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The close interconnection of plants with rhizosphere- and root-associated microorganisms is well recognized, and high expectations are raised for considering their symbioses in the breeding of future crop varieties. However, it is unclear how consistently plant-mediated selection, a potential target in crop breeding, influences microbiome members compared to selection imposed by the agricultural environment. Landraces may have traits shaping their microbiome, which were lost during the breeding of modern varieties, but knowledge about this is scarce. We investigated prokaryotic community composition along the radial root axis of two European maize (Zea mays L.) landraces. A sampling gradient included bulk soil, a distal and proximal rhizosphere fraction, and the root compartment. Our study was replicated at two field locations with differing edaphic and climatic conditions. Further, we tested for differences between two plant developmental stages and two precipitation treatments. Community data were generated by metabarcoding of the V4 SSU rRNA region. While communities were generally distinct between field sites, the effects of landrace variety, developmental stage, and precipitation treatment were comparatively weak and not statistically significant. Under all conditions, patterns in community composition corresponded strongly to the distance to the root. Changes in a- and b-diversity, as well as abundance shifts of many taxa along this gradient, were similar for both landraces and field locations. Most affected taxa belonged to a core microbiome present in all investigated samples. Remarkably, we observed consistent enrichment of Actinobacteriota (particularly Streptomyces, Lechevalieria) and Pseudomonadota (particularly Sphingobium) toward the root. Further, we report a depletion of ammoniaoxidizers along this axis at both field sites. We identified clear enrichment and depletion patterns in microbiome composition along the radial root axis of Z. mays. Many of these were consistent across two distinct field locations, plant developmental stages, precipitation treatments, and for both landraces. This suggests a considerable influence of plant-mediated effects on the microbiome. We propose that the affected taxa have key roles in the rhizosphere and root microbiome of Z. mays. Understanding the functions of these taxa appears highly relevant for the development of methods aiming to promote microbiome services for crops. [ABSTRACT FROM AUTHOR]

A holistic understanding of plant strategies to acquire soil resources is pivotal in achieving sustainable food security. However, we lack knowledge about variety‐specific root and rhizosphere traits for resource acquisition, their plasticity and adaptation to drought. We conducted a greenhouse experiment to phenotype root and rhizosphere traits (mean root diameter [Root D], specific root length [SRL], root tissue density, root nitrogen content, specific rhizosheath mass [SRM], arbuscular mycorrhizal fungi [AMF] colonization) of 16 landraces and 22 modern cultivars of temperate maize (Zea mays L.). Our results demonstrate that landraces and modern cultivars diverge in their root and rhizosphere traits. Although landraces follow a 'do‐it‐yourself' strategy with high SRLs, modern cultivars exhibit an 'outsourcing' strategy with increased mean Root Ds and a tendency towards increased root colonization by AMF. We further identified that SRM indicates an 'outsourcing' strategy. Additionally, landraces were more drought‐responsive compared to modern cultivars based on multitrait response indices. We suggest that breeding leads to distinct resource acquisition strategies between temperate maize varieties. Future breeding efforts should increasingly target root and rhizosphere economics, with SRM serving as a valuable proxy for identifying varieties employing an outsourcing resource acquisition strategy. Summary statement: This study provides novel insights into the diversity of soil resource acquisition strategies within a single plant species. By comparing landraces and modern cultivars of maize and considering plant‐soil‐microbe interactions, we reveal functional differences in root and rhizosphere economics and trait plasticity that shed light on their ecological role and breeding relevance. [ABSTRACT FROM AUTHOR]

Cable bacteria are living electric wires, exerting remarkable effects on their surroundings. Despite still refusing pure culture isolation, their biological conductivity and their impact on aquatic sediments give rise to interesting application prospects in environmental and bio-engineering, including bioremediation, the mitigation of greenhouse gas emissions, and bioelectronics. Here, we summarize how research is currently striving to realize the very versatile application potential of these fascinating bacteria. [ABSTRACT FROM AUTHOR]

Background and Aims Stomatal regulation allows plants to promptly respond to water stress. However, our understanding of the impact of above and belowground hydraulic traits on stomatal regulation remains incomplete. The objective of this study was to investigate how key plant hydraulic traits impact transpiration of maize during soil drying. We hypothesize that the stomatal response to soil drying is related to a loss in soil hydraulic conductivity at the root–soil interface, which in turn depends on plant hydraulic traits. Methods We investigate the response of 48 contrasting maize (Zea mays) genotypes to soil drying, utilizing a novel phenotyping facility. In this context, we measure the relationship between leaf water potential, soil water potential, soil water content and transpiration, as well as root, rhizosphere and aboveground plant traits. Key Results Genotypes differed in their responsiveness to soil drying. The critical soil water potential at which plants started decreasing transpiration was related to a combination of above and belowground traits: genotypes with a higher maximum transpiration and plant hydraulic conductance as well as a smaller root and rhizosphere system closed stomata at less negative soil water potentials. Conclusions Our results demonstrate the importance of belowground hydraulics for stomatal regulation and hence drought responsiveness during soil drying. Furthermore, this finding supports the hypothesis that stomata start to close when soil hydraulic conductivity drops at the root–soil interface. [ABSTRACT FROM AUTHOR]

Cable bacteria (CB) perform electrogenic sulfur oxidation (e-SOx) by spatially separating redox half reactions over centimetre distances. For freshwater systems, the ecology of CB is not yet well understood, partly because they proved difficult to cultivate. This study introduces a new 'agar pillar' approach to selectively enrich and investigate CB populations. Within sediment columns, a central agar pillar is embedded, providing a sediment-free gradient system in equilibrium with the surrounding sediment. We incubated freshwater sediments from a streambed, a sulfidic lake and a hydrocarbon-polluted aquifer in such agar pillar columns. Microprofiling revealed typical patterns of e-SOx, such as the development of a suboxic zone and the establishment of electric potentials. The bacterial communities in the sediments and agar pillars were analysed over depth by PacBio near-full-length 16S rRNA gene amplicon sequencing, allowing for a precise phylogenetic placement of taxa detected. The selective niche of the agar pillar was preferentially colonized by CB related to Candidatus Electronema for surface water sediments, including several potentially novel species, but not for putative groundwater CB affiliated with Desulfurivibrio spp. The presence of CB was seemingly linked to co-enriched fermenters, hinting at a possible role of e-SOx populations as an electron sink for heterotrophic microbes. These findings add to our current understanding of the diversity and ecology of CB in freshwater systems, and to a discrimination of CB from surface and groundwater sediments. The agar pillar approach provides a new strategy that may facilitate the cultivation of redox gradient-dependent microorganisms, including previously unrecognized CB populations. [ABSTRACT FROM AUTHOR]

