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The ecophysiology of complete ammonia-oxidizing bacteria (CMX) of the genus Nitrospiraand their widespread occurrence in groundwater suggests that CMX bacteria have a competitive advantage over ammonia-oxidizing bacteria (AOB) and archaea (AOA) in these environments. However, the specific contribution of their activity to nitrification processes has remained unclear. We aimed to disentangle the contribution of CMX, AOA and AOB to nitrification and to identify the environmental drivers of their niche differentiation at different levels of ammonium and oxygen in oligotrophic carbonate rock aquifers. CMX ammonia monooxygenase sub-unit A (amoA) genes accounted on average for 16 to 75% of the total groundwater amoAgenes detected. Nitrification rates were positively correlated to CMX clade A associated phylotypes and AOB affiliated with Nitrosomonas ureae. Short-term incubations amended with the nitrification inhibitors allylthiourea and chlorate suggested that AOB contributed a large fraction to overall ammonia oxidation, while metaproteomics analysis confirmed an active role of CMX in both ammonia and nitrite oxidation. Ecophysiological niche differentiation of CMX clades A and B, AOB and AOA was linked to their requirements for ammonium, oxygen tolerance, and metabolic versatility. Our results demonstrate that despite numerical predominance of CMX, the first step of nitrification in oligotrophic groundwater appears to be primarily governed by AOB. Higher growth yields at lower ammonia turnover rates and energy derived from nitrite oxidation most likely enable CMX to maintain consistently high populations.

Carbon cycling is one of the major biogeochemical processes driven by bacteria. Autotrophic bacteria convert carbon dioxide (CO2) into organic compounds that are used by heterotrophs. Mixotrophic bacteria can employ both autotrophy and heterotrophy for growth. The characterization of the lifestyle of individual cells is essential to understand the microbial activity and thus reveal the implication of bacteria in the carbon flux. In this study, we used groundwater bacteria to investigate the potential of Raman-D2O labeling in combination with chemometrics to identify the carbon assimilation strategies of bacteria. Classification models were built using principal component analysis (PCA) followed by linear discriminant analysis (LDA). Autotrophs assimilated a significantly higher amount (mean C–D ratio between 16.63 and 21.69%) of deuterium than heterotrophs. The C–D signal only provides information about the activity since it appears in the Raman-silent region, where no interference with the taxonomic information is expected. The classification between autotrophs and heterotrophs achieved an overall accuracy of 96.3%. In the validation step with an independent dataset containing species not included in the model, the PCA-LDA model achieved 100% accuracy. This demonstrated that the C–D signal contributed to the identification of autotrophic and heterotrophic bacterial cells. This work reports a robust, rapid, and nondestructive approach for the identification of single cells based on their carbon acquisition strategies. The present study foresees the potential of Raman-D2O labeling as a promising method for automated discrimination of in situfunctional activities of bacteria in environmental systems.

Current understanding of organic carbon inputs into ecosystems lacking photosynthetic primary production is predicated on data and inferences derived almost entirely from metagenomic analyses. The elevated abundances of putative chemolithoautotrophs in groundwaters suggest that dark CO2fixation is an integral component of subsurface trophic webs. To understand the impact of autotrophically fixed carbon, the flux of CO2-derived carbon through various populations of subsurface microbiota must first be resolved, both quantitatively and temporally. Here we implement novel Stable Isotope Cluster Analysis to render a time-resolved and quantitative evaluation of 13CO2-derived carbon flow through a groundwater community in microcosms stimulated with reduced sulfur compounds. We demonstrate that mixotrophs, not strict autotrophs, were the most abundant active organisms in groundwater microcosms. Species of Hydrogenophaga, Polaromonas, Dechloromonas, and other metabolically versatile mixotrophs drove the production and remineralization of organic carbon. Their activity facilitated the replacement of 43% and 80% of total microbial carbon stores in the groundwater microcosms with 13C in just 21 and 70 days, respectively. The mixotrophs employed different strategies for satisfying their carbon requirements by balancing CO2fixation and uptake of available organic compounds. These different strategies might provide fitness under nutrient-limited conditions, explaining the great abundances of mixotrophs in other oligotrophic habitats, such as the upper ocean and boreal lakes.

