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Incubation experiments with 15N-labeled compounds (NO3- and NH4+) were performed during three cruises (2002, 2004, and 2005) to study the loss of inorganic N as dinitrogen gas (N3) via denitrification and anammox in the water column of the Gotland Deep (central Baltic Sea). 15N incubations did not provide evidence for direct conversion of NO3- reduction to N3 (heterotrophic denitrification) in the suboxic (O3 < 10 µmol L-1) sulfide-free waters. Substantial denitrification rates (up to 2.7 µmol L-1 d-1 N2) were measured in water samples collected from the NO3--H2S interface (redoxcline) in 2002 and in water from the sulfidic zone in 2004, which indicates chemolithotrophic denitrification as the dominant N-loss process in both years. Massive inflows of oxygenated North Sea water from 2002 to 2003 caused a complete ventilation of the Baltic Sea with high oxygen concentrations in the Gotland Deep bottom water. After the reestablishment of the redoxcline in 2004, a newly formed suboxic zone above sulfidic waters-with NO3-, NO2-, and NH&sub4;+ at the detection limit-was observed in spring 2005. The development of this zone was associated with a several-fold increase in reduced and oxidized manganese and with a shift from denitrification to anammox as the main N-loss process. Fluorescence in situ hybridization analysis confirmed the presence of anammox bacteria and the number of anammox cells was consistent with the observed N2 production rates in 2005 [ABSTRACT FROM AUTHOR]

Fixation of atmospheric N 2 by free-living diazotrophs accounts for an important proportion of nitrogen naturally introduced to temperate grasslands. The effect of plants or fertilization on the general microbial community has been extensively studied, yet an understanding of the potential combinatorial effects on the community structure and activity of free-living diazotrophs is lacking. In this study we provide a multilevel assessment of the single and interactive effects of different long-term fertilization treatments, plant species and vicinity to roots on the free-living diazotroph community in relation to the general microbial community in grassland soils. We sequenced the dinitrogenase reductase (nifH) and the 16S rRNA genes of bulk soil and root-associated compartments (rhizosphere soil, rhizoplane and root) of two grass species (Arrhenatherum elatius and Anthoxanthum odoratum) and two herb species (Galium album and Plantago lanceolata) growing in Austrian grassland soils treated with different fertilizers (N, P, NPK) since 1960. Overall, fertilization has the strongest effect on the diazotroph and general microbial community structure, however with vicinity to the root, the plant effect increases. Despite the long-term fertilization, plants strongly influence the diazotroph communities emphasizing the complexity of soil microbial communities' responses to changing nutrient conditions in temperate grasslands., (© 2024. The Author(s).)

Background: Fungi and ants belong to the most important organisms in terrestrial ecosystems on Earth. In nutrient-poor niches of tropical rainforests, they have developed steady ecological relationships as a successful survival strategy. In tropical ant-plant mutualisms worldwide, where resident ants provide the host plants with defense and nutrients in exchange for shelter and food, fungi are regularly found in the ant nesting space, inhabiting ant-made dark-colored piles ("patches"). Unlike the extensively investigated fungus-growing insects, where the fungi serve as the primary food source, the purpose of this ant-fungi association is less clear. To decipher the roles of fungi in these structures within ant nests, it is crucial to first understand the dynamics and drivers that influence fungal patch communities during ant colony development., Results: In this study, we investigated how the ant colony age and the ant-plant species affect the fungal community in the patches. As model we selected one of the most common mutualisms in the Tropics of America, the Azteca-Cecropia complex. By amplicon sequencing of the internal transcribed spacer 2 (ITS2) region, we analyzed the patch fungal communities of 93 Azteca spp. colonies inhabiting Cecropia spp. trees. Our study demonstrates that the fungal diversity in patches increases as the ant colony grows and that a change in the prevalent fungal taxa occurs between initial and established patches. In addition, the ant species significantly influences the composition of the fungal community in established ant colonies, rather than the host plant species., Conclusions: The fungal patch communities become more complex as the ant colony develops, due to an acquisition of fungi from the environment and a substrate diversification. Our results suggest a successional progression of the fungal communities in the patches during ant colony growth and place the ant colony as the main driver shaping such communities. The findings of this study demonstrate the unexpectedly complex nature of ant-plant mutualisms in tropical regions at a micro scale., (© 2024. The Author(s).)

Microbial activity in drylands tends to be confined to rare and short periods of rain. Rapid growth should be key to the maintenance of ecosystem processes in such narrow activity windows, if desiccation and rehydration cause widespread cell death due to osmotic stress. Here, simulating rain with 2 H 2 O followed by single-cell NanoSIMS, we show that biocrust microbial communities in the Negev Desert are characterized by limited productivity, with median replication times of 6 to 19 days and restricted number of days allowing growth. Genome-resolved metatranscriptomics reveals that nearly all microbial populations resuscitate within minutes after simulated rain, independent of taxonomy, and invest their activity into repair and energy generation. Together, our data reveal a community that makes optimal use of short activity phases by fast and universal resuscitation enabling the maintenance of key ecosystem functions. We conclude that desert biocrust communities are highly adapted to surviving rapid changes in soil moisture and solute concentrations, resulting in high persistence that balances limited productivity., (© 2024. The Author(s).)

The omnipresence of ants is commonly attributed to their eusocial organization and division of labor, however, bacteria in their nests may facilitate their success. Like many other arboreal ants living in plant-provided cavities, Azteca ants form dark-colored "patches" in their nesting space inside Cecropia host plants. These patches are inhabited by bacteria, fungi and nematodes and appear to be essential for ant colony development. Yet, detailed knowledge of the microbial community composition and its consistency throughout the life cycle of ant colonies was lacking. Amplicon sequencing of the microbial 16S rRNA genes in patches from established ant colonies reveals a highly diverse, ant species-specific bacterial community and little variation within an individual ant colony, with Burkholderiales, Rhizobiales and Chitinophagales being most abundant. In contrast, bacterial communities of early ant colony stages show low alpha diversity and no ant species-specific community composition. We suggest a substrate-caused bottleneck after vertical transmission of the bacterial patch community from mother to daughter colonies. The subsequent ecological succession is driven by environmental parameters and influenced by ant behavior. Our study provides key information for future investigations determining the functions of these bacteria, which is essential to understand the ubiquity of such patches among arboreal ants., (© 2023. The Author(s).)