Sulfate-reducing microorganisms (SRMs) often compete with methanogens for common substrates. Due to thermodynamic reasons, SRMs should outcompete methanogens in the presence of sulfate. However, many studies have documented coexistence of these microbial groups in natural environments, suggesting that thermodynamics alone cannot explain the interactions among them. In this study, we investigated how SRMs compete with the established methanogenic communities in sediment from a long-term, electron acceptor-depleted, asphalt-exposed ecosystem and how they affect the composition of the organic material. We hypothesized that, upon addition of sulfate, SRMs (i) outcompete the methanogenic communities and (ii) markedly contribute to transformations of the organic material. We sampled sediments from the test and proximate control sites under anoxic conditions and incubated them in seawater medium with or without sulfate. Abundance and activity pattern of SRMs and methanogens, as well as the total prokaryotic community, were followed for 6 weeks by using qPCR targeting selected marker genes. Some of these genes were also subjected to amplicon sequencing to assess potential shifts in diversity patterns. Alterations of the organic material in the microcosms were determined by mass spectrometry. Our results indicate that the competition of SRMs with methanogens upon sulfate addition strongly depends on the environment studied and the starting microbiome composition. In the asphalt-free sediments (control), the availability of easily degradable organic material (mainly plant-derived) allows SRMs to use a larger variety of substrates, reducing interspecies competition with methanogens. In contrast, the abundant presence of recalcitrant compounds in the asphalt-exposed sediment was associated with a strong competition between SRMs and methanogens, ultimately detrimental for the latter. Our data underpin the importance of the quality of bioavailable organic materials in anoxic environments as a driver for microbial community structure and function. [ABSTRACT FROM AUTHOR]

Microorganisms are essential in the degradation of environmental pollutants. Aromatic hydrocarbons, e.g., benzene, toluene, ethylbenzene, and xylene (BTEX), are common aquifer contaminants, whose degradation in situ is often limited by the availability of electron acceptors. It is clear that different electron acceptors such as nitrate, iron, or sulfate support the activity of distinct degraders. However, this has not been demonstrated for the availability of nitrate vs. nitrite, both of which can be respired in reductive nitrogen cycling. Here via DNA-stable isotope probing, we report that nitrate and nitrite provided as electron acceptors in different concentrations and ratios not only modulated the microbial communities responsible for toluene degradation but also influenced how nitrate reduction proceeded. Zoogloeaceae members, mainly Azoarcus spp., were the key toluene degraders with nitrate-only, or both nitrate and nitrite as electron acceptors. In addition, a shift within Azoarcus degrader populations was observed on the amplicon sequence variant (ASV) level depending on electron acceptor ratios. In contrast, members of the Sphingomonadales were likely the most active toluene degraders when only nitrite was provided. Nitrate reduction did not proceed beyond nitrite in the nitrate-only treatment, while it continued when nitrite was initially also present in the microcosms. Likely, this was attributed to the fact that different microbial communities were stimulated and active in different microcosms. Together, these findings demonstrate that the availability of nitrate and nitrite can define degrader community selection and N-reduction outcomes. It also implies that nitrate usage efficiency in bioremediation could possibly be enhanced by an initial co-supply of nitrite, via modulating the active degrader communities. [ABSTRACT FROM AUTHOR]

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 N2 and O2. 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 × 106–3.2 × 107 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. [ABSTRACT FROM AUTHOR]

Zoogloea oleivorans, capable of using toluene as a sole source of carbon and energy, was earlier found to be an active degrader under microaerobic conditions in aquifer samples. To uncover the genetic background of the ability of microaerobic toluene degradation in Z. oleivorans, the whole-genome sequence of the type strain BucT was revealed. Metatranscriptomic sequence reads, originated from a previous SIP study on microaerobic toluene degradation, were mapped on the genome. The genome (5.68 Mb) had a mean G + C content of 62.5%, 5005 protein coding gene sequences and 80 RNA genes. Annotation predicted that 66 genes were involved in the metabolism of aromatic compounds. Genome analysis revealed the presence of a cluster with genes coding for a multicomponent phenol-hydroxylase system and a complete catechol meta-cleavage pathway. Another cluster flanked by mobile-element protein coding genes coded a partial catechol meta-cleavage pathway including a subfamily I.2.C-type extradiol dioxygenase. Analysis of metatranscriptomic data of a microaerobic toluene-degrading enrichment, containing Z. oleivorans as an active-toluene degrader revealed that a toluene dioxygenase-like enzyme was responsible for the ring-hydroxylation, while enzymes of the partial catechol meta-cleavage pathway coding cluster were responsible for further degradation of the aromatic ring under microaerobic conditions. This further advances our understanding of aromatic hydrocarbon degradation between fully oxic and strictly anoxic conditions. [ABSTRACT FROM AUTHOR]

Oxygenic denitrification represents a new route in reductive nitrogen turnover which differs from canonical denitrification in how nitric oxide (NO) is transformed into dinitrogen gas. Instead of NO reduction via N2O to N2, NO is proposed to be directly disproportionated into N2 and O2 in oxygenic denitrification, catalyzed by the putative NO dismutase (Nod). Although a high diversity of nod genes has been recovered from various environments, still little is known about the niche partitioning and ecophysiology of oxygenic denitrifiers. One constraint is that nod as a functional marker for oxygenic denitrifiers is not well established. To address this issue, we compared the diversity and phylogeny of nod , 16S rRNA and pmoA gene sequences of four NC10 enrichments that are capable of methane-driven oxygenic denitrification and one environmental sample. The phylogenies of nod , 16S rRNA and pmoA genes of these cultures were generally congruent. The diversity of NC10 bacteria inferred from different genes was also similar in each sample. A new set of NC10-specific nod primers was developed and used in qPCR. The abundance of NC10 bacteria inferred from nod genes was constantly lower than via 16S rRNA genes, but the difference was within one order of magnitude. These results suggest that nod is a suitable molecular marker for studying the diversity and phylogeny of methane-driven oxygenic denitrifiers, the further investigation of which may be of value to develop enhanced strategies for sustainable nitrogen or methane removal. [ABSTRACT FROM AUTHOR]

While most studies using RNA-stable isotope probing (SIP) to date have focused on ribosomal RNA, the detection of 13C-labeled mRNA has rarely been demonstrated. This approach could alleviate some of the major caveats of current non-target environmental "omics." Here, we demonstrate the feasibility of total RNA-SIP in an experiment where hydrocarbon-degrading microbes from a BTEX-contaminated aquifer were studied in microcosms with 13C-labeled toluene under microoxic conditions. From the total sequencing reads (∼30 mio. reads per density-resolved RNA fraction), an average of 1.2% of reads per sample were identified as non-rRNA, including mRNA. Members of the Rhodocyclaceae (including those related to Quatrionicoccus spp.) were most abundant and enriched in 13C-rRNA, while well-known aerobic degraders such as Pseudomonas spp. remained unlabeled. Transcripts related to cell motility, secondary metabolite formation and xenobiotics degradation were highly labeled with 13C. mRNA of phenol hydroxylase genes were highly labeled and abundant, while other transcripts of toluene-activation were not detected. Clear labeling of catechol 2,3-dioxygenase transcripts supported previous findings that some of these extradiol dioxygenases were adapted to low oxygen concentrations. We introduce a novel combination of total RNA-SIP with calculation of transcript-specific enrichment factors (EFs) in 13C-RNA, enabling a targeted approach to process-relevant gene expression in complex microbiomes. [ABSTRACT FROM AUTHOR]