The terrestrial subsurface contains nearly all of Earth’s freshwater reserves and harbours the majority of our planet’s total prokaryotic biomass. Although genetic surveys suggest these organisms rely on in situ carbon fixation, rather than the photosynthetically derived organic carbon transported from surface environments, direct measurements of carbon fixation in the subsurface are absent. Using an ultra-low level 14C-labelling technique, we estimate in situ carbon fixation rates in a carbonate aquifer. We find these rates are similar to those measured in oligotrophic marine surface waters and up to six-fold greater than those observed in the lower euphotic zone. Our empirical carbon fixation rates agree with nitrification rate data. Metagenomic analyses reveal abundant putative chemolithoautotrophic members of an uncharacterized order of Nitrospiria that may be behind the carbon fixation. On the basis of our determined carbon fixation rates, we conservatively extrapolate global primary production in carbonate groundwaters (10% of global reserves) to be 0.11 Pg carbon per year. These rates fall within the range found for oligotrophic marine surface waters, indicating a substantial contribution of in situ primary production to subsurface ecosystem processes. We further suggest that, just as phototrophs are for marine biogeochemical cycling, such subsurface carbon fixation is potentially foundational to subsurface trophic webs.

Raman-stable isotope labeling using heavy water (Raman-D2O) is attracting great interest as a fast technique with various applications ranging from the identification of pathogens in medical samples to the determination of microbial activity in the environment. Despite its widespread applications, little is known about the fundamental processes of hydrogen–deuterium (H/D) exchange, which are crucial for understanding molecular interactions in microorganisms. By combining two-dimensional (2D) correlation spectroscopy and Raman deuterium labeling, we have investigated H/D exchange in bacterial cells under time dependence. Most C–H stretching signals decreased in intensity over time, prior to the formation of the C–D stretching vibration signals. The intensity of the C–D signal gradually increased over time, and the shape of the C–D signal was more uniform after longer incubation times. Deuterium uptake showed high variability between the bacterial genera and mainly led to an observable labeling of methylene and methyl groups. Thus, the C–D signal encompassed a combination of symmetric and antisymmetric CD2and CD3stretching vibrations, depending on the bacterial genera. The present study allowed for the determination of the sequential order of deuterium incorporation into the functional groups of proteins, lipids, and nucleic acids and hence understanding the process of biomolecule synthesis and the growth strategies of different bacterial taxa. We present the combination of Raman-D2O labeling and 2D correlation spectroscopy as a promising approach to gain a fundamental understanding of molecular interactions in biological systems.

Current understanding of organic carbon inputs into ecosystems lacking photosynthetic primary production is predicated on data and inferences derived almost entirely from metagenomic analyses. The elevated abundances of putative chemolithoautotrophs in groundwaters suggest that dark CO2fixation is an integral component of subsurface trophic webs. To understand the impact of autotrophically fixed carbon, the flux of CO2-derived carbon through various populations of subsurface microbiota must first be resolved, both quantitatively and temporally. Here we implement novel Stable Isotope Cluster Analysis to render a time-resolved and quantitative evaluation of 13CO2-derived carbon flow through a groundwater community in microcosms stimulated with reduced sulfur compounds. We demonstrate that mixotrophs, not strict autotrophs, were the most abundant active organisms in groundwater microcosms. Species of Hydrogenophaga, Polaromonas, Dechloromonas, and other metabolically versatile mixotrophs drove the production and remineralization of organic carbon. Their activity facilitated the replacement of 43% and 80% of total microbial carbon stores in the groundwater microcosms with 13C in just 21 and 70 days, respectively. The mixotrophs employed different strategies for satisfying their carbon requirements by balancing CO2fixation and uptake of available organic compounds. These different strategies might provide fitness under nutrient-limited conditions, explaining the great abundances of mixotrophs in other oligotrophic habitats, such as the upper ocean and boreal lakes.