Bacteria colonize plant roots and engage in reciprocal interactions with their hosts. However, the contribution of individual taxa or groups of bacteria to plant nutrition and fitness is not well characterized due to a lack of in situ evidence of bacterial activity. To address this knowledge gap, we developed an analytical approach that combines the identification and localization of individual bacteria on root surfaces via gold-based in situ hybridization with correlative NanoSIMS imaging of incorporated stable isotopes, indicative of metabolic activity. We incubated Kosakonia strain DS-1-associated, gnotobiotically grown rice plants with 15 N-N 2 gas to detect in situ N 2 fixation activity. Bacterial cells along the rhizoplane showed heterogeneous patterns of 15 N enrichment, ranging from the natural isotope abundance levels up to 12.07 at% 15 N (average and median of 3.36 and 2.85 at% 15 N, respectively, n = 697 cells). The presented correlative optical and chemical imaging analysis is applicable to a broad range of studies investigating plant-microbe interactions. For example, it enables verification of the in situ metabolic activity of host-associated commercialized strains or plant growth-promoting bacteria, thereby disentangling their role in plant nutrition. Such data facilitate the design of plant-microbe combinations for improvement of crop management., (© 2023 The Authors New Phytologist © 2023 New Phytologist Foundation.)

Ectomycorrhizal fungi live in close association with their host plants and form complex interactions with bacterial/archaeal communities in soil. We investigated whether abundant or rare ectomycorrhizal fungi on root-tips of young beech trees (Fagus sylvatica) shape bacterial/archaeal communities. We sequenced 16S rRNA genes and fungal internal transcribed spacer regions of individual root-tips and used ecological networks to detect the tendency of certain assemblies of fungal and bacterial/archaeal taxa to inhabit the same root-tip (i.e. modularity). Individual ectomycorrhizal root-tips hosted distinct fungal communities associated with unique bacterial/archaeal communities. The structure of the fungal-bacterial/archaeal association was determined by both, dominant and rare fungi. Integrating our data in a conceptual framework suggests that the effect of rare fungi on the bacterial/archaeal communities of ectomycorrhizal root-tips contributes to assemblages of bacteria/archaea on root-tips. This highlights the potential impact of complex fine-scale interactions between root-tip associated fungi and other soil microorganisms for the ectomycorrhizal symbiosis., (© 2022. The Author(s).)

Research needs a balance of risk-taking in "breakthrough projects" and gradual progress. For building a sustainable knowledge base, it is indispensable to provide support for both., (© 2022 The Authors. Published under the terms of the CC BY 4.0 license.)

Background: Symbiotic ant-plant associations, in which ants live on plants, feed on plant-provided food, and protect host trees against threats, are ubiquitous across the tropics, with the Azteca-Cecropia associations being amongst the most widespread interactions in the Neotropics. Upon colonization of Cecropia's hollow internodes, Azteca queens form small patches with plant parenchyma, which are then used as waste piles when the colony grows. Patches-found in many ant-plant mutualisms-are present throughout the colony life cycle and may supplement larval food. Despite their initial nitrogen (N)-poor substrate, patches in Cecropia accommodate fungi, nematodes, and bacteria. In this study, we investigated the atmospheric N 2 fixation as an N source in patches of early and established ant colonies., Results: Via 15 N 2 tracer assays, N 2 fixation was frequently detected in all investigated patch types formed by three Azteca ant species. Quantified fixation rates were similar in early and established ant colonies and higher than in various tropical habitats. Based on amplicon sequencing, the identified microbial functional guild-the diazotrophs-harboring and transcribing the dinitrogenase reductase (nifH) gene was highly diverse and heterogeneous across Azteca colonies. The community composition differed between early and established ant colonies and partly between the ant species., Conclusions: Our data show that N 2 fixation can result in reasonable amounts of N in ant colonies, which might not only enable bacterial, fungal, and nematode growth in the patch ecosystems but according to our calculations can even support the growth of ant populations. The diverse and heterogeneous diazotrophic community implies a functional redundancy, which could provide the ant-plant-patch system with a higher resilience towards changing environmental conditions. Hence, we propose that N 2 fixation represents a previously unknown potential to overcome N limitations in arboreal ant colonies., (© 2022. The Author(s).)

Grassland ecosystems cover around 37% of the ice-free land surface on Earth and have critical socioeconomic importance globally. As in many terrestrial ecosystems, biological dinitrogen (N 2 ) fixation represents an essential natural source of nitrogen (N). The ability to fix atmospheric N 2 is limited to diazotrophs, a diverse guild of bacteria and archaea. To elucidate the abiotic (climatic, edaphic), biotic (vegetation), and spatial factors that govern diazotrophic community composition in global grassland soils, amplicon sequencing of the dinitrogenase reductase gene- nifH -was performed on samples from a replicated standardized nutrient [N, phosphorus (P)] addition experiment in 23 grassland sites spanning four continents. Sites harbored distinct and diverse diazotrophic communities, with most of reads assigned to diazotrophic taxa within the Alphaproteobacteria (e.g., Rhizobiales ), Cyanobacteria (e.g., Nostocales ), and Deltaproteobacteria (e.g., Desulforomonadales ) groups. Likely because of the wide range of climatic and edaphic conditions and spatial distance among sampling sites, only a few of the taxa were present at all sites. The best model describing the variation among soil diazotrophic communities at the OTU level combined climate seasonality (temperature in the wettest quarter and precipitation in the warmest quarter) with edaphic (C:N ratio, soil texture) and vegetation factors (various perennial plant covers). Additionally, spatial variables (geographic distance) correlated with diazotrophic community variation, suggesting an interplay of environmental variables and spatial distance. The diazotrophic communities appeared to be resilient to elevated nutrient levels, as 2-4 years of chronic N and P additions had little effect on the community composition. However, it remains to be seen, whether changes in the community composition occur after exposure to long-term, chronic fertilization regimes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Nepel, Angel, Borer, Frey, MacDougall, McCulley, Risch, Schütz, Seabloom and Woebken.)

Ectomycorrhizal plants trade plant-assimilated carbon for soil nutrients with their fungal partners. The underlying mechanisms, however, are not fully understood. Here we investigate the exchange of carbon for nitrogen in the ectomycorrhizal symbiosis of Fagus sylvatica across different spatial scales from the root system to the cellular level. We provided 15 N-labelled nitrogen to mycorrhizal hyphae associated with one half of the root system of young beech trees, while exposing plants to a 13 CO 2 atmosphere. We analysed the short-term distribution of 13 C and 15 N in the root system with isotope-ratio mass spectrometry, and at the cellular scale within a mycorrhizal root tip with nanoscale secondary ion mass spectrometry (NanoSIMS). At the root system scale, plants did not allocate more 13 C to root parts that received more 15 N. Nanoscale secondary ion mass spectrometry imaging, however, revealed a highly heterogenous, and spatially significantly correlated distribution of 13 C and 15 N at the cellular scale. Our results indicate that, on a coarse scale, plants do not allocate a larger proportion of photoassimilated C to root parts associated with N-delivering ectomycorrhizal fungi. Within the ectomycorrhizal tissue, however, recently plant-assimilated C and fungus-delivered N were spatially strongly coupled. Here, NanoSIMS visualisation provides an initial insight into the regulation of ectomycorrhizal C and N exchange at the microscale., (© 2021 The Authors. New Phytologist © 2021 New Phytologist Foundation.)