The availability of oxygen is often a limiting factor for the degradation of aromatic hydrocarbons in subsurface environments. However, while both aerobic and anaerobic degraders have been intensively studied, degradation betwixt, under micro- or hypoxic conditions has rarely been addressed. It is speculated that in environments with limited, but sustained oxygen supply, such as in the vicinity of groundwater monitoring wells, hypoxic degradation may take place. A large diversity of subfamily I.2.C extradiol dioxygenase genes has been previously detected in a BTEX-contaminated aquifer in Hungary. Older literature suggests that such catabolic potentials could be associated to hypoxic degradation. Bacterial communities dominated by members of the Rhodocyclaceae were found, but the majority of the detected C23O genotypes could not be affiliated to any known bacterial degrader lineages. To address this, a stable isotope probing (SIP) incubation of site sediments with 13C7-toluene was performed under microoxic conditions. A combination of 16S rRNA gene amplicon sequencing and T-RFLP fingerprinting of C23O genes from SIP gradient fractions revealed the central role of degraders within the Rhodocyclaceae in hypoxic toluene degradation. The main assimilators of 13C were identified as members of the genera Quatrionicoccus and Zoogloea, and a yet uncultured group of the Rhodocyclaceae. [ABSTRACT FROM AUTHOR]

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, 13C-CO2 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 13C enrichment pattern across abundant operational taxonomic units (OTUs). Whereas, OTUs linked to Proteobacteria were enriched in 13C mainly in the topsoil, in both subsoil layers OTUs related to Cohnella, Paenibacillus, Flavobacterium showed a clear 13C signal, indicating an important, so far overseen role of Firmicutes and Bacteriodetes in the subsoil rhizosphere. [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]

The degradation of benzene, toluene, ethylbenzene and xylene (BTEX) contaminants in groundwater relies largely on anaerobic processes. While the physiology and biochemistry of selected relevant microbes have been intensively studied, research has now started to take the generated knowledge back to the field, in order to trace the populations truly responsible for the anaerobic degradation of BTEX hydrocarbons in situ and to unravel their ecology in contaminated aquifers. Here, recent advances in our knowledge of the identity, diversity and ecology of microbes involved in these important ecosystem services are discussed. At several sites, distinct lineages within the Desulfobulbaceae, the Rhodocyclaceae and the Gram-positive Peptococcaceae have been shown to dominate the degradation of different BTEX hydrocarbons. Especially for the functional guild of anaerobic toluene degraders, specific molecular detection systems have been developed, allowing researchers to trace their diversity and distribution in contaminated aquifers. Their populations appear enriched in hot spots of biodegradation in situ. 13C-labelling experiments have revealed unexpected pathways of carbon sharing and obligate syntrophic interactions to be relevant in degradation. Together with feedback mechanisms between abiotic and biotic habitat components, this promotes an enhanced ecological perspective of the anaerobic degradation of BTEX hydrocarbons, as well as its incorporation into updated concepts for site monitoring and bioremediation. [ABSTRACT FROM AUTHOR]

Background and aims: Forest ecosystems may act as sinks for or source of atmospheric CO. While inorganic nitrogen (N) fertilization increases aboveground tree biomass, the effects on soil and rhizosphere microorganisms are less clear, indicating potentially unpredictable changes in nutrient cycling processes maintaining ecosystem functioning. Although plant-derived carbon (C) is the main C source in soils during the vegetation period, information on the response of rhizosphere bacteria assimilating rhizodeposits to increased soil N availability mainly for trees is missing. Methods: We performed a greenhouse experiment with C-CO labelled young beech seedlings grown under different N fertilization levels. DNA Stable Isotope Probing (DNA-SIP) in combination with TRFLP and pyrosequencing enabled us to identify bacteria assimilating plant-derived C and to assess the main responders phylogenetically. Results: Although above- and belowground allocation of recently fixed photosynthates remained unchanged, microbial rhizosphere community composition was clearly affected by fertilization. Besides, we found lower C incorporation into microbial biomass in fertilized soil. Moreover, it could be shown that only a small subset of the rhizosphere microbiome incorporated recently fixed C into its DNA, dominated by Proteobacteria (Alpha- and Betaproteobacteria) and Actinobacteria (Actinomycetales). Conclusions: Our results suggest that N fertilization may change both the diversity of bacterial communities using rhizodeposits and assimilation rates of recently fixed photosynthates. Given the close interaction of beneficial and/or deleterious microbes and plants in the rhizosphere, this could potentially have positive or negative implications for plant performance on long-term. [ABSTRACT FROM AUTHOR]

The flow of plant-derived carbon in soil is a key component of global carbon cycling. Conceptual models of trophic carbon fluxes in soil have assumed separate bacterial and fungal energy channels in the detritusphere, controlled by both substrate complexity and recalcitrance. However, detailed understanding of the key populations involved and niche-partitioning between them is limited. Here, a microcosm experiment was performed to trace the flow of detritusphere C from substrate analogs (glucose, cellulose) and plant biomass amendments (maize leaves, roots) in an agricultural soil. Carbon flow was traced by rRNA stable isotope probing and amplicon sequencing across three microbial kingdoms. Distinct lineages within the Actinobacteria, Bacteroidetes, Gammaproteobacteria, Basidiomycota, Ascomycota as well as Peronosporomycetes were identified as important primary substrate consumers. A dynamic succession of primary consumers was observed especially in the cellulose treatments, but also in plant amendments over time. While intra-kingdom niche partitioning was clearly observed, distinct bacterial and fungal energy channels were not apparent. Furthermore, while the diversity of primary substrate consumers did not notably increase with substrate complexity, consumer succession and secondary trophic links to bacterivorous and fungivorous microbes resulted in increased food web complexity in the more recalcitrant substrates. This suggests that rather than substrate-defined energy channels, consumer succession as well as intra- and inter-kingdom cross-feeding should be considered as mechanisms supporting food web complexity in the detritusphere. [ABSTRACT FROM AUTHOR]

Stable isotope probing (SIP) techniques have become stateof- the-art in microbial ecology over the last 10 years, allowing for the targeted detection and identification of organisms, metabolic pathways and elemental fluxes active in specific processes within complex microbial communities. For studying anaerobic hydrocarbon-degrading microbial communities, four stable isotope techniques have been used so far: DNA/RNA-SIP, PLFA (phospholipid-derived fatty acids)-SIP, protein-SIP, and single-cell-SIP by nanoSIMS (nanoscale secondary ion mass spectrometry) or confocal Raman microscopy. DNA/RNA-SIP techniques are most frequently applied due to their most meaningful phylogenetic resolution. Especially using 13 C-labeled benzene and toluene as model substrates, many new hydrocarbon degraders have been identified by SIP under various electron acceptor conditions. This has extended the current perspective of the true diversity of anaerobic hydrocarbon degraders relevant in the environment. Syntrophic hydrocarbon degradation was found to be a common mechanism for various electron acceptors. Fundamental concepts and recent advances in SIP are reflected here. A discussion is presented concerning how these techniques generate direct insights into intrinsic hydrocarbon degrader populations in environmental systems and how useful they are for more integrated approaches in the monitoring of contaminated sites and for bioremediation. [ABSTRACT FROM AUTHOR]