Coexistence of microaerophilic Fe(II)-oxidizers and anaerobic Fe(III)-reducers in environments with fluctuating redox conditions is a prime example of mutualism, in which both partners benefit from the sustained Fe-pool. Consequently, the Fe-cycling machineries (i.e., metal-reducing or –oxidizing pathways) should be most affected during co-cultivation. However, contrasting growth requirements impeded systematic elucidation of their interactions. To disentangle underlying interaction mechanisms, we established a suboxic co-culture system of Sideroxydanssp. CL21 and Shewanella oneidensis. We showed that addition of the partner’s cell-free supernatant enhanced both growth and Fe(II)-oxidizing or Fe(III)-reducing activity of each partner. Metabolites of the exometabolome of Sideroxydanssp. CL21 are generally upregulated if stimulated with the partner´s spent medium, while S. oneidensisexhibits a mixed metabolic response in accordance with a balanced response to the partner. Surprisingly, RNA-seq analysis revealed genes involved in Fe-cycling were not differentially expressed during co-cultivation. Instead, the most differentially upregulated genes included those encoding for biopolymer production, lipoprotein transport, putrescine biosynthesis, and amino acid degradation suggesting a regulated inter-species biofilm formation. Furthermore, the upregulation of hydrogenases in Sideroxydanssp. CL21 points to competition for H2as electron donor. Our findings reveal that a complex metabolic and transcriptomic response, but not accelerated formation of Fe-end products, drive interactions of Fe-cycling microorganisms.

A rapid and reliable method for the differentiation between active and inactive bacteria at single cell level is urgently needed in many fields including clinical diagnosis and environmental microbiology, to understand the contribution of metabolically active bacteria in fundamental processes triggering environmental and public health risks. Here, using heavy water (D2O) with Raman-stable isotope labeling (Raman-D2O), we evaluated the reliability of the quantification of deuterium uptake, a well-known indicator for the general metabolic activity of bacteria. For this purpose, we based our study on the quantification of deuterium assimilation from heavy water into single bacterial cells to check the influence of carbon source and bacterial identity on the deuterium uptake. We show that compared to complex carbon substrates, the deuterium assimilation is higher in the presence of simpler substrates such as sugars but differs significantly among bacterial isolates. Despite this variability, the developed classification models could differentiate deuterium labeled and nonlabeled single cells with high sensitivity and specificity. Highlighting the variability between single bacterial cells, the study emphasizes the challenges in establishing a threshold in terms of deuterium uptake to distinguish deuterium labeled and nonlabeled cells. Overall, we show that the Raman-D2O approach, when coupled with chemometrics, constitutes a powerful approach for monitoring single bacterial cells.