High-affinity terminal oxidases (TOs) are believed to permit microbial respiration at low oxygen (O 2 ) levels. Genes encoding such oxidases are widespread, and their existence in microbial genomes is taken as an indicator for microaerobic respiration. We combined respiratory kinetics determined via highly sensitive optical trace O 2 sensors, genomics, and transcriptomics to test the hypothesis that high-affinity TOs are a prerequisite to respire micro- and nanooxic concentrations of O 2 in environmentally relevant model soil organisms: acidobacteria. Members of the Acidobacteria harbor branched respiratory chains terminating in low-affinity ( caa 3 -type cytochrome c oxidases) as well as high-affinity ( cbb 3 -type cytochrome c oxidases and/or bd -type quinol oxidases) TOs, potentially enabling them to cope with varying O 2 concentrations. The measured apparent K m ( K m (app) ) values for O 2 of selected strains ranged from 37 to 288 nmol O 2 liter -1 , comparable to values previously assigned to low-affinity TOs. Surprisingly, we could not detect the expression of the conventional high-affinity TO ( cbb 3 type) at micro- and nanomolar O 2 concentrations but detected the expression of low-affinity TOs. To the best of our knowledge, this is the first observation of microaerobic respiration imparted by low-affinity TOs at O 2 concentrations as low as 1 nM. This challenges the standing hypothesis that a microaerobic lifestyle is exclusively imparted by the presence of high-affinity TOs. As low-affinity TOs are more efficient at generating ATP than high-affinity TOs, their utilization could provide a great benefit, even at low-nanomolar O 2 levels. Our findings highlight energy conservation strategies that could promote the success of Acidobacteria in soil but might also be important for as-yet-unrevealed microorganisms. IMPORTANCE Low-oxygen habitats are widely distributed on Earth, ranging from the human intestine to soils. Microorganisms are assumed to have the capacity to respire low O 2 concentrations via high-affinity terminal oxidases. By utilizing strains of a ubiquitous and abundant group of soil bacteria, the Acidobacteria , and combining respiration kinetics, genomics, and transcriptomics, we provide evidence that these microorganisms use the energetically more efficient low-affinity terminal oxidases to respire low-nanomolar O 2 concentrations. This questions the standing hypothesis that the ability to respire traces of O 2 stems solely from the activity of high-affinity terminal oxidases. We propose that this energetically efficient strategy extends into other, so-far-unrevealed microbial clades. Our findings also demonstrate that physiological predictions regarding the utilization of different O 2 concentrations based solely on the presence or absence of terminal oxidases in bacterial genomes can be misleading.

Hypersaline microbial mats are dense microbial ecosystems capable of performing complete element cycling and are considered analogs of early Earth and hypothetical extraterrestrial ecosystems. We studied the functionality and limits of key biogeochemical processes, such as photosynthesis, aerobic respiration, and sulfur cycling, in salt crust-covered microbial mats from a tidal flat at the coast of Oman. We measured light, oxygen, and sulfide microprofiles as well as sulfate reduction rates at salt saturation and in flood conditions and determined fine-scale stratification of pigments, biomass, and microbial taxa in the resident microbial community. The salt crust did not protect the mats against irradiation or evaporation. Although some oxygen production was measurable at salinities of ≤30% (wt/vol) in situ , at saturation-level salinity (40%), oxygenic photosynthesis was completely inhibited and only resumed 2 days after reducing the porewater salinity to 12%. Aerobic respiration and active sulfur cycling occurred at low rates under salt saturation and increased strongly upon salinity reduction. Apart from high relative abundances of Chloroflexi , photoheterotrophic Alphaproteobacteria , Bacteroidetes , and Archaea , the mat contained a distinct layer harboring filamentous Cyanobacteria , which is unusual for such high salinities. Our results show that the diverse microbial community inhabiting this salt flat mat ultimately depends on periodic salt dilution to be self-sustaining and is rather adapted to merely survive salt saturation than to thrive under the salt crust. IMPORTANCE Due to their abilities to survive intense radiation and low water availability, hypersaline microbial mats are often suggested to be analogs of potential extraterrestrial life. However, even the limitations imposed on microbial processes by saturation-level salinity found on Earth have rarely been studied in situ . While abundance and diversity of microbial life in salt-saturated environments are well documented, most of our knowledge on process limitations stems from culture-based studies, few in situ studies, and theoretical calculations. In particular, oxygenic photosynthesis has barely been explored beyond 5 M NaCl (28% wt/vol). By applying a variety of biogeochemical and molecular methods, we show that despite abundance of photoautotrophic microorganisms, oxygenic photosynthesis is inhibited in salt-crust-covered microbial mats at saturation salinities, while rates of other energy generation processes are decreased several-fold. Hence, the complete element cycling required for self-sustaining microbial communities only occurs at lower salt concentrations.

Significant rates of atmospheric dihydrogen (H 2 ) consumption have been observed in temperate soils due to the activity of high-affinity enzymes, such as the group 1h [NiFe]-hydrogenase. We designed broadly inclusive primers targeting the large subunit gene (hhyL) of group 1h [NiFe]-hydrogenases for long-read sequencing to explore its taxonomic distribution across soils. This approach revealed a diverse collection of microorganisms harboring hhyL, including previously unknown groups and taxonomically not assignable sequences. Acidobacterial group 1h [NiFe]-hydrogenase genes were abundant and expressed in temperate soils. To support the participation of acidobacteria in H 2 consumption, we studied two representative mesophilic soil acidobacteria, which expressed group 1h [NiFe]-hydrogenases and consumed atmospheric H 2 during carbon starvation. This is the first time mesophilic acidobacteria, which are abundant in ubiquitous temperate soils, have been shown to oxidize H 2 down to below atmospheric concentrations. As this physiology allows bacteria to survive periods of carbon starvation, it could explain the success of soil acidobacteria. With our long-read sequencing approach of group 1h [NiFe]-hydrogenase genes, we show that the ability to oxidize atmospheric levels of H 2 is more widely distributed among soil bacteria than previously recognized and could represent a common mechanism enabling bacteria to persist during periods of carbon deprivation.

