Cell-specific rates of sulfate reduction and fermentation in the sub-seafloor biosphere.

Microorganisms in subsurface sediments live from recalcitrant organic matter deposited thousands or millions of years ago. Their catabolic activities are low, but the deep biosphere is of global importance due to its volume. The stability of deeply buried sediments provides a natural laboratory where prokaryotic communities that live in steady state with their environments can be studied over long time scales. We tested if a balance is established between the flow of energy, the microbial community size, and the basal power requirement needed to maintain cells in sediments buried meters below the sea floor. We measured rates of carbon oxidation by sulfate reduction and counted the microbial cells throughout ten carefully selected sediment cores with ages from years to millions of years. The rates of carbon oxidation were converted to power (J s-1 i.e., Watt) using the Gibbs free energy of the anaerobic oxidation of complex organic carbon. We separated energy dissipation by fermentation from sulfate reduction. Similarly, we separated the community into sulfate reducers and non-sulfate reducers based on the dsrB gene, so that sulfate reduction could be related to sulfate reducers. We found that the per-cell sulfate reduction rate was stable near 10-2 fmol C cell-1 day-1 right below the zone of bioturbation and did not decrease with increasing depth and sediment age. The corresponding power dissipation rate was 10-17 W sulfate-reducing cell-1. The cell-specific power dissipation of sulfate reducers in old sediments was similar to the slowest growing anaerobic cultures. The energy from mineralization of organic matter that was not dissipated by sulfate reduction was distributed evenly to all cells that did not possess the dsrB gene, i.e., cells operationally defined as fermenting. In contrast to sulfate reducers, the fermenting cells had decreasing catabolism as the sediment aged. A vast difference in power requirement between fermenters and sulfate reducers caused the microbial community in old sediments to consist of a minute fraction of sulfate reducers and a vast majority of fermenters.

Methane production controls in a young thermokarst lake formed by abrupt permafrost thaw.

Methane (CH4 ) release to the atmosphere from thawing permafrost contributes significantly to global CH4 emissions. However, constraining the effects of thaw that control the production and emission of CH4 is needed to anticipate future Arctic emissions. Here are presented robust rate measurements of CH4 production and cycling in a region of rapidly degrading permafrost. Big Trail Lake, located in central Alaska, is a young, actively expanding thermokarst lake. The lake was investigated by taking two 1 m cores of sediment from different regions. Two independent methods of measuring microbial CH4  production, long term (CH4 accumulation) and short term (14 C tracer), produced similar average rates of 11 ± 3.5 and 9 ± 3.6 nmol cm-3  d-1 , respectively. The rates had small variations between the different lithological units, indicating homogeneous CH4 production despite heterogeneous lithology in the surface ~1 m of sediment. To estimate the total CH4 production, the CH4 production rates were multiplied through the 10-15 m deep talik (thaw bulb). This estimate suggests that CH4  production is higher than emission by a maximum factor of ~2, which is less than previous estimates. Stable and radioactive carbon isotope measurements showed that 50% of dissolved CH4 in the first meter was produced further below. Interestingly, labeled 14 C incubations with 2-14 C acetate and 14 C CO2 indicate that variations in the pathway used by microbes to produce CH4 depends on the age and type of organic matter in the sediment, but did not appear to influence the rates at which CH4  was produced. This study demonstrates that at least half of the CH4 produced by microbial breakdown of organic matter in actively expanding thermokarst is emitted to the atmosphere, and that the majority of this CH4 is produced in the deep sediment.

Potentially bioavailable iron produced through benthic cycling in glaciated Arctic fjords of Svalbard.

The Arctic has the highest warming rates on Earth. Glaciated fjord ecosystems, which are hotspots of carbon cycling and burial, are extremely sensitive to this warming. Glaciers are important for the transport of iron from land to sea and supply this essential nutrient to phytoplankton in high-latitude marine ecosystems. However, up to 95% of the glacially-sourced iron settles to sediments close to the glacial source. Our data show that while 0.6-12% of the total glacially-sourced iron is potentially bioavailable, biogeochemical cycling in Arctic fjord sediments converts the glacially-derived iron into more labile phases, generating up to a 9-fold increase in the amount of potentially bioavailable iron. Arctic fjord sediments are thus an important source of potentially bioavailable iron. However, our data suggests that as glaciers retreat onto land the flux of iron to the sediment-water interface may be reduced. Glacial retreat therefore likely impacts iron cycling in coastal marine ecosystems.

Physicochemical and biological controls of sulfide accumulation in a high temperature oil reservoir.