Volatile sulfur compounds (VSCs) contribute to acid rain, cloud formation, and albedo, and thus influence the climate. Their global emissions are quite uncertain, especially contributions from freshwater wetlands. We investigated the processes leading to hydrogen sulfide (H2S), methanethiol (MeSH), and dimethyl sulfide (Me2S) emissions in a slightly acidic peatland and found multiple indications that organic matter degradation rather than sulfate reduction is the main driver for Me2S emissions in this system. Evidence includes: the lack of labeled Me234S production after addition of Na234SO4despite high emissions of Me34SH and H234S, and increased emission rates when soils were amended with organic substrates containing thiol groups (H2S emissions), methylthiols (MeSH), and dimethyl sulfonio groups (Me2S). VSC precursors were identified from an Untargeted Metabolomics data set from the same soil. The abundance of sulfur cycling microbes like AcidobacteriaSD 1 and Desulfosporosinuscorrelated with VSC emissions. We conclude that organic matter degradation is more important than sulfate reduction as a source of Me2S in our peatland system, and potentially also in other organic and wetland soils. Sulfur gases influence acid rain and the way clouds form. In peats and swamps, there are two mechanisms to form sulfur gases. First, when the soils are flooded soil microbes use sulfate for respiration as an alternative to oxygen for the degradation of organic matter. During this process, microbes can convert sulfate to the sulfur gases hydrogen sulfide and methanethiol. In some wetland soils, methanethiol can then be converted to dimethyl sulfide, but we found that this only happens very slowly in our peatland soil. Second, soil microbes degrade organic sulfur compounds and produce sulfur gases as waste even in conditions where O2is present. Which sulfur gas is produced depends on the structure of the organic sulfur compound that serves as a substrate. Compounds with a free ‐S‐H group form hydrogen sulfide (H‐S‐H), compounds with a ‐S‐CH3group form methanethiol (CH3‐S‐H), compounds with a ‐S(‐CH3)2group form dimethyl sulfide (H3C‐S‐CH3), while compounds without those groups do not produce sulfur gases. We also found microbes that could be feeding on these VSC precursors. This means that emissions of sulfur gases from wetlands are caused not just by the reduction of sulfate under anoxic conditions, but directly from the degradation of organic matter, even in oxic soils outside of wetlands. Organic matter decomposition is a main source of volatile sulfur compound (VSC) emitted from organic soils and wetlandsSulfate reduction is also an important source of methanethiol and hydrogen sulfide but not dimethyl sulfideInterconversion of volatile sulfur compounds is slower than formation and emission Organic matter decomposition is a main source of volatile sulfur compound (VSC) emitted from organic soils and wetlands Sulfate reduction is also an important source of methanethiol and hydrogen sulfide but not dimethyl sulfide Interconversion of volatile sulfur compounds is slower than formation and emission

Determining the biogeochemical pathways utilized by microbes living in groundwater is essential for understanding the subsurface C cycle and the fate of organic compounds, including pollutants. The radiocarbon signature (Δ14C) of fatty acid methyl esters derived from microbial phospholipids (PLFA) provides useful information for differentiating microbial C sources and infering microbial metabolism. However, in subsurface environments, those analyses remain challenging. Here we present a method combining large volume groundwater filtration (up to 10,000 L) and PLFA purification for subsequent compound‐specific radiocarbon analyses. The analytical method involves conventional chemical extraction of PLFA followed by purification of individual compounds by semipreparative high‐performance liquid chromatography. Different saturated PLFA in amounts of up to 10 μg each can be simultaneously separated on a C18high‐load column using a mixture of MeOH/water and acetonitrile as the mobile phase. Our procedure introduced dead‐Cextcontaminations of 0.57 ± 0.29 and 0.35 ± 0.18 μg for the high‐performance liquid chromatography and combustion/graphitization steps of the sample preparation, respectively. However, tests on different high‐performance liquid chromatography C18columns revealed a large difference in dead Cextassociated with column bleed. Modern Cextin the amount of 0.40 ± 0.20 μg was introduced by the combustion/graphitization step of the sample preparation, but other steps did not add modern Cext.The entire method recovered ∼50% of the purified compounds on average, but this did not affect their 14C content. This method will allow routine analysis of the Δ14C of PLFA isolated from groundwaters or other sample types, revealing the relationships between microbial and soil‐derived C, sedimentary or dissolved C sources. Fatty acid methyl esters derived from microbial phospholipids were purified and concentrated for compound‐specific radiocarbon analysesThe method involves chemical extraction followed by semipreparative high‐performance liquid chromatography of diverse saturated FAMELarge differences in the degree of extraneous C contamination were observed depending on the HPLC columns used for separations