Desert surface soils devoid of plant cover are populated by a variety of microorganisms, many with yet unresolved physiologies and lifestyles. Nevertheless, a common feature vital for these microorganisms inhabiting arid soils is their ability to survive long drought periods and reactivate rapidly in rare incidents of rain. Chemolithotrophic processes such as oxidation of atmospheric hydrogen and carbon monoxide are suggested to be a widespread energy source to support dormancy and resuscitation in desert soil microorganisms. Here, we assessed the distribution of chemolithotrophic, phototrophic, and desiccation-related metabolic potential among microbial populations in arid biological soil crusts (BSCs) from the Negev Desert, Israel, via population-resolved metagenomic analysis. While the potential to utilize light and atmospheric hydrogen as additional energy sources was widespread, carbon monoxide oxidation was less common than expected. The ability to utilize continuously available energy sources might decrease the dependency of mixotrophic populations on organic storage compounds and carbon provided by the BSC-founding cyanobacteria. Several populations from five different phyla besides the cyanobacteria encoded CO 2 fixation potential, indicating further potential independence from photoautotrophs. However, we also found population genomes with a strictly heterotrophic genetic repertoire. The highly abundant Rubrobacteraceae ( Actinobacteriota ) genomes showed particular specialization for this extreme habitat, different from their closest cultured relatives. Besides the ability to use light and hydrogen as energy sources, they encoded extensive O 2 stress protection and unique DNA repair potential. The uncovered differences in metabolic potential between individual, co-occurring microbial populations enable predictions of their ecological niches and generation of hypotheses on the dynamics and interactions among them. IMPORTANCE This study represents a comprehensive community-wide genome-centered metagenome analysis of biological soil crust (BSC) communities in arid environments, providing insights into the distribution of genes encoding different energy generation mechanisms, as well as survival strategies, among populations in an arid soil ecosystem. It reveals the metabolic potential of several uncultured and previously unsequenced microbial genera, families, and orders, as well as differences in the metabolic potential between the most abundant BSC populations and their cultured relatives, highlighting once more the danger of inferring function on the basis of taxonomy. Assigning functional potential to individual populations allows for the generation of hypotheses on trophic interactions and activity patterns in arid soil microbial communities and represents the basis for future resuscitation and activity studies of the system, e.g., involving metatranscriptomics., (Copyright © 2021 Meier et al.)

Microbial life is surprisingly abundant and diverse in global desert ecosystems. In these environments, microorganisms endure a multitude of physicochemical stresses, including low water potential, carbon and nitrogen starvation, and extreme temperatures. In this review, we summarize our current understanding of the energetic mechanisms and trophic dynamics that underpin microbial function in desert ecosystems. Accumulating evidence suggests that dormancy is a common strategy that facilitates microbial survival in response to water and carbon limitation. Whereas photoautotrophs are restricted to specific niches in extreme deserts, metabolically versatile heterotrophs persist even in the hyper-arid topsoils of the Atacama Desert and Antarctica. At least three distinct strategies appear to allow such microorganisms to conserve energy in these oligotrophic environments: degradation of organic energy reserves, rhodopsin- and bacteriochlorophyll-dependent light harvesting, and oxidation of the atmospheric trace gases hydrogen and carbon monoxide. In turn, these principles are relevant for understanding the composition, functionality, and resilience of desert ecosystems, as well as predicting responses to the growing problem of desertification., Competing Interests: Conflict of Interest Disclosures for the Authors: Pok Man Leung has nothing to disclose. Sean K. Bay has nothing to disclose. Dimitri V. Meier has nothing to disclose. Eleonora Chiri has nothing to disclose. Don A. Cowan has nothing to disclose. Osnat Gillor has nothing to disclose. Dagmar Woebken has nothing to disclose. Chris Greening has nothing to disclose. Conflict of Interest Disclosures for the Editor: James C. Stegen has nothing to disclose., (Copyright © 2020 Leung et al.)

We report eight genomes from representatives of the phylum Acidobacteria subdivisions 1 and 3, isolated from soils. The genome sizes range from 4.9 to 6.7 Mb. Genomic analysis reveals putative genes for low- and high-affinity respiratory oxygen reductases, high-affinity hydrogenases, and the capacity to use a diverse collection of carbohydrates., (Copyright © 2020 Eichorst et al.)

Ammonia-oxidizing microorganisms perform the first step of nitrification, the oxidation of ammonia to nitrite. The bacterium Nitrosomonas europaea is the best-characterized ammonia oxidizer to date. Exposure to hypoxic conditions has a profound effect on the physiology of N. europaea , e.g., by inducing nitrifier denitrification, resulting in increased nitric and nitrous oxide production. This metabolic shift is of major significance in agricultural soils, as it contributes to fertilizer loss and global climate change. Previous studies investigating the effect of oxygen limitation on N. europaea have focused on the transcriptional regulation of genes involved in nitrification and nitrifier denitrification. Here, we combine steady-state cultivation with whole-genome transcriptomics to investigate the overall effect of oxygen limitation on N. europaea Under oxygen-limited conditions, growth yield was reduced and ammonia-to-nitrite conversion was not stoichiometric, suggesting the production of nitrogenous gases. However, the transcription of the principal nitric oxide reductase (cNOR) did not change significantly during oxygen-limited growth, while the transcription of the nitrite reductase-encoding gene ( nirK ) was significantly lower. In contrast, both heme-copper-containing cytochrome c oxidases encoded by N. europaea were upregulated during oxygen-limited growth. Particularly striking was the significant increase in transcription of the B-type heme-copper oxidase, proposed to function as a nitric oxide reductase (sNOR) in ammonia-oxidizing bacteria. In the context of previous physiological studies, as well as the evolutionary placement of N. europaea's sNOR with regard to other heme-copper oxidases, these results suggest sNOR may function as a high-affinity terminal oxidase in N. europaea and other ammonia-oxidizing bacteria. IMPORTANCE Nitrification is a ubiquitous microbially mediated process in the environment and an essential process in engineered systems such as wastewater and drinking water treatment plants. However, nitrification also contributes to fertilizer loss from agricultural environments, increasing the eutrophication of downstream aquatic ecosystems, and produces the greenhouse gas nitrous oxide. As ammonia-oxidizing bacteria are the most dominant ammonia-oxidizing microbes in fertilized agricultural soils, understanding their responses to a variety of environmental conditions is essential for curbing the negative environmental effects of nitrification. Notably, oxygen limitation has been reported to significantly increase nitric oxide and nitrous oxide production during nitrification. Here, we investigate the physiology of the best-characterized ammonia-oxidizing bacterium, Nitrosomonas europaea , growing under oxygen-limited conditions., (Copyright © 2020 Sedlacek et al.)