In order to maintain the reservoir pressure during secondary oil production large volumes of seawater are injected into reservoirs. This practice introduces high concentrations of sulfate into the reservoir promoting the growth of sulfate-reducing microorganisms (SRM) and results in the production of an increasing volume of produced water (PW) that needs to be discharged. SRM reduce sulfate to sulfide causing reservoir souring and as a mitigation strategy nitrate is injecting along with the seawater into the reservoir. We used PW from the Halfdan oil field (North Sea) to set up microcosms to determine the best reinjection strategy in order to inhibit SRM activity and minimize the environmental impact of PW during secondary oil production. We discuss the effect of temperature, electron donor, and sulfate and nitrate availability on sulfide production and microbial community composition. Temperature and the terminal electron acceptor played a key role in shaping the microbial community of the microcosms. PW reinjection at 62 °C inhibited SRM activity due to nitrite toxicity by encouraging nitrate reduction to nitrite by thermophilic nitrate reducers, while at 74 °C we observed complete absence of any microbial activity over the course of 150 days. KEY POINTS: • Temperature and the presence/ absence of nitrate shaped the microbial community structure. • Thermophilic nitrate reducers convert nitrate to ammonia with the accumulation of nitrite that inhibits sulfide production. • Nitrite inhibition is the most effective nitrate-based souring mitigation mechanisms. • The reinjection of hot produced water to oil reservoirs is a promising souring mitigation approach.

Macrofaunal control of microbial community structure in continental margin sediments.

Through a process called "bioturbation," burrowing macrofauna have altered the seafloor habitat and modified global carbon cycling since the Cambrian. However, the impact of macrofauna on the community structure of microorganisms is poorly understood. Here, we show that microbial communities across bioturbated, but geochemically and sedimentologically divergent, continental margin sites are highly similar but differ clearly from those in nonbioturbated surface and underlying subsurface sediments. Solid- and solute-phase geochemical analyses combined with modeled bioturbation activities reveal that dissolved O2 introduction by burrow ventilation is the major driver of archaeal community structure. By contrast, solid-phase reworking, which regulates the distribution of fresh, algal organic matter, is the main control of bacterial community structure. In nonbioturbated surface sediments and in subsurface sediments, bacterial and archaeal communities are more divergent between locations and appear mainly driven by site-specific differences in organic carbon sources.

Constraints on CaCO3 precipitation in superabsorbent polymer by aerobic bacteria.

Microbially induced CaCO3 precipitation (MICP) can give concrete self-healing properties. MICP agents are typically bacterial endospores which are coated into shelled granules, infused into expanded clay, or embedded into superabsorbent polymer (SAP). When small cracks appear in the cured concrete, the encapsulation is broken and the metabolic CO2 production from the germinated bacteria causes healing of the cracks by precipitation of CaCO3. Such systems are being tested empirically at large scales, but survival of endospores through preparation and application, as well as germination and growth kinetics of the germinated vegetative cells, remains poorly resolved. We encapsulated endospores of Bacillus subtilis and Bacillus alkalinitrilicus in crosslinked acrylamide-based SAP and quantified their germination, growth, and, in the case of B. alkalinitrilicus, CaCO3 precipitation potential. The endospores survived crosslinking and desiccation inside the polymer matrix. Microcalorimetry and microscopy showed that ~ 80% of the encapsulated endospores of both strains readily germinated after rehydration of freeze-dried SAP. Germinated cells grew into dense colonies of cells inside the SAP, and those of B. alkalinitrilicus calcified with up to 0.3 g CaCO3 produced per g desiccated SAP when incubated aerobically. Measurements by planar optodes indicated that the precipitation rates were inherently oxygen limited due to diffusional constraints, rather than limited by electron donor or Ca2+ availability. Such oxygen limitation will limit MICP in all water-saturated and oxygen-dependent systems, and MICP agents based on anaerobic bacteria, e.g., nitrate reducers, should be developed to broaden the applicability of bioactive self-healing concretes to wet and waterlogged environments.

Optical Sensing of pH and O2 in the Evaluation of Bioactive Self-Healing Cement.

Leakage from cementitious structures with a retaining function can have devastating environmental consequences. Leaks can originate from cracks within the hardened cementitious material that is supposed to seal the structure off from the surrounding environment. Bioactive self-healing concretes containing bacteria capable of microbially inducing CaCO3 precipitation have been suggested to mitigate the healing of such cracks before leaking occurs. An important parameter determining the biocompatibility of concretes and cements is the pH environment. Therefore, a novel ratiometric pH optode imaging system based on an inexpensive single-lens reflex (SLR) camera was used to characterize the pH of porewater within cracks of submerged hydrated oil and gas well cement. This enabled the imaging of pH with a spatial distribution in high resolution (50 µm per pixel) and a gradient of 1.4 pH units per 1 mm. The effect of fly ash substitution and hydration time on the pH of the cement surface was evaluated by this approach. The results show that pH is significantly reduced from pH >11 to below 10 with increasing fly ash content as well as hydration time. The assessment of bioactivity in the cement was evaluated by introducing superabsorbent polymers with encapsulated Bacillus alkalinitrilicus endospores into the cracks. The bacterial activity was measured using oxygen optodes, which showed the highest bacterial activity with increasing amounts of fly ash substitution in the cement, correlating with the decrease in the pH. Overall, our results demonstrate that the pH of well cements can be reliably measured and modified to sustain the microbial activity.