Despite the global significance of the subsurface biosphere, the degree to which it depends on surface organic carbon (OC) is still poorly understood. Here, we compare stable and radiogenic carbon isotope compositions of microbial phospholipid fatty acids (PLFAs) with those of in situ potential microbial C sources to assess the major C sources for subsurface microorganisms in biogeochemical distinct shallow aquifers (Critical Zone Exploratory, Thuringia Germany). Despite the presence of younger OC, the microbes assimilated 14C‐free OC to varying degrees; ~31% in groundwater within the oxic zone, ~47% in an iron reduction zone, and ~70% in a sulfate reduction/anammox zone. The persistence of trace amounts of mature and partially biodegraded hydrocarbons suggested that autochthonous petroleum‐derived hydrocarbons were a potential 14C‐free C source for heterotrophs in the oxic zone. In this zone, Δ14C values of dissolved inorganic carbon (−366 ± 18‰) and 11MeC16:0 (−283 ± 32‰), an important component in autotrophic nitrite oxidizers, were similar enough to indicate that autotrophy is an important additional C fixation pathway. In anoxic zones, methane as an important C source was unlikely since the 13C‐fractionations between the PLFAs and CH4were inconsistent with kinetic isotope effects associated with methanotrophy. In the sulfate reduction/anammox zone, the strong 14C‐depletion of 10MeC16:0 (−942 ± 22‰), a PLFA common in sulfate reducers, indicated that those bacteria were likely to play a critical part in 14C‐free sedimentary OC cycling. Results indicated that the 14C‐content of microbial biomass in shallow sedimentary aquifers results from complex interactions between abundance and bioavailability of naturally occurring OC, hydrogeology, and specific microbial metabolisms. Microbial lipids (PLFAs) and C sources in aquifers with different subsurface‐surface relationships were analyzed for their 14C‐contentSubsurface microbes incorporated 14C‐free C in proportions depending on the physiological strategies of the microorganism communityAutotrophic nitrite oxidation occurred in oxic zone, whereas heterotrophy on sedimentary C dominated in sulfate reduction/anammox zone

Chemosynthetic Fe-oxidizing communities are common at diffuse-flow hydrothermal vents throughout the world’s oceans. The foundational members of these communities are the Zetaproteobacteria, a class of Proteobacteria that is primarily associated with ecosystems fueled by ferrous iron, Fe(II). We report here the discovery of two new isolates of Zetaproteobacteria isolated from the Mid-Atlantic Ridge (TAG-1), and the Mariana back-arc (SV-108), that are unique in that they can utilize either Fe(II) or molecular hydrogen (H2) as sole electron donor and oxygen as terminal electron acceptor for growth. Both strains precipitated Fe-oxyhydroxides as amorphous particulates. The cell doubling time on H2vs Fe(II) for TAG-1 was 14.1 vs 21.8 h, and for SV-108 it was 16.3 vs 20 h, and it appeared both strains could use either H2or Fe(II) simultaneously. The strains were close relatives, based on genomic analysis, and both possessed genes for the uptake NiFe-hydrogenase required for growth on H2. These two strains belong to Zetaproteobacteria operational taxonomic unit 9 (ZetaOTU9). A meta-analysis of public databases found ZetaOTU9 was only associated with Fe(II)-rich habitats, and not in other environments where known H2-oxidizers exist. These results expand the metabolic repertoire of the Zetaproteobacteria, yet confirm that Fe(II) metabolism is the primary driver of their physiology and ecology.

Marine and lake snow is a continuous shower of mixed organic and inorganic aggregates falling from the upper water where primary production is substantial. These pelagic aggregates provide a niche for microbes that can exploit these physical structures and resources for growth, thus are local hot spots for microbial activity. However, processes underlying their formation remain unknown. Here, we investigated the role of chemical signaling between two co-occurring bacteria that each make up more than 10% of the community in iron-rich lakes aggregates (iron snow). The filamentous iron-oxidizing Acidithrix strain showed increased rates of Fe(II) oxidation when incubated with cell-free supernatant of the heterotrophic iron-reducing Acidiphilium strain. Amendment of Acidithrix supernatant to motile cells of Acidiphilium triggered formation of cell aggregates displaying similar morphology to those of iron snow. Comparative metabolomics enabled the identification of the aggregation-inducing signal, 2-phenethylamine, which also induced faster growth of Acidiphilium. We propose a model that shows rapid iron snow formation, and ultimately energy transfer from the photic zone to deeper water layers, is controlled via a chemically mediated interplay.

Rare species are increasingly recognized as crucial, yet vulnerable components of Earth’s ecosystems. This is also true for microbial communities, which are typically composed of a high number of relatively rare species. Recent studies have demonstrated that rare species can have an over-proportional role in biogeochemical cycles and may be a hidden driver of microbiome function. In this review, we provide an ecological overview of the rare microbial biosphere, including causes of rarity and the impacts of rare species on ecosystem functioning. We discuss how rare species can have a preponderant role for local biodiversity and species turnover with rarity potentially bound to phylogenetically conserved features. Rare microbes may therefore be overlooked keystone species regulating the functioning of host-associated, terrestrial and aquatic environments. We conclude this review with recommendations to guide scientists interested in investigating this rapidly emerging research area.

We demonstrate in this study that natural green rust nanoparticles and their synthetic analogs can be complex polycrystalline phases composed of crystallites only a few nanometers in size, and they often include nanometer-sized regions of amorphous material. The natural green rusts are Zn-bearing pseudo-hexagonal platelets previously identified by us in the contaminated mine drainage of the former Ronneburg uranium mine in Germany (Johnson et al. 2014). We also identified Ni- and Cubearing green rust platelets in the sediment underlying the drainage outflow 20 m downstream, and, using dark-field transmission electron microscopy (DF-TEM), found that these natural green rusts are not usually structurally coherent single crystals. Synthetic sulfate green rusts are also polycrystalline and composed of crystallites of only a few nanometers in size, though different synthesis conditions produced different patterns of polycrystallinity. While pseudo-hexagonal platelets are the typical morphology of green rust, we also synthesized green rust nanorods, which have not previously been reported. In addition to the known characteristics of green rusts (including a very large aspect ratio and surface area to volume ratio, and the redox properties allowed by the structural mixture of Fe2+and Fe3+), these polycrystalline platelets exhibit a high abundance of defect sites and likely a rough surface topography. The combination of these characteristics has important implications for the reactivity of green rust with biogeochemical interfaces in natural and anthropogenic systems.

Environmental context Legacy contamination from mining operations is a serious and complex environmental problem. We examine a former uranium mine where groundwater leaving the site enters a stream with chemically dramatic effects resulting in a fundamental change in the way contaminant metals are transported to the surface environment. The results are important for our understanding of how these contaminants are dispersed, and how they could interact with the biosphere. Abstract In this study of iron- and silica-bearing nanoparticle and colloid aggregates in slightly acidic mine drainage, we combined bulk scale geochemistry techniques with detailed nanoscale analyses using high-resolution transmission electron microscopy (HR-TEM) to demonstrate the complexity of iron oxide formation and transformation at a steep redox gradient (groundwater outflow into a stream), and the resulting role in metal(loid) uptake. We also identified pseudohexagonal nanosheets of Zn-bearing green rust in outflowing groundwater using HR-TEM. This is only the second study where green rust was identified in groundwater, and the second to examine naturally occurring green rust with analytical TEM. In aerated downstream waters, we found aggregates of poorly crystalline iron oxide particles (20–200nm in diameter). Inductively coupled plasma–mass spectrometry (ICP-MS) analysis of water fractions shows that most elements such as Ni and Zn were found almost exclusively in the dissolved–nanoparticulate (<0.1μm) fraction, whereas Cu and As were primarily associated with suspended particles. In the underlying sediments composed of deposited particles, goethite nanoneedles formed on the ferrihydrite surfaces of larger aggregated particles (100–1000nm), resulting in more reactive surface area for metal(loid) uptake. Sequential extraction of sediments showed that many metal(loid)s, particularly As and Zn, were associated with iron oxides identified as ferrihydrite, goethite and possibly schwertmannite. Amorphous silica co-precipitation with iron oxides was prevalent at all sampling sites, but its effect on metal(loid) sorption is unknown. Fine-grained iron oxide sediments are easily remobilised during turbulent flow events, adding to the mobility of the associated metals.

Acetogens reduce CO2to acetate via the acetyl-CoA pathway and have been classically thought of as obligately anaerobic bacteria. Nearly 100 acetogenic species from 20 different genera have been isolated to date. These isolates are able to use very diverse electron donors and acceptors, and it is thus very likely that the in situactivities of acetogens are very diverse and not restricted to acetogenesis. Since acetogens constitute a very phylogenetically diverse bacteriological group, it should be anticipated that they can inhabit, and have impact on, diverse habitats. Indeed, they have been isolated from a broad range of habitats, including oxic soils and other habitats not generally regarded as suitable for acetogens. Although the ecological impact of acetogens is determined by the in situmanifestation of their physiological potentials, assessing their in situactivities is difficult due to their physiological and phylogenetic diversities. This mini-review will highlight a few of the physiological and ecological realities of acetogens, and will focus on: (i) metabolic diversities and regulation, (ii) phylogenetic diversity and molecular ecology, and (iii) the capacity of acetogens to cope with oxic conditions under both laboratory and in situconditions.

Although anaerobic microorganisms can be isolated from well-drained soils, the qualitative and quantitative composition of the anaerobic populations potentially involved in the turnover of carbon in oxic soils is poorly resolved. In the present study, most probable number (MPN) estimates demonstrated that the number of anaerobes that were cultured from both forest (beech) mineral soil and litter approximated 107to 108(g dry wt. soil or litter)−1, a value that also approximated the number of acetate-producing anaerobes. When a complex mixture of substrates was provided, the substrate-product profiles of the MPN series indicated that the substrates that yielded the highest numbers of microbes were: yeast extract>sugars>H2>organic acids and alcohols>methoxylated aromatic compounds. Except for yeast extract, this sequence also reflected the temporal consumption of these substrates in the MPN series. The cultured anaerobic population was dominated by members of the Enterobacteriaceae and other facultative anaerobes; the facultative microorganisms appeared to determine the initial anaerobic activities of both forest soil and litter. Compared to mineral soil, litter contained more sugar-consuming anaerobes but lower numbers of butyrate-forming anaerobes. H2, ethanol, butanediol, and aromatic methoxyl groups were utilized by acetogenic bacteria. H2-utilizing acetogens were a dominant group of obligate anaerobes, attesting to the ability of acetogens to survive transient or sustained periods of aeration in habitats such as leaf litter. The numbers of cultured methanogens were negligible. Together with previous findings, these results (i) demonstrate that the formation of acetate in soils is dependent on the combined activity of facultative and obligately anaerobic microorganisms, and (ii) emphasize the potential importance of acetate as an intermediate during the turnover of carbon in habitats subject to steep oxygen gradients.

Groundwater environments provide habitats for diverse microbial communities, and although Archaea usually represent a minor fraction of communities, they are involved in key biogeochemical cycles. We analysed the archaeal diversity within a mixed carbonate-rock/siliciclastic-rock aquifer system, vertically from surface soils to subsurface groundwater including aquifer and aquitard rocks. Archaeal diversity was also characterized along a monitoring well transect that spanned surface land uses from forest/woodland to grassland and cropland. Sequencing of 16S rRNA genes showed that only a few surface soil-inhabiting Archaea were present in the groundwater suggesting a restricted input from the surface. Dominant groups in the groundwater belonged to the marine group I (MG-I) Thaumarchaeota and the Woesearchaeota. Most of the groups detected in the aquitard and aquifer rock samples belonged to either cultured or predicted lithoautotrophs (e.g., Thaumarchaeota or Hadesarchaea). Furthermore, to target autotrophs, a series of 13CO2 stable isotope-probing experiments were conducted using filter pieces obtained after filtration of 10,000 L of groundwater to concentrate cells. These incubations identified the SAGMCG Thaumarchaeota and Bathyarchaeota as groundwater autotrophs. Overall, the results suggest that the majority of Archaea on rocks are fixing CO2, while archaeal autotrophy seems to be limited in the groundwater.