Microorganisms are critical in mediating carbon (C) and nitrogen (N) cycling processes in soils. Yet, it has long been debated whether the processes underlying biogeochemical cycles are affected by the composition and diversity of the soil microbial community or not. The composition and diversity of soil microbial communities can be influenced by various environmental factors, which in turn are known to impact biogeochemical processes. The objectives of this study were to test effects of multiple edaphic drivers individually and represented as the multivariate soil environment interacting with microbial community composition and diversity, and concomitantly on multiple soil functions (i.e. soil enzyme activities, soil C and N processes). We employed high-throughput sequencing (Illumina MiSeq) to analyze bacterial/archaeal and fungal community composition by targeting the 16S rRNA gene and the ITS1 region of soils collected from three land uses (cropland, grassland and forest) deriving from two bedrock forms (silicate and limestone). Based on this data set we explored single and combined effects of edaphic variables on soil microbial community structure and diversity, as well as on soil enzyme activities and several soil C and N processes. We found that both bacterial/archaeal and fungal communities were shaped by the same edaphic factors, with most single edaphic variables and the combined soil environment representation exerting stronger effects on bacterial/archaeal communities than on fungal communities, as demonstrated by (partial) Mantel tests. We also found similar edaphic controls on the bacterial/archaeal/fungal richness and diversity. Soil C processes were only directly affected by the soil environment but not affected by microbial community composition. In contrast, soil N processes were significantly related to bacterial/archaeal community composition and bacterial/archaeal/fungal richness/diversity but not directly affected by the soil environment. This indicates direct control of the soil environment on soil C processes and indirect control of the soil environment on soil N processes by structuring the microbial communities. The study further highlights the importance of edaphic drivers and microbial communities (i.e. composition and diversity) on important soil C and N processes., Competing Interests: Conflicts of interest The authors declare no competing interests.

Legume-rhizobia symbioses play a major role in food production for an ever growing human population. In this symbiosis, dinitrogen is reduced ("fixed") to ammonia by the rhizobial nitrogenase enzyme complex and is secreted to the plant host cells, whereas dicarboxylic acids derived from photosynthetically produced sucrose are transported into the symbiosomes and serve as respiratory substrates for the bacteroids. The symbiosome membrane contains high levels of SST1 protein, a sulfate transporter. Sulfate is an essential nutrient for all living organisms, but its importance for symbiotic nitrogen fixation and nodule metabolism has long been underestimated. Using chemical imaging, we demonstrate that the bacteroids take up 20-fold more sulfate than the nodule host cells. Furthermore, we show that nitrogenase biosynthesis relies on high levels of imported sulfate, making sulfur as essential as carbon for the regulation and functioning of symbiotic nitrogen fixation. Our findings thus establish the importance of sulfate and its active transport for the plant-microbe interaction that is most relevant for agriculture and soil fertility., (© 2018 The Authors Plant, Cell & Environment Published by John Wiley & Sons Ltd.)

Plant roots release recent photosynthates into the rhizosphere, accelerating decomposition of organic matter by saprotrophic soil microbes ("rhizosphere priming effect") which consequently increases nutrient availability for plants. However, about 90% of all higher plant species are mycorrhizal, transferring a significant fraction of their photosynthates directly to their fungal partners. Whether mycorrhizal fungi pass on plant-derived carbon (C) to bacteria in root-distant soil areas, i.e., incite a "hyphosphere priming effect," is not known. Experimental evidence for C transfer from mycorrhizal hyphae to soil bacteria is limited, especially for ectomycorrhizal systems. As ectomycorrhizal fungi possess enzymatic capabilities to degrade organic matter themselves, it remains unclear whether they cooperate with soil bacteria by providing photosynthates, or compete for available nutrients. To investigate a possible C transfer from ectomycorrhizal hyphae to soil bacteria, and its response to changing nutrient availability, we planted young beech trees ( Fagus sylvatica ) into "split-root" boxes, dividing their root systems into two disconnected soil compartments. Each of these compartments was separated from a litter compartment by a mesh penetrable for fungal hyphae, but not for roots. Plants were exposed to a 13 C-CO 2 -labeled atmosphere, while 15 N-labeled ammonium and amino acids were added to one side of the split-root system. We found a rapid transfer of recent photosynthates via ectomycorrhizal hyphae to bacteria in root-distant soil areas. Fungal and bacterial phospholipid fatty acid (PLFA) biomarkers were significantly enriched in hyphae-exclusive compartments 24 h after 13 C-CO 2 -labeling. Isotope imaging with nanometer-scale secondary ion mass spectrometry (NanoSIMS) allowed for the first time in situ visualization of plant-derived C and N taken up by an extraradical fungal hypha, and in microbial cells thriving on hyphal surfaces. When N was added to the litter compartments, bacterial biomass, and the amount of incorporated 13 C strongly declined. Interestingly, this effect was also observed in adjacent soil compartments where added N was only available for bacteria through hyphal transport, indicating that ectomycorrhizal fungi were acting on soil bacteria. Together, our results demonstrate that (i) ectomycorrhizal hyphae rapidly transfer plant-derived C to bacterial communities in root-distant areas, and (ii) this transfer promptly responds to changing soil nutrient conditions.

Soil microorganisms control carbon losses from soils to the atmosphere1-3, yet their responses to climate warming are often short-lived and unpredictable4-7. Two mechanisms, microbial acclimation and substrate depletion, have been proposed to explain temporary warming effects on soil microbial activity8-10. However, empirical support for either mechanism is unconvincing. Here we used geothermal temperature gradients (> 50 years of field warming)11 and a short-term experiment to show that microbial activity (gross rates of growth, turnover, respiration and carbon uptake) is intrinsically temperature sensitive and does not acclimate to warming (+ 6 ºC) over weeks or decades. Permanently accelerated microbial activity caused carbon loss from soil. However, soil carbon loss was temporary because substrate depletion reduced microbial biomass and constrained the influence of microbes over the ecosystem. A microbial biogeochemical model12-14 showed that these observations are reproducible through a modest, but permanent, acceleration in microbial physiology. These findings reveal a mechanism by which intrinsic microbial temperature sensitivity and substrate depletion together dictate warming effects on soil carbon loss via their control over microbial biomass. We thus provide a framework for interpreting the links between temperature, microbial activity and soil carbon loss on timescales relevant to Earth's climate system., Competing Interests: The authors declare no competing financial interests.

Plastic materials are widely used in agricultural applications to achieve food security for the growing world population. The use of biodegradable instead of nonbiodegradable polymers in single-use agricultural applications, including plastic mulching, promises to reduce plastic accumulation in the environment. We present a novel approach that allows tracking of carbon from biodegradable polymers into CO 2 and microbial biomass. The approach is based on 13 C-labeled polymers and on isotope-specific analytical methods, including nanoscale secondary ion mass spectrometry (NanoSIMS). Our results unequivocally demonstrate the biodegradability of poly(butylene adipate- co -terephthalate) (PBAT), an important polyester used in agriculture, in soil. Carbon from each monomer unit of PBAT was used by soil microorganisms, including filamentous fungi, to gain energy and to form biomass. This work advances both our conceptual understanding of polymer biodegradation and the methodological capabilities to assess this process in natural and engineered environments.

Sulfur-cycling microorganisms impact organic matter decomposition in wetlands and consequently greenhouse gas emissions from these globally relevant environments. However, their identities and physiological properties are largely unknown. By applying a functional metagenomics approach to an acidic peatland, we recovered draft genomes of seven novel Acidobacteria species with the potential for dissimilatory sulfite (dsrAB, dsrC, dsrD, dsrN, dsrT, dsrMKJOP) or sulfate respiration (sat, aprBA, qmoABC plus dsr genes). Surprisingly, the genomes also encoded DsrL, which so far was only found in sulfur-oxidizing microorganisms. Metatranscriptome analysis demonstrated expression of acidobacterial sulfur-metabolism genes in native peat soil and their upregulation in diverse anoxic microcosms. This indicated an active sulfate respiration pathway, which, however, might also operate in reverse for dissimilatory sulfur oxidation or disproportionation as proposed for the sulfur-oxidizing Desulfurivibrio alkaliphilus. Acidobacteria that only harbored genes for sulfite reduction additionally encoded enzymes that liberate sulfite from organosulfonates, which suggested organic sulfur compounds as complementary energy sources. Further metabolic potentials included polysaccharide hydrolysis and sugar utilization, aerobic respiration, several fermentative capabilities, and hydrogen oxidation. Our findings extend both, the known physiological and genetic properties of Acidobacteria and the known taxonomic diversity of microorganisms with a DsrAB-based sulfur metabolism, and highlight new fundamental niches for facultative anaerobic Acidobacteria in wetlands based on exploitation of inorganic and organic sulfur molecules for energy conservation.

Members of the phylum Acidobacteria are abundant and ubiquitous across soils. We performed a large-scale comparative genome analysis spanning subdivisions 1, 3, 4, 6, 8 and 23 (n = 24) with the goal to identify features to help explain their prevalence in soils and understand their ecophysiology. Our analysis revealed that bacteriophage integration events along with transposable and mobile elements influenced the structure and plasticity of these genomes. Low- and high-affinity respiratory oxygen reductases were detected in multiple genomes, suggesting the capacity for growing across different oxygen gradients. Among many genomes, the capacity to use a diverse collection of carbohydrates, as well as inorganic and organic nitrogen sources (such as via extracellular peptidases), was detected - both advantageous traits in environments with fluctuating nutrient environments. We also identified multiple soil acidobacteria with the potential to scavenge atmospheric concentrations of H 2 , now encompassing mesophilic soil strains within the subdivision 1 and 3, in addition to a previously identified thermophilic strain in subdivision 4. This large-scale acidobacteria genome analysis reveal traits that provide genomic, physiological and metabolic versatility, presumably allowing flexibility and versatility in the challenging and fluctuating soil environment., (© 2018 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

Investigating active participants in the fixation of dinitrogen gas is vital as N is often a limiting factor for primary production. Biological nitrogen fixation is performed by a diverse guild of bacteria and archaea (diazotrophs), which can be free-living or symbionts. Free-living diazotrophs are widely distributed in the environment, yet our knowledge about their identity and ecophysiology is still limited. A major challenge in investigating this guild is inferring activity from genetic data as this process is highly regulated. To address this challenge, we evaluated and improved several 15 N-based methods for detecting N 2 fixation activity (with a focus on soil samples) and studying active diazotrophs. We compared the acetylene reduction assay and the 15 N 2 tracer method and demonstrated that the latter is more sensitive in samples with low activity. Additionally, tracing 15 N into microbial RNA provides much higher sensitivity compared to bulk soil analysis. Active soil diazotrophs were identified with a 15 N-RNA-SIP approach optimized for environmental samples and benchmarked to 15 N-DNA-SIP. Lastly, we investigated the feasibility of using SIP-Raman microspectroscopy for detecting 15 N-labelled cells. Taken together, these tools allow identifying and investigating active free-living diazotrophs in a highly sensitive manner in diverse environments, from bulk to the single-cell level., (© 2017 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

The combined approach of incubating environmental samples with stable isotope-labeled substrates followed by single-cell analyses through high-resolution secondary ion mass spectrometry (NanoSIMS) or Raman microspectroscopy provides insights into the in situ function of microorganisms. This approach has found limited application in soils presumably due to the dispersal of microbial cells in a large background of particles. We developed a pipeline for the efficient preparation of cell extracts from soils for subsequent single-cell methods by combining cell detachment with separation of cells and soil particles followed by cell concentration. The procedure was evaluated by examining its influence on cell recoveries and microbial community composition across two soils. This approach generated a cell fraction with considerably reduced soil particle load and of sufficient small size to allow single-cell analysis by NanoSIMS, as shown when detecting active N2-fixing and cellulose-responsive microorganisms via (15)N2 and (13)C-UL-cellulose incubations, respectively. The same procedure was also applicable for Raman microspectroscopic analyses of soil microorganisms, assessed via microcosm incubations with a (13)C-labeled carbon source and deuterium oxide (D2O, a general activity marker). The described sample preparation procedure enables single-cell analysis of soil microorganisms using NanoSIMS and Raman microspectroscopy, but should also facilitate single-cell sorting and sequencing., (© FEMS 2015.)