Glacial Runoff Promotes Deep Burial of Sulfur Cycling-Associated Microorganisms in Marine Sediments.

Marine fjords with active glacier outlets are hot spots for organic matter burial in the sediments and subsequent microbial mineralization. Here, we investigated controls on microbial community assembly in sub-arctic glacier-influenced (GI) and non-glacier-influenced (NGI) marine sediments in the Godthåbsfjord region, south-western Greenland. We used a correlative approach integrating 16S rRNA gene and dissimilatory sulfite reductase (dsrB) amplicon sequence data over six meters of depth with biogeochemistry, sulfur-cycling activities, and sediment ages. GI sediments were characterized by comparably high sedimentation rates and had "young" sediment ages of <500 years even at 6 m sediment depth. In contrast, NGI stations reached ages of approximately 10,000 years at these depths. Sediment age-depth relationships, sulfate reduction rates (SRR), and C/N ratios were strongly correlated with differences in microbial community composition between GI and NGI sediments, indicating that age and diagenetic state were key drivers of microbial community assembly in subsurface sediments. Similar bacterial and archaeal communities were present in the surface sediments of all stations, whereas only in GI sediments were many surface taxa also abundant through the whole sediment core. The relative abundance of these taxa, including diverse Desulfobacteraceae members, correlated positively with SRRs, indicating their active contributions to sulfur-cycling processes. In contrast, other surface community members, such as Desulfatiglans, Atribacteria, and Chloroflexi, survived the slow sediment burial at NGI stations and dominated in the deepest sediment layers. These taxa are typical for the energy-limited marine deep biosphere and their relative abundances correlated positively with sediment age. In conclusion, our data suggests that high rates of sediment accumulation caused by glacier runoff and associated changes in biogeochemistry, promote persistence of sulfur-cycling activity and burial of a larger fraction of the surface microbial community into the deep subsurface.

The Polyextremophilic Bacterium Clostridium paradoxum Attains Piezophilic Traits by Modulating Its Energy Metabolism and Cell Membrane Composition.

In polyextremophiles, i.e., microorganisms growing preferentially under multiple extremes, synergistic effects may allow growth when application of the same extremes alone would not. High hydrostatic pressure (HP) is rarely considered in studies of polyextremophiles, and its role in potentially enhancing tolerance to other extremes remains unclear. Here, we investigated the HP-temperature response in Clostridium paradoxum, a haloalkaliphilic moderately thermophilic endospore-forming bacterium, in the range of 50 to 70°C and 0.1 to 30 MPa. At ambient pressure, growth limits were extended from the previously reported 63°C to 70°C, defining C. paradoxum as an actual thermophile. Concomitant application of high HP and temperature compared to standard conditions (i.e., ambient pressure and 50°C) remarkably enhanced growth, with an optimum growth rate observed at 22 MPa and 60°C. HP distinctively defined C. paradoxum physiology, as at 22 MPa biomass, production increased by 75% and the release of fermentation products per cell decreased by >50% compared to ambient pressure. This metabolic modulation was apparently linked to an energy-preserving mechanism triggered by HP, involving a shift toward pyruvate as the preferred energy and carbon source. High HPs decreased cell damage, as determined by Syto9 and propidium iodide staining, despite no organic solute being accumulated intracellularly. A distinct reduction in carbon chain length of phospholipid fatty acids (PLFAs) and an increase in the amount of branched-chain PLFAs occurred at high HP. Our results describe a multifaceted, cause-and-effect relationship between HP and cell metabolism, stressing the importance of applying HP to define the boundaries for life under polyextreme conditions.IMPORTANCE Hydrostatic pressure (HP) is a fundamental parameter influencing biochemical reactions and cell physiology; however, it is less frequently applied than other factors, such as pH, temperature, and salinity, when studying polyextremophilic microorganisms. In particular, how HP affects microbial tolerance to other and multiple extremes remains unclear. Here, we show that under polyextreme conditions of high pH and temperature, Clostridium paradoxum demonstrates a moderately piezophilic nature as cultures grow to highest cell densities and most efficiently at a specific combination of temperature and HP. Our results highlight the importance of considering HP when exploring microbial physiology under extreme conditions and thus have implications for defining the limits for microbial life in nature and for optimizing industrial bioprocesses occurring under multiple extremes.

Marine Deep Biosphere Microbial Communities Assemble in Near-Surface Sediments in Aarhus Bay.

Analyses of microbial diversity in marine sediments have identified a core set of taxa unique to the marine deep biosphere. Previous studies have suggested that these specialized communities are shaped by processes in the surface seabed, in particular that their assembly is associated with the transition from the bioturbated upper zone to the nonbioturbated zone below. To test this hypothesis, we performed a fine-scale analysis of the distribution and activity of microbial populations within the upper 50 cm of sediment from Aