taken over 6 months. Surprisingly, the formation of [(13)C]methane was linked to acetoclastic methanogenesis in both the [(13)C]acetate- and the H(2)-(13)CO(2)-amended cultures of coal and timber. H(2)-(13)CO(2) was used mainly by acetogens related to Pelobacter acetylenicus and Clostridium species. Active methanogens, closely affiliated with Methanosarcina barkeri, utilized the readily available acetate rather than the thermodynamically more favorable hydrogen. Thus, the methanogenic microbial community appears to be highly adapted to the low-H(2) conditions found in coal mines.

The understanding of microbial interactions and trophic networks is a prerequisite for the elucidation of the turnover and transformation of organic materials in soils. To elucidate the incorporation of biomass carbon into a soil microbial food web, we added 13C-labeled Escherichia coli biomass to an agricultural soil and identified those indigenous microbes that were specifically active in its mineralization and carbon sequestration. rRNA stable isotope probing (SIP) revealed that uncultivated relatives of distinct groups of gliding bacterial micropredators (Lysobacter spp., Myxococcales, and the Bacteroidetes) lead carbon sequestration and mineralization from the added biomass. In addition, fungal populations within the Microascaceae were shown to respond to the added biomass after only 1 h of incubation and were thus surprisingly reactive to degradable labile carbon. This RNA-SIP study identifies indigenous microbes specifically active in the transformation of a nondefined complex carbon source, bacterial biomass, directly in a soil ecosystem.

Propionate is an important intermediate of the degradation of organic matter in many anoxic environments. In methanogenic environments, due to thermodynamic constraints, the oxidation of propionate requires syntrophic cooperation of propionate-fermenting proton-reducing bacteria and H(2)-consuming methanogens. We have identified here microorganisms that were active in syntrophic propionate oxidation in anoxic paddy soil by rRNA-based stable-isotope probing (SIP). After 7 weeks of incubation with [(13)C]propionate (<10 mM) and the oxidation of approximately 30 micromol of (13)C-labeled substrate per g dry weight of soil, we found that archaeal nucleic acids were (13)C labeled to a larger extent than those of the bacterial partners. Nevertheless, both terminal restriction fragment length polymorphism and cloning analyses revealed Syntrophobacter spp., Smithella spp., and the novel Pelotomaculum spp. to predominate in "heavy" (13)C-labeled bacterial rRNA, clearly showing that these were active in situ in syntrophic propionate oxidation. Among the Archaea, mostly Methanobacterium and Methanosarcina spp. and also members of the yet-uncultured "rice cluster I" lineage had incorporated substantial amounts of (13)C label, suggesting that these methanogens were directly involved in syntrophic associations and/or thriving on the [(13)C]acetate released by the syntrophs. With this first application of SIP in an anoxic soil environment, we were able to clearly demonstrate that even guilds of microorganisms growing under thermodynamic constraints, as well as phylogenetically diverse syntrophic associations, can be identified by using SIP. This approach holds great promise for determining the structure and function relationships of further syntrophic or other nutritional associations in natural environments and for defining metabolic functions of yet-uncultivated microorganisms.

Stable isotope probing (SIP) is a novel technique to characterize structure and in situ function of active microbial populations, which is based on the incorporation of 13C-labelled substrates into nucleic acids. Here, we have traced methylotrophic members of a rice field soil microbial community, which became active upon continuous addition of 13C-methanol (< 22 mM) as studied in microcosms. By combining rRNA- and DNA-based SIP, as well as domain-specific real-time PCR detection of templates in fractions of centrifugation gradients, we were able to detect 13C-labelled bacterial rRNA after 6 days of incubation. Fingerprinting and comparative sequence analysis of 'heavy' bacterial rRNA showed that mostly members of the Methylobacteriaceae and a novel clade within the Methylophilaceae formed part of the indigenous methylotrophic community. Over time, however, the Methylophilaceae were enriched. Unexpectedly, nucleic acids of eukaryotic origin were detected, mostly in intermediately 13C-labelled gradient fractions. These eukaryotes were identified as fungi mostly related to Fusarium and Aspergillus spp., and also Cercozoa, known as predatory soil flagellates. The detection of fungi and protozoa in 13C-enriched nucleic acid fractions suggests a possible involvement in either direct assimilation of label by the fungi, or a food web, i.e. that primary 13C-methanol consuming methylotrophs were decomposed by fungi and grazed by protozoa.

Methane emission from paddy fields may be reduced by the addition of electron acceptors to stimulate microbial populations competitive to methanogens. We have studied the effects of ferrihydrite and gypsum (CaSO(4). 2H(2)O) amendment on methanogenesis and population dynamics of methanogens after flooding of Italian rice field soil slurries. Changes in methanogen community structure were followed by archaeal small subunit (SSU) ribosomal DNA (rDNA)- and rRNA-based terminal restriction fragment length polymorphism analysis and by quantitative SSU rRNA hybridization probing. Under ferrihydrite amendment, acetate was consumed efficiently (<60 microM) and a rapid but incomplete inhibition of methanogenesis occurred after 3 days. In contrast to unamended controls, the dynamics of Methanosarcina populations were largely suppressed as indicated by rDNA and rRNA analysis. However, the low acetate availability was still sufficient for activation of Methanosaeta spp., as indicated by a strong increase of SSU rRNA but not of relative rDNA frequencies. Unexpectedly, rRNA amounts of the novel rice cluster I (RC-I) methanogens increased significantly, while methanogenesis was low, which may be indicative of transient energy conservation coupled to Fe(III) reduction by these methanogens. Under gypsum addition, hydrogen was rapidly consumed to low levels ( approximately 0.4 Pa), indicating the presence of a competitive population of hydrogenotrophic sulfate-reducing bacteria (SRB). This was paralleled by a suppressed activity of the hydrogenotrophic RC-I methanogens as indicated by the lowest SSU rRNA quantities detected in all experiments. Full inhibition of methanogenesis only became apparent when acetate was depleted to nonpermissive thresholds (<5 microM) after 10 days. Apparently, a competitive, acetotrophic population of SRB was not present initially, and hence, acetotrophic methanosarcinal populations were less suppressed than under ferrihydrite amendment. In conclusion, although methane production was inhibited effectively under both mitigation regimens, different methanogenic populations were either suppressed or stimulated, which demonstrates that functionally similar disturbances of an ecosystem may result in distinct responses of the populations involved.

Substantial amounts of organic matter are mobilized from upper soil layers during extreme precipitation events. This results in considerable fluxes of carbon from plant-associated topsoil to deeper mineral soil and to groundwater. Microbes constitute an important part of this mobile organic matter (MOM) pool. Previous work has shown that specific bacteria associated with the rhizosphere of decaying maize roots were selectively transported with seepage water upon snowmelt in winter. However, effective mechanisms of mobilization and also possible distinctions to microbial transport for living root systems remain poorly understood. In the present study, bacteria in seepage water were sampled from lysimeters at an experimental maize field after extreme rain events in summer. We show that a distinctive subset of rhizoplane-associated bacterial populations was mobilized after summer rain, especially including abundant members of the Bacteroidetes , representing a microbial conduit for fresh plant-derived carbon inputs into deeper soil layers. Marked distinctions of seepage communities were not observed between lysimeters with a different relative contribution of preferential vs. matrix flow. Time-resolved analyses of seepage water during an artificial rain event revealed temporal patterns in the mobilization of certain lineages, with members of the Chitinophagaceae , Sphingomonadaceae , and Bradyrhizobiaceae preferentially mobilized in early and late seepage fractions, and members of the candidate phyla Parcubacteria and Microgenomates mobilized mostly in intermediate fractions. While average bacterial cell counts were at ∼10 7  ml −1 in seepage water, the recovery of amended fluorescently labeled cells of Arthrobacter globiformis was low (0.2–0.6%) over seepage events. Still, mobilized bacteria clearly have the potential to influence bacterial activities and communities in subsoils. These findings demonstrate that dynamic hydraulic events must be considered for a better understanding of the connectivities between microbial populations and communities in soil, as well as of the links between distinct carbon pools over depth. [ABSTRACT FROM AUTHOR]

Predation is a fundamental mechanism of all food webs, but its drivers and organismic connectivities, especially at microbial level, are still poorly understood. Specifically, competitive carbon flows in the presence of multiple micropredators, as well as trophic links within and between microbial kingdoms have rarely been resolved. Here, using maize-planted agricultural soil as a model system, we have investigated the predation of amended bacterial prey by both prokaryotic and eukaryotic micropredators. We have queried how soil compartment (rhizosphere vs bulk soil) and nature of prey (Gram-positive vs Gram-negative) influence predation outcomes. We added 13C-labelled biomass of Pseudomonas putida and Arthrobacter globiformis to soil microcosms and found that P. putida was consumed much more rapidly. Bacteria and microeukaryotes specifically responsive to the biomass amendments were identified by RNA-stable isotope probing. Amongst the bacteria, only a few myxobacteria sequestered C from A. globiformis, whereas a considerable diversity of predatory bacteria incorporated C derived from P. putida. Diverse groups of heterotrophic protists, especially amoeba including Glaeseria, Hartmanella and Vahlkampfia spp., were observed to incorporate 13C from both amendments, but with pronounced niche differentiation between rhizosphere and bulk soil. This provides novel insights into niche partitioning between bacterial and eukaryotic micropredators in soil, driven not only by the nature of bacterial prey itself, but also by soil compartments. [ABSTRACT FROM AUTHOR]

Bioavailability of electron acceptors is probably the most limiting factor in the restoration of anoxic, contaminated environments. The oxidation of contaminants such as aromatic hydrocarbons, particularly in aquifers, often depends on the reduction of ferric iron or sulphate. We have previously detected a highly active fringe zone beneath a toluene plume at a tar-oil-contaminated aquifer in Germany, where a specialized community of contaminant degraders codominated by Desulfobulbaceae and Geobacteraceae had established. Although on-site geochemistry links degradation to sulphidogenic processes, dominating catabolic (benzylsuccinate synthase α-subunit, bssA) genes detected in situ appeared to be more related to those of Geobacter spp. Therefore, a stable isotope probing (SIP) incubation of sediment samples with 13C7-toluene and comparative electron acceptor amendment was performed. We introduce pyrosequencing of templates from SIP microcosms as a powerful new strategy in SIP gradient interpretation (Pyro-SIP). Our results reveal the central role of Desulfobulbaceae in sulphidogenic toluene degradation in situ, and affiliate the detected bssA genes to this lineage. This and the absence of 13C-labelled DNA of Geobacter spp. in SIP gradients preclude their relevance as toluene degraders in situ. In contrast, Betaproteobacteria related to Georgfuchsia spp. became labelled under iron-reducing conditions. Furthermore, secondary toluene degraders belonging to the Peptococcaceae detected in both treatments suggest the possibility of functional redundancy among anaerobic toluene degraders on site. [ABSTRACT FROM AUTHOR]

Examines the methanogen-specific methyl-coenzyme M reductase alpha-subunit (mcrA) genes in Italian rice field soil. Sequences of the mcrA; Detection of the SSU rRNA of rice cluster through cloning and terminal restriction fragment length polymorphism; Significance of the catabolic gene in demonstrating the genotype and phenotype of RC-I Archea.

Plants introduce abundant carbon into soils, where it is mineralised and sequestered. Proportions of this fresh organic carbon introduced to top soils can be relocated to deeper soil layers and even to groundwater by event-driven transport upon heavy rainfalls or after snowmelt. It is assumed that a significant fraction of this flux involves biocolloids and possibly microbial biomass itself. However, the nature of such transported microbes, their origin and the mechanisms of their mobilisation are still poorly understood. Here, we provide primary evidence that specific microbial populations are exported from top soils upon seepage events. At an experimental maize field, we have analysed the composition of mobilised bacterial communities collected in seepage water directly after snowmelt in winter at different depths (35 and 65 cm), and compared them to the corresponding bulk soil microbiota. Using T-RFLP fingerprinting and pyrotag sequencing, we reveal that mostly members of the Betaproteobacteria (Methylophilaceae, Oxalobacteraceae, Comamonadaceae), the Alphaproteobacteria (Sphingomonadaceae, Bradyrhizobiaceae), the Gammaproteobacteria (Legionellaceae) and the Bacteroidetes (Sphingobacteriaceae) were mobilised, all characteristic taxa for the rhizoplane. This highlights the importance of preferential flow along root channels for the vertical mobilisation and transport of microbes. Although the estimated quantitative fluxes of bacterial biomass carbon appeared low, our study allows for an improved understanding of the links between top soil, subsoil, and groundwater microbiota, as well as carbon fluxes between soil compartments. [ABSTRACT FROM AUTHOR]