Photosynthetic microbial mats are complex, stratified ecosystems in which high rates of primary production create a demand for nitrogen, met partially by N₂ fixation. Dinitrogenase reductase (nifH) genes and transcripts from Cyanobacteria and heterotrophic bacteria (for example, Deltaproteobacteria) were detected in these mats, yet their contribution to N2 fixation is poorly understood. We used a combined approach of manipulation experiments with inhibitors, nifH sequencing and single-cell isotope analysis to investigate the active diazotrophic community in intertidal microbial mats at Laguna Ojo de Liebre near Guerrero Negro, Mexico. Acetylene reduction assays with specific metabolic inhibitors suggested that both sulfate reducers and members of the Cyanobacteria contributed to N₂ fixation, whereas (15)N₂ tracer experiments at the bulk level only supported a contribution of Cyanobacteria. Cyanobacterial and nifH Cluster III (including deltaproteobacterial sulfate reducers) sequences dominated the nifH gene pool, whereas the nifH transcript pool was dominated by sequences related to Lyngbya spp. Single-cell isotope analysis of (15)N₂-incubated mat samples via high-resolution secondary ion mass spectrometry (NanoSIMS) revealed that Cyanobacteria were enriched in (15)N, with the highest enrichment being detected in Lyngbya spp. filaments (on average 4.4 at% (15)N), whereas the Deltaproteobacteria (identified by CARD-FISH) were not significantly enriched. We investigated the potential dilution effect from CARD-FISH on the isotopic composition and concluded that the dilution bias was not substantial enough to influence our conclusions. Our combined data provide evidence that members of the Cyanobacteria, especially Lyngbya spp., actively contributed to N₂ fixation in the intertidal mats, whereas support for significant N₂ fixation activity of the targeted deltaproteobacterial sulfate reducers could not be found.

Microbial communities are essential to the function of virtually all ecosystems and eukaryotes, including humans. However, it is still a major challenge to identify microbial cells active under natural conditions in complex systems. In this study, we developed a new method to identify and sort active microbes on the single-cell level in complex samples using stable isotope probing with heavy water (D2O) combined with Raman microspectroscopy. Incorporation of D2O-derived D into the biomass of autotrophic and heterotrophic bacteria and archaea could be unambiguously detected via C-D signature peaks in single-cell Raman spectra, and the obtained labeling pattern was confirmed by nanoscale-resolution secondary ion MS. In fast-growing Escherichia coli cells, label detection was already possible after 20 min. For functional analyses of microbial communities, the detection of D incorporation from D2O in individual microbial cells via Raman microspectroscopy can be directly combined with FISH for the identification of active microbes. Applying this approach to mouse cecal microbiota revealed that the host-compound foragers Akkermansia muciniphila and Bacteroides acidifaciens exhibited distinctive response patterns to amendments of mucin and sugars. By Raman-based cell sorting of active (deuterated) cells with optical tweezers and subsequent multiple displacement amplification and DNA sequencing, novel cecal microbes stimulated by mucin and/or glucosamine were identified, demonstrating the potential of the nondestructive D2O-Raman approach for targeted sorting of microbial cells with defined functional properties for single-cell genomics.

To study how microbes establish themselves in a mammalian gut environment, we colonized germ-free mice with microbial communities from human, zebrafish, and termite guts, human skin and tongue, soil, and estuarine microbial mats. Bacteria from these foreign environments colonized and persisted in the mouse gut; their capacity to metabolize dietary and host carbohydrates and bile acids correlated with colonization success. Cohousing mice harboring these xenomicrobiota or a mouse cecal microbiota, along with germ-free "bystanders," revealed the success of particular bacterial taxa in invading guts with established communities and empty gut habitats. Unanticipated patterns of ecological succession were observed; for example, a soil-derived bacterium dominated even in the presence of bacteria from other gut communities (zebrafish and termite), and human-derived bacteria colonized germ-free bystander mice before mouse-derived organisms. This approach can be generalized to address a variety of mechanistic questions about succession, including succession in the context of microbiota-directed therapeutics., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Past studies of hydrogen cycling in hypersaline microbial mats have shown an active nighttime cycle, with production largely from Cyanobacteria and consumption from sulfate-reducing bacteria (SRB). However, the mechanisms and magnitude of hydrogen cycling have not been extensively studied. Two mats types near Guerrero Negro, Mexico-permanently submerged Microcoleus microbial mat (GN-S), and intertidal Lyngbya microbial mat (GN-I)-were used in microcosm diel manipulation experiments with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), molybdate, ammonium addition, and physical disruption to understand the processes responsible for hydrogen cycling between mat microbes. Across microcosms, H2 production occurred under dark anoxic conditions with simultaneous production of a suite of organic acids. H2 production was not significantly affected by inhibition of nitrogen fixation, but rather appears to result from constitutive fermentation of photosynthetic storage products by oxygenic phototrophs. Comparison to accumulated glycogen and to CO2 flux indicated that, in the GN-I mat, fermentation released almost all of the carbon fixed via photosynthesis during the preceding day, primarily as organic acids. Across mats, although oxygenic and anoxygenic phototrophs were detected, cyanobacterial [NiFe]-hydrogenase transcripts predominated. Molybdate inhibition experiments indicated that SRBs from a wide distribution of DsrA phylotypes were responsible for H2 consumption. Incubation with (13)C-acetate and NanoSIMS (secondary ion mass-spectrometry) indicated higher uptake in both Chloroflexi and SRBs relative to other filamentous bacteria. These manipulations and diel incubations confirm that Cyanobacteria were the main fermenters in Guerrero Negro mats and that the net flux of nighttime fermentation byproducts (not only hydrogen) was largely regulated by the interplay between Cyanobacteria, SRBs, and Chloroflexi.

The nonheterocystous filamentous cyanobacterium strain ESFC-1 has recently been isolated from a marine microbial mat system, where it was identified as belonging to a recently discovered lineage of active nitrogen-fixing microorganisms. Here, we report the draft genome sequence of this isolate. The assembly consists of 3 scaffolds and contains 5,632,035 bp with a GC content of 46.5%.

Anaerobic ammonium-oxidizing (anammox) bacteria are responsible for a significant portion of the loss of fixed nitrogen from the oceans, making them important players in the global nitrogen cycle. To date, marine anammox bacteria found in marine water columns and sediments worldwide belong almost exclusively to the 'Candidatus Scalindua' species, but the molecular basis of their metabolism and competitive fitness is presently unknown. We applied community sequencing of a marine anammox enrichment culture dominated by 'Candidatus Scalindua profunda' to construct a genome assembly, which was subsequently used to analyse the most abundant gene transcripts and proteins. In the S. profunda assembly, 4756 genes were annotated, and only about half of them showed the highest identity to the only other anammox bacterium of which a metagenome assembly had been constructed so far, the freshwater 'Candidatus Kuenenia stuttgartiensis'. In total, 2016 genes of S. profunda could not be matched to the K. stuttgartiensis metagenome assembly at all, and a similar number of genes in K.stuttgartiensis could not be found in S. profunda. Most of these genes did not have a known function but 98 expressed genes could be attributed to oligopeptide transport, amino acid metabolism, use of organic acids and electron transport. On the basis of the S. profunda metagenome, and environmental metagenome data, we observed pronounced differences in the gene organization and expression of important anammox enzymes, such as hydrazine synthase (HzsAB), nitrite reductase (NirS) and inorganic nitrogen transport proteins. Adaptations of Scalindua to the substrate limitation of the ocean may include highly expressed ammonium, nitrite and oligopeptide transport systems and pathways for the transport, oxidation, and assimilation of small organic compounds that may allow a more versatile lifestyle contributing to the competitive fitness of Scalindua in the marine realm., (© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.)

Photosynthetic microbial mats possess extraordinary phylogenetic and functional diversity that makes linking specific pathways with individual microbial populations a daunting task. Close metabolic and spatial relationships between Cyanobacteria and Chloroflexi have previously been observed in diverse microbial mats. Here, we report that an expressed metabolic pathway for the anoxic catabolism of photosynthate involving Cyanobacteria and Chloroflexi in microbial mats can be reconstructed through metatranscriptomic sequencing of mats collected at Elkhorn Slough, Monterey Bay, CA, USA. In this reconstruction, Microcoleus spp., the most abundant cyanobacterial group in the mats, ferment photosynthate to organic acids, CO2 and H2 through multiple pathways, and an uncultivated lineage of the Chloroflexi take up these organic acids to store carbon as polyhydroxyalkanoates. The metabolic reconstruction is consistent with metabolite measurements and single cell microbial imaging with fluorescence in situ hybridization and NanoSIMS.

N(2) fixation is a key process in photosynthetic microbial mats to support the nitrogen demands associated with primary production. Despite its importance, groups that actively fix N(2) and contribute to the input of organic N in these ecosystems still remain largely unclear. To investigate the active diazotrophic community in microbial mats from the Elkhorn Slough estuary, Monterey Bay, CA, USA, we conducted an extensive combined approach, including biogeochemical, molecular and high-resolution secondary ion mass spectrometry (NanoSIMS) analyses. Detailed analysis of dinitrogenase reductase (nifH) transcript clone libraries from mat samples that fixed N(2) at night indicated that cyanobacterial nifH transcripts were abundant and formed a novel monophyletic lineage. Independent NanoSIMS analysis of (15)N(2)-incubated samples revealed significant incorporation of (15)N into small, non-heterocystous cyanobacterial filaments. Mat-derived enrichment cultures yielded a unicyanobacterial culture with similar filaments (named Elkhorn Slough Filamentous Cyanobacterium-1 (ESFC-1)) that contained nifH gene sequences grouping with the novel cyanobacterial lineage identified in the transcript clone libraries, displaying up to 100% amino-acid sequence identity. The 16S rRNA gene sequence recovered from this enrichment allowed for the identification of related sequences from Elkhorn Slough mats and revealed great sequence diversity in this cluster. Furthermore, by combining (15)N(2) tracer experiments, fluorescence in situ hybridization and NanoSIMS, in situ N(2) fixation activity by the novel ESFC-1 group was demonstrated, suggesting that this group may be the most active cyanobacterial diazotroph in the Elkhorn Slough mat. Pyrotag sequences affiliated with ESFC-1 were recovered from mat samples throughout 2009, demonstrating the prevalence of this group. This work illustrates that combining standard and single-cell analyses can link phylogeny and function to identify previously unknown key functional groups in complex ecosystems.

Hydrogen (H(2)) release from photosynthetic microbial mats has contributed to the chemical evolution of Earth and could potentially be a source of renewable H(2) in the future. However, the taxonomy of H(2)-producing microorganisms (hydrogenogens) in these mats has not been previously determined. With combined biogeochemical and molecular studies of microbial mats collected from Elkhorn Slough, Monterey Bay, California, we characterized the mechanisms of H(2) production and identified a dominant hydrogenogen. Net production of H(2) was observed within the upper photosynthetic layer (0-2 mm) of the mats under dark and anoxic conditions. Pyrosequencing of rRNA gene libraries generated from this layer demonstrated the presence of 64 phyla, with Bacteriodetes, Cyanobacteria and Proteobacteria dominating the sequences. Sequencing of rRNA transcripts obtained from this layer demonstrated that Cyanobacteria dominated rRNA transcript pyrotag libraries. An OTU affiliated to Microcoleus spp. was the most abundant OTU in both rRNA gene and transcript libraries. Depriving mats of sunlight resulted in an order of magnitude decrease in subsequent nighttime H(2) production, suggesting that newly fixed carbon is critical to H(2) production. Suppression of nitrogen (N(2))-fixation in the mats did not suppress H(2) production, which indicates that co-metabolic production of H(2) during N(2)-fixation is not an important contributor to H(2) production. Concomitant production of organic acids is consistent with fermentation of recently produced photosynthate as the dominant mode of H(2) production. Analysis of rRNA % transcript:% gene ratios and H(2)-evolving bidirectional [NiFe] hydrogenase % transcript:% gene ratios indicated that Microcoelus spp. are dominant hydrogenogens in the Elkhorn Slough mats.

An anaerobic nitrate-reducing enrichment culture was established with a cyclic saturated petroleum hydrocarbon, cyclohexane, the fate of which in anoxic environments has been scarcely investigated. GC-MS showed cyclohexylsuccinate as a metabolite, in accordance with an anaerobic enzymatic activation of cyclohexane by carbon-carbon addition to fumarate. Furthermore, long-chain cyclohexyl-substituted cell fatty acids apparently derived from cyclohexane were detected. Nitrate reduction was not only associated with cyclohexane utilization but also with striking depletion of added ammonium ions. Significantly more ammonium was consumed than could be accounted for by assimilation. This indicated the occurrence of anaerobic ammonium oxidation (anammox) with nitrite from cyclohexane-dependent nitrate reduction. Indeed, nitrite depletion was stimulated upon further addition of ammonium. Analysis of 16S rRNA genes and subsequent cell hybridization with specific probes showed that approximately 75% of the bacterial cells affiliated with the Geobacteraceae and approximately 18% with Candidatus 'Brocadia anammoxidans' (member of the Planctomycetales), an anaerobic ammonium oxidizer. These results and additional quantitative growth experiments indicated that the member of the Geobacteraceae reduced nitrate with cyclohexane to nitrite and some ammonium; the latter two and ammonium added to the medium were scavenged by anammox bacteria to yield dinitrogen. A model was established to quantify the partition of each microorganism in the overall process. Such hydrocarbon oxidation by an alleged 'denitrification' ('pseudo-denitrification'), which in reality is a dissimilatory loop through anammox, can in principle also occur in other microbial systems with nitrate-dependent hydrocarbon attenuation.