Anaerobic degradation is a key process in many environments either naturally or anthropogenically exposed to petroleum hydrocarbons. Considerable advances into the biochemistry and physiology of selected anaerobic degraders have been achieved over the last decades, especially for the degradation of aromatic hydrocarbons. However, researchers have only recently begun to explore the ecology of complex anaerobic hydrocarbon degrader communities directly in their natural habitats, as well as in complex laboratory systems using tools of molecular biology. These approaches have mainly been facilitated by the establishment of a suite of targeted marker gene assays, allowing for rapid and directed insights into the diversity as well as the identity of intrinsic degrader populations and degradation potentials established at hydrocarbon-impacted sites. These are based on genes encoding either peripheral or central key enzymes in aromatic compound breakdown, such as fumarate-adding benzylsuccinate synthases or dearomatizing aryl-coenzyme A reductases, or on aromatic ring-cleaving hydrolases. Here, we review recent advances in this field, explain the different detection methodologies applied, and discuss how the detection of site-specific catabolic gene markers has improved the understanding of processes at contaminated sites. Functional marker gene-based strategies may be vital for the development of a more elaborate population-based assessment and prediction of aromatic degradation potentials in hydrocarbon-impacted environments. [ABSTRACT FROM AUTHOR]

Hydrocarbons are abundant in anoxic environments and pose biochemical challenges to their anaerobic degradation by microorganisms. Within the framework of the Priority Program 1319, investigations funded by the Deutsche Forschungsgemeinschaft on the anaerobic microbial degradation of hydrocarbons ranged from isolation and enrichment of hitherto unknown hydrocarbon-degrading anaerobic microorganisms, discovery of novel reactions, detailed studies of enzyme mechanisms and structures to process-oriented in situ studies. Selected highlights from this program are collected in this synopsis, with more detailed information provided by theme-focused reviews of the special topic issue on 'Anaerobic biodegradation of hydrocarbons' [this issue, pp. 1-244]. The interdisciplinary character of the program, involving microbiologists, biochemists, organic chemists and environmental scientists, is best exemplified by the studies on alkyl-/arylalkylsuccinate synthases. Here, research topics ranged from in-depth mechanistic studies of archetypical toluene-activating benzylsuccinate synthase, substrate-specific phylogenetic clustering of alkyl-/arylalkylsuccinate synthases (toluene plus xylenes, p -cymene, p -cresol, 2-methylnaphthalene, n -alkanes), stereochemical and co-metabolic insights into n -alkane-activating (methylalkyl)succinate synthases to the discovery of bacterial groups previously unknown to possess alkyl-/arylalkylsuccinate synthases by means of functional gene markers and in situ field studies enabled by state-of-the-art stable isotope probing and fractionation approaches. Other topics are Mo-cofactor-dependent dehydrogenases performing O 2 -independent hydroxylation of hydrocarbons and alkyl side chains (ethylbenzene, p -cymene, cholesterol, n -hexadecane), degradation of p -alkylated benzoates and toluenes, glycyl radical-bearing 4-hydroxyphenylacetate decarboxylase, novel types of carboxylation reactions (for acetophenone, acetone, and potentially also benzene and naphthalene), W-cofactor-containing enzymes for reductive dearomatization of benzoyl-CoA (class II benzoyl-CoA reductase) in obligate anaerobes and addition of water to acetylene, fermentative formation of cyclohexanecarboxylate from benzoate, and methanogenic degradation of hydrocarbons. [ABSTRACT FROM AUTHOR]

Slow sand filtration (SSF) is an effective low-tech water treatment method for pathogen and particle removal. Yet despite its application for centuries, it has been uncertain to which extent pathogenic microbes are removed by mechanical filtration or due to ecological interactions such as grazing and competition for nutrients. In this study, we quantified the removal of bacterial faecal indicators, Escherichia coli and Enterococcus faecalis, from secondary effluent of a wastewater treatment plant and analysed the microbial community composition in compartments of laboratory model SSF columns. The columns were packed with different sand grain sizes and eliminated 1.6-2.3 log units of faecal indicators, which translated into effluents of bathing water quality according to the EU directive (<500 colony forming units of E. coli per 100 ml) for columns with small grain size. Most of that removal occurred in the upper filter area, the Schmutzdecke. Within that same zone, total bacterial numbers increased however, thus suggesting a specific elimination of the faecal indicators. The analysis of the microbial communities also revealed that some taxa were removed more from the wastewater than others. These results accentuate the contribution of biological mechanisms to water purification in SSF. [ABSTRACT FROM AUTHOR]

The use of colloidal iron oxide (FeOx) in the bioremediation of groundwater contamination implies its increasing release into the environment and requires an assessment of its ecotoxicological risk. Therefore, microcosm experiments were carried out to investigate the impact of ferrihydrite colloids on the bacterial and meiofaunal communities of pristine freshwater sediments. The effects of ferrihydrite colloids were compared with those of ferrihydrite macroaggregates to discriminate between colloid-specific and general FeOx impacts. The influence of ferrihydrite colloids on the toxicity of sediment-bound fluoranthene was also considered. At high concentrations (496 mg Fe kg−1 sediment dry wt), ferrihydrite colloids had a significant, but transient impact on bacterial and meiofaunal communities. Although bacterial community composition specifically responded to ferrihydrite colloids, a more general FeOx effect was observed for meiofauna. Bacterial activity responded most sensitively (already at 55 mg Fe kg−1 dry wt) without the potential of recovery. Ferrihydrite colloids did not influence the toxicity of sediment-bound fluoranthene. Significant correlations between bacterial activity and meiofaunal abundances were indicative of trophic interactions between bacteria and meiofauna and therefore of the contribution of indirect food web effects to the observed impacts. The results suggest that the application of ferrihydrite colloids for remediation purposes in the field poses no risk for benthic communities, given that, with the exception of generic bacterial activity, any negative effects on communities were reversible. Environ Toxicol Chem 2015;34:2660-2669. © 2015 SETAC [ABSTRACT FROM AUTHOR]

The emerging interest in using stable bedrock formations for industrial purposes, e.g., nuclear waste disposal, has increased the need for understanding microbiological and geochemical processes in deep crystalline rock environments, including the carbon cycle. Considering the origin and evolution of life on Earth, these environments may also serve as windows to the past. Various geological, chemical, and biological processes can influence the deep carbon cycle. Conditions of CH4 formation, available substrates and time scales can be drastically different from surface environments. This paper reviews the origin, source, and cycling of methane in deep terrestrial crystalline bedrock with an emphasis on microbiology. In addition to potential formation pathways of CH4, microbial consumption of CH4 is also discussed. Recent studies on the origin of CH4 in continental bedrock environments have shown that the traditional separation of biotic and abiotic CH4 by the isotopic composition can be misleading in substrate-limited environments, such as the deep crystalline bedrock. Despite of similarities between Precambrian continental sites in Fennoscandia, South Africa and North America, where deep methane cycling has been studied, common physicochemical properties which could explain the variation in the amount of CH4 and presence or absence of CH4 cycling microbes were not found. However, based on their preferred carbon metabolism, methanogenic microbes appeared to have similar spatial distribution among the different sites. [ABSTRACT FROM AUTHOR]

Biogeochemical and microbiological data indicate that the anaerobic oxidation of non-methane hydrocarbons by sulfate-reducing bacteria (SRB) has an important role in carbon and sulfur cycling at marine seeps. Yet, little is known about the bacterial hydrocarbon degraders active in situ. Here, we provide the link between previous biogeochemical measurements and the cultivation of degraders by direct identification of SRB responsible for butane and dodecane degradation in complex on-site microbiota. Two contrasting seep sediments from Mediterranean Amon mud volcano and Guaymas Basin (Gulf of California) were incubated with 13C-labeled butane or dodecane under sulfate-reducing conditions and analyzed via complementary stable isotope probing (SIP) techniques. Using DNA- and rRNA-SIP, we identified four specialized clades of alkane oxidizers within Desulfobacteraceae to be distinctively active in oxidation of short- and long-chain alkanes. All clades belong to the Desulfosarcina/Desulfococcus (DSS) clade, substantiating the crucial role of these bacteria in anaerobic hydrocarbon degradation at marine seeps. The identification of key enzymes of anaerobic alkane degradation, subsequent β-oxidation and the reverse Wood-Ljungdahl pathway for complete substrate oxidation by protein-SIP further corroborated the importance of the DSS clade and indicated that biochemical pathways, analog to those discovered in the laboratory, are of great relevance for natural settings. The high diversity within identified subclades together with their capability to initiate alkane degradation and growth within days to weeks after substrate amendment suggest an overlooked potential of marine benthic microbiota to react to natural changes in seepage, as well as to massive hydrocarbon input, for example, as encountered during anthropogenic oil spills. [ABSTRACT FROM AUTHOR]

Hydrocarbon contaminants in groundwater can be degraded by microbes under different redox settings, forming hot spots of degradation especially at the fringes of contaminant plumes. At a tar-oil-contaminated aquifer in Germany, it was previously shown that the distribution of anaerobic toluene degraders as traced via catabolic and ribosomal marker genes is highly correlated to zones of increased anaerobic degradation at the lower fringe of the plume. Here, we trace the respective distribution of aerobic toluene degraders over a fine-scale depth transect of sediments taken at the upper fringe of the plume and below, based on the analysis of 16 S rRNA genes as well as catabolic markers in intervals of 3-10 cm. Well-defined small-scale distribution maxima of typical aerobic degrader lineages within the Pseudomonadaceae, Comamonadaceae and Burkholderiaceae are revealed over the redox gradient. An unexpected maximal abundance of 9.2 × 106 toluene monooxygenase ( tmoA) genes per g of sediment was detected in the strongly reduced plume core, and gene counts did not increase towards the more oxidized upper plume fringe. This may point towards unusual ecological controls of these yet unidentified aerobic degraders, and indicates that competitive niche partitioning between aerobic and anaerobic hydrocarbon degraders in the field is not yet fully understood. These findings demonstrate the potential of catabolic marker gene assays in elaborating the ecology of contaminant plumes, which is a prerequisite for developing integrated monitoring strategies for natural attenuation. [ABSTRACT FROM AUTHOR]

The characterization of microbial community structure via 16S rRNA gene profiling has been greatly advanced in recent years by the introduction of amplicon pyrosequencing. The possibility of barcoding gives the opportunity to massively screen multiple samples from environmental or clinical sources for community details. However, an on-going debate questions the reproducibility and semi-quantitative rigour of pyrotag sequencing, similar to the early days of community fingerprinting. In this study we demonstrate the reproducibility of bacterial 454 pyrotag sequencing over biological and technical replicates of aquifer sediment bacterial communities. Moreover, we explore the potential of recovering specific template ratios via quantitatively defined template spiking to environmental DNA. We sequenced pyrotag libraries of triplicate sediment samples taken in annual sampling campaigns at a tar oil contaminated aquifer in Düsseldorf, Germany. The abundance of dominating lineages was highly reproducible with a maximal standard deviation of ∼4% read abundance across biological, and ∼2% across technical replicates. Our workflow also allows for the linking of read abundances within defined assembled pyrotag contigs to that of specific 'in vivo' fingerprinting signatures. Thus we demonstrate that both terminal restriction fragment length polymorphism (T-RFLP) analysis and pyrotag sequencing are capable of recovering highly comparable community structure. Overall diversity was roughly double in amplicon sequencing. Pyrotag libraries were also capable of linearly recovering increasing ratios (up to 20%) of 16S rRNA gene amendments from a pure culture of Aliivibrio fisheri spiked to sediment DNA. Our study demonstrates that 454 pyrotag sequencing is a robust and reproducible method, capable of reliably recovering template abundances and overall community structure within natural microbial communities. [ABSTRACT FROM AUTHOR]

A novel approach was developed to follow the successive utilization of organic carbon under anoxic conditions by microcalorimetry, chemical analyses of fermentation products and stable-isotope probing ( SIP). The fermentation of 13 C-labeled glucose was monitored over 4 weeks by microcalorimetry in a stimulation experiment with tidal-flat sediments. Based on characteristic heat production phases, time points were selected for quantifying fermentation products and identifying substrate-assimilating bacteria by the isolation of intact ribosomes prior to rRNA- SIP. The preisolation of ribosomes resulted in rRNA with an excellent quality. Glucose was completely consumed within 2 days and was mainly fermented to acetate. Ethanol, formate, and hydrogen were detected intermittently. The amount of propionate that was built within the first 3 days stayed constant. Ribosome-based SIP of fully labeled and unlabeled rRNA was used for fingerprinting the glucose-degrading species and the inactive background community. The most abundant actively degrading bacterium was related to Psychromonas macrocephali (similarity 99%) as identified by DGGE and sequencing. The disappearance of Desulfovibrio-related bands in labeled rRNA after 3 days indicated that this group was active during the first degradation phase only. In summary, ribosome-based SIP in combination with microcalorimetry allows dissecting distinct phases in substrate turnover in a very sensitive manner. [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]

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Global groundwater resources are constantly challenged by a multitude of contaminants such as aromatic hydrocarbons. Especially in anaerobic habitats, a large diversity of unrecognized microbial populations may be responsible for their degradation. Still, our present understanding of the respective microbiota and their ecophysiology is almost exclusively based on a small number of cultured organisms, mostly within the Proteobacteria. Here, by DNA-based stable isotope probing (SIP), we directly identified the most active sulfate-reducing toluene degraders in a diverse sedimentary microbial community originating from a tar-oil-contaminated aquifer at a former coal gasification plant. On incubation of fresh sediments with 13C7-toluene, the production of both sulfide and 13CO2 was clearly coupled to the 13C-labeling of DNA of microbes related to Desulfosporosinus spp. within the Peptococcaceae (Clostridia). The screening of labeled DNA fractions also suggested a novel benzylsuccinate synthase alpha-subunit (bssA) sequence type previously only detected in the environment to be tentatively affiliated with these degraders. However, carbon flow from the contaminant into degrader DNA was only ∼50%, pointing toward high ratios of heterotrophic CO2-fixation during assimilation of acetyl-CoA originating from the contaminant by these degraders. These findings demonstrate that the importance of non-proteobacterial populations in anaerobic aromatics degradation, as well as their specific ecophysiology in the subsurface may still be largely ungrasped. [ABSTRACT FROM AUTHOR]

High-resolution depth-resolved monitoring was applied to groundwater and sediments samples in a tar oil contaminated aquifer. Today, it is not fully clear, whether groundwater-based lines of evidence are always sufficient to adequately assess natural attenuation (NA) potentials and processes going on in situ. Our data unveiled small-scale heterogeneities, steep physical-chemical and microbial gradients, as well as hot spots of contaminants and biodegradation in the supposedly homogeneous sandy aquifer. The comparison of basic geochemical data revealed a fairly good agreement between sediment and groundwater samples. Nevertheless, a comprehensive understanding of both BTEX and PAH distribution, as well as redox processes involving insoluble electron acceptors, i.e., iron reduction, clearly asks for consideration of both, sediment and groundwater analysis. A thin BTEX plume right below the groundwater table was visible only in groundwater, while significant amounts of PAHs were present in sediments from deeper zones of the aquifer. Indications for sulfate reduction as a dominant process involved in BTEX degradation were largely obtained from groundwater, while the role of iron reduction in degradation and possible sulfide oxidation at the capillary fringe and the upper BTEX plume fringe, as well as in deeper PAH-contaminated zones was evident from sediments. Moreover, sediment analyses were essential to meaningfully recover cell abundances, distribution, activity and composition of the bacterial community. Sediments harbored > 97.7% of bacterial cells and displayed enzyme activities 5 to 6 orders of magnitude higher than groundwater samples. Bacterial community T-RFLP fingerprints revealed important distinctions, but also similarities in depth-resolved microbial community distribution in sediments and water. An apparently highly specialized degrader population was found to dominate the lower BTEX plume fringe. However, even though sediment data seemed to comprise most community information found also in groundwater, this relation did not apply vice versa. In summary, our results show that groundwater sampled at an appropriate scale may contain sufficient information to identify and localize dominant redox reactions, but clearly fails to unravel natural attenuation potentials. This clearly emphasizes the importance of both groundwater and sediment samples for truly assessing natural attenuation potentials and activities at organically contaminated aquifers. [ABSTRACT FROM AUTHOR]

The flow of carbon under sulfate-reducing conditions within a benzene-mineralizing enrichment culture was analysed using fully labelled [13C6]-benzene. Over 180 days of incubation, 95% of added 13C-benzene was released as 13C-carbon dioxide. DNA extracted from cultures that had degraded different amounts of unlabelled or 13C-labelled benzene was centrifuged in CsCl density gradients to identify 13C-benzene-assimilating organisms by density-resolved terminal restriction fragment length polymorphism analysis and cloning of 16S rRNA gene fragments. Two phylotypes showed significantly increased relative abundance of their terminal restriction fragments in ‘heavy’ fractions of 13C-benzene-incubated microcosms compared with a 12C-benzene-incubated control: a member of the Cryptanaerobacter/ Pelotomaculum group within the Peptococcaceae, and a phylotype belonging to the Epsilonproteobacteria. The Cryptanaerobacter/Pelotomaculum phylotype was the most frequent sequence type. A small amount of 13C-methane was aceticlastically produced, as concluded from the linear relationship between methane production and benzene degradation and the detection of Methanosaetaceae as the only methanogens present. Other phylotypes detected but not 13C-labelled belong to several genera of sulfate-reducing bacteria, that may act as hydrogen scavengers for benzene oxidation. Our results strongly support the hypothesis that benzene is mineralized by a consortium consisting of syntrophs, hydrogenotrophic sulfate reducers and to a minor extent of aceticlastic methanogens. [ABSTRACT FROM AUTHOR]

A toluene-degrading microbial consortium was enriched directly in a BTEX-contaminated aquifer under sulfate-reducing conditions using in situ microcosms consisting of toluene-loaded activated carbon pellets. Degradation of toluene and concomitant sulfide production by the consortium was subsequently demonstrated in laboratory microcosms. The consortium was physiologically and phylogenetically characterized by isotope tracer experiments using nonlabeled toluene, [13C]-α-toluene or [13C7]-toluene as growth substrates. Cells incubated with [13C]-α-toluene or [13C7]-toluene incorporated 8–15 at.%13C and 51–57 at.%13C into total lipid fatty acids, respectively, indicating a lower specific incorporation of 13C from [13C7]-toluene. In order to identify the toluene-assimilating bacteria, the incorporation of carbon from both [13C]-α-toluene and [13C7]-toluene into rRNA was analyzed by stable isotope probing. Time and buoyant density-resolved 16S rRNA gene-based terminal restriction fragment length polymorphism profiles, combined with cloning and sequencing, revealed that an uncultured bacterium (99% sequence similarity) related to the genus Desulfocapsa was the main toluene-degrading organism in the consortium. The ratio of the respective terminal restriction fragments changed over time, indicating trophic interactions within this consortium. [ABSTRACT FROM AUTHOR]

The use of groundwater as a carrier of thermal energy is an important source of sustainable heating and cooling. However, the effects of thermal use on geochemical and biological aquifer characteristics are poorly understood. Here, we have assessed the impacts of heat discharge on an uncontaminated, shallow aquifer by monitoring the hydrogeochemical, bacterial and faunal parameters at an active thermal discharge facility. The observed variability between wells was considerable. Yet, no significant temperature impacts on bacterial or faunal abundance and on bacterial productivity were observed. Also, we did not observe an improved survival or growth of coliforms with temperature. In contrast, the diversity of bacterial terminal restriction fragment (T-RF) length polymorphism fingerprints and faunal populations was either positively or negatively affected by temperature, respectively, and the abundance of selected T-RFs was clearly temperature dependent. Canonical correspondence analysis indicated that both the impact of temperature and of surface water from a nearby river, were important drivers of aquifer biotic variability. These results demonstrate that aquifer thermal energy discharge can affect aquifer bacteria and fauna, while at the same time controlling only a minor part of the total seasonal and spatial variability and therefore posing no likely threat to ecosystem functioning and drinking water protection in uncontaminated, shallow aquifers. [ABSTRACT FROM AUTHOR]

• A 13CO2 (99 atom-%, 350 ppm) incubation experiment was performed to identify active bacterial endophytes in two cultivars of Solanum tuberosum, cultivars Desirée and Merkur. We showed that after the assimilation and photosynthetic transformation of 13CO2 into 13C-labeled metabolites by the plant, the most directly active, cultivar-specific heterotrophic endophytic bacteria that consume these labeled metabolites can be identified by DNA stable isotope probing (DNA-SIP). • Density-resolved DNA fractions obtained from SIP were subjected to 16S rRNA gene-based community analysis using terminal restriction fragment length polymorphism analysis and sequencing of generated gene libraries. • Community profiling revealed community compositions that were dominated by plant chloroplast and mitochondrial 16S rRNA genes for the ‘light’ fractions of 13CO2-incubated potato cultivars and of potato cultivars not incubated with 13CO2. In the ‘heavy’ fractions of the 13CO2-incubated endophyte DNA, a bacterial 492-bp terminal restriction fragment became abundant, which could be clearly identified as Acinetobacter and Acidovorax spp. in cultivars Merkur and Desirée, respectively, indicating cultivar-dependent distinctions in 13C-label flow. These two species represent two common potato endophytes with known plant-beneficial activities. • The approach demonstrated the successful detection of active bacterial endophytes in potato. DNA-SIP therefore offers new opportunities for exploring the complex nature of plant–microbe interactions and plant-dependent microbial metabolisms within the endosphere. [ABSTRACT FROM AUTHOR]

Marine pelagic redoxclines are zones of high dark CO2 fixation rates, which can correspond up to 30% of the surface primary production. However, despite this significant contribution to the pelagic carbon cycle, the identity of most chemolithoautotrophic organisms is still unknown. Therefore, the aim of this study was to directly link the dark CO2 fixation capacity of a pelagic redoxcline in the central Baltic Sea (Landsort Deep) with the identity of the main chemolithoautotrophs involved. Our approach was based on the analysis of natural carbon isotope signatures in fatty acid methyl esters (FAMEs) and on measurements of CO2 incorporation in 13C-bicarbonate pulse experiments. The incorporation of 13C into chemolithoautotrophic cells was investigated by rRNA-based stable isotope probing (RNA-SIP) and FAME analysis after incubation for 24 and 72 h under in situ conditions. Our results demonstrated that fatty acids indicative of Proteobacteria were significantly enriched in 13C slightly below the chemocline. RNA-SIP analyses revealed that two different Gammaproteobacteria and three closely related Epsilonproteobacteria of the Sulfurimonas cluster were active dark CO2-fixing microorganisms, with a time-dependent community shift between these groups. Labelling of Archaea was not detectable, but after 72 h of incubation the 13C-label had been transferred to a potentially bacterivorous ciliate related to Euplotes sp. Thus, RNA-SIP provided direct evidence for the contribution of chemolithoautotrophic production to the microbial food web in this marine pelagic redoxcline, emphasizing the importance of dark CO2-fixing Proteobacteria within this habitat. [ABSTRACT FROM AUTHOR]

Here, we present a detailed functional and phylogenetic characterization of an iron-reducing enrichment culture maintained in our lab with benzene as sole carbon and energy source. We used DNA-stable isotope probing to identify microbes within the enrichment most active in the assimilation of 13C-label. When 12C6- and 13C6-benzene were added as comparative substrates, marked differences in the quantitative buoyant density distribution became apparent especially for uncultured microbes within the Gram-positive Peptococcaceae, closely related to environmental clones retrieved from contaminated aquifers world wide and only distantly related to cultured representatives of the genus Thermincola. Prominent among the other constituents of the enrichment were uncultured Deltaproteobacteria, as well as members of the Actinobacteria. Although their presence within the enrichment seems to be stable they did not assimilate 13C-label as significantly as the Clostridia within the time course of our experiment. We hypothesize that benzene degradation in our enrichment involves an unusual syntrophy, where members of the Clostridia primarily oxidize benzene. Electrons from the contaminant are both directly transferred to ferric iron by the primary oxidizers, but also partially shared with the Desulfobulbaceae as syntrophic partners. Alternatively, electrons may also be quantitatively transferred to the partners, which then reduce the ferric iron. Thus our results provide evidence for the importance of a novel clade of Gram-positive iron-reducers in anaerobic benzene degradation, and a role of syntrophic interactions in this process. These findings shed a totally new light on the factors controlling benzene degradation in anaerobic contaminated environments.The ISME Journal (2007) 1, 643–653; doi:10.1038/ismej.2007.73; published online 13 September 2007 [ABSTRACT FROM AUTHOR]

Benzylsuccinate synthase (Bss) is the key enzyme of anaerobic toluene degradation and has been found in all anaerobic toluene degrading bacterial isolates tested. However, only a few pure cultures capable of anaerobic toluene oxidation are available to date, and it is important to understand the relevance of these model organisms for in situ bioremediation of hydrocarbon-contaminated aquifers. Due to their phylogenetic dispersal, it is not possible to specifically target anaerobic toluene degraders using marker rRNA genes. We therefore established an assay targeting a ∼794 bp fragment within the Bss alpha-subunit ( bssA) gene, which allows for the specific detection and affiliation of both known and unknown anaerobic degraders. Three distinct tar-oil-contaminated aquifer sites were screened for intrinsic bssA gene pools in order to identify and compare the diversity of hydrocarbon degraders present at these selected sites. We were able to show that local diversity patterns of degraders were entirely distinct, apparently highly specialized and well-adapted to local biogeochemical settings. Discovered at one of the sites were bssA genes closely related to that of Geobacter spp., which provides evidence for an importance of iron reduction for toluene degradation in these sediments. Retrieved from the other two sites, dominated by sulfate reduction, were previously unidentified bssA genes and also deeply branching putative bssA homologues. We provide evidence for a previously unrecognized diversity of anaerobic toluene degraders and also of other hydrocarbon degraders using fumarate-adding key reactions in contaminated aquifers. These findings enhance our current understanding of intrinsic hydrocarbon-degrading microbial communities in perturbed aquifers and may have potential for the future assessment and prediction of natural attenuation based on degradation genes. [ABSTRACT FROM AUTHOR]

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-13C]acetate we found that archaeal 16S rRNA was 13C-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’13C-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 13C-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 13C-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. [ABSTRACT FROM AUTHOR]

Detailed studies on the relation of structure and function of microbial communities in a sediment depth profile scarcely exist. We determined as functional aspect the vertical distribution of the acetotrophic and hydrogenotrophic CH4 production activity by measuring production rates and stable 13C/12C-isotopic signatures of CH4 in the profundal sediment of Lake Dagow. The structural aspect was determined by the composition of the methanogenic community by quantifying the abundance of different archaeal groups using ‘real-time’ polymerase chain reaction and analysis of terminal restriction fragment length polymorphism (T-RFLP). Methane production rates in the surface sediment (0–3 cm depth) were higher in August than in May, but strongly decreased with depth (down to 20 cm). The δ13C of the produced CH4 and CO2 indicated an increase in isotopic fractionation with sediment depth. The relative contribution of hydrogenotrophic to total methanogenesis, which was calculated from the isotopic signatures, increased with depth from about 22% to 38%. Total numbers of microorganisms were higher in August than in May, but strongly decreased with depth. The increase of microorganisms from May to August mainly resulted from Bacteria. The Archaea, on the other hand, exhibited a rather constant abundance, but also decreased with depth from about 1 × 108 copies of the archaeal 16S rRNA gene per gram of dry sediment at the surface to 4 × 107 copies per gram at 15–20 cm depth. T-RFLP analysis combined with phylogenetic analysis of cloned sequences of the archaeal 16S rRNA genes showed that the methanogenic community consisted mainly of Methanomicrobiales and Methanosaetaceae. The relative abundance of Methanosaetaceae decreased with depth, whereas that of Methanomicrobiales slightly increased. Hence, the vertical distribution of the functional characteristics (CH4 production from acetate versus H2/CO2) was reflected in the structure of the community consisting of acetotrophic ( Methanosaetaceae) versus hydrogenotrophic ( Methanomicrobiales) phenotypes. [ABSTRACT FROM AUTHOR]

Methane is formed on rice roots mainly by CO2 reduction. The present study aimed to identify the active methanogenic populations responsible for this process. Soil-free rice roots were incubated anaerobically under an atmosphere of H2/13CO2 or N2/13CO2 with phosphate or carbonate (marble) as buffer medium. Nucleic acids were extracted and fractionated by caesium trifluoroacetate equilibrium density gradient centrifugation after 16-day incubation. Community analyses were performed for gradient fractions using terminal restriction fragment polymorphism analysis (T-RFLP) and sequencing of the 16S rRNA genes. In addition, rRNA was extracted and analysed at different time points to trace the community change during the 16-day incubation. TheMethanosarcinaceaeand the yet-uncultured archaeal lineage Rice Cluster-I (RC-I) were predominant in the root incubations when carbonate buffer and N2 headspace were used. The analysis of[13C]DNA showed that the relative 16S rRNA gene abundance of RC-I increased whereas that of theMethanosarcinaceaedecreased with increasing DNA buoyant density, indicating that members of RC-I were more active than theMethanosarcinaceae. However, an unexpected finding was that RC-I was suppressed in the presence of high H2 concentrations (80%, v/v), which during the early incubation period caused a lower CH4 production compared with that with N2 in the headspace. Eventually, however, CH4 production increased, probably because of the activity ofMethanosarcinaceae,which became prevalent. Phosphate buffer appeared to inhibit the activity of theMethanosarcinaceae, resulting in lower CH4 production as compared with carbonate buffer. Under these conditions,Methanobacteriaceaewere the prevalent methanogens. Our study suggests that the active methanogenic populations on rice roots change in correspondence to the presence of H2 (80%, v/v) and the type of buffer used in the system. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Stable isotope probing (SIP) of nucleic acids allows the detection and identification of active members of natural microbial populations that are involved in the assimilation of an isotopically labelled compound into nucleic acids. SIP is based on the separation of isotopically labelled DNA or rRNA by isopycnic density gradient centrifugation. We have developed a highly sensitive protocol for the detection of ‘light’ and ‘heavy’ nucleic acids in fractions of centrifugation gradients. It involves the fluorometric quantification of total DNA or rRNA, and the quantification of either 16S rRNA genes or 16S rRNA in gradient fractions by real-time PCR with domain-specific primers. Using this approach, we found that fully 13C-labelled DNA or rRNA of Methylobacterium extorquens was quantitatively resolved from unlabelled DNA or rRNA of Methanosarcina barkeri by cesium chloride or cesium trifluoroacetate density gradient centrifugation respectively. However, a constant low background of unspecific nucleic acids was detected in all DNA or rRNA gradient fractions, which is important for the interpretation of environmental SIP results. Consequently, quantitative analysis of gradient fractions provides a higher precision and finer resolution for retrieval of isotopically enriched nucleic acids than possible using ethidium bromide or gradient fractionation combined with fingerprinting analyses. This is a prerequisite for the fine-scale tracing of microbial populations metabolizing 13C-labelled compounds in natural ecosystems. [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.

Plastics entering the environment can not only undergo physical degradation and fragmentation processes, but they also tend to be colonized by microorganisms. Microbial colonization and the subsequent biofilm formation on plastics can alter their palatability to organisms and result in a higher ingestion as compared to pristine plastics. To date, the early stage of biofilm formation on plastic materials has not been investigated in context of the environmental medium and polymer properties. We explored the early-stage biofilm formation on polyamide (PA), polyethylene terephthalate (PET), and polyvinyl chloride (PVC) after incubation in freshwater and artificial seawater and categorized the structural diversity on images obtained via scanning electron microscopy. Furthermore, by the measurement of the initial ζ-potential of the plastic materials, we found that PA with the highest negative ζ-potential tended to have the highest structural diversity, followed by PET and PVC after incubation in freshwater. However, PVC with the lowest negative ζ-potential showed the highest structural diversity after incubation in seawater, indicating that the structural diversity is additionally dependent on the incubation medium. Our results give insights into how the incubation medium and polymer properties can influence the early-stage biofilm formation of just recently environmentally exposed microplastics. These differences are responsible for whether organisms may ingest microplastic particles with their food or not. [ABSTRACT FROM AUTHOR]

Many ecological and evolutionary processes in animals depend upon microbial symbioses. In spiders, the role of the microbiome in these processes remains mostly unknown. We compared the microbiome between populations, individuals, and tissue types of a range-expanding spider, using 16S rRNA gene sequencing. Our study is one of the first to go beyond targeting known endosymbionts in spiders and characterizes the total microbiome across different body compartments (leg, prosoma, hemolymph, book lungs, ovaries, silk glands, midgut, and fecal pellets). Overall, the microbiome differed significantly between populations and individuals, but not between tissue types. The microbiome of the wasp spider Argiope bruennichi features a novel dominant bacterial symbiont, which is abundant in every tissue type in spiders from geographically distinct populations and that is also present in offspring. The novel symbiont is affiliated with the Tenericutes, but has low sequence identity (<85%) to all previously named taxa, suggesting that the novel symbiont represents a new bacterial clade. Its presence in offspring implies that it is vertically transmitted. Our results shed light on the processes that shape microbiome differentiation in this species and raise several questions about the implications of the novel dominant bacterial symbiont on the biology and evolution of its host. [ABSTRACT FROM AUTHOR]

In this article, the author talks about the life in deep subsurface discussing topics such as the microorganisms there including the subsurface bacteria, iron oxidation in microbial species, and the metabolic functions in these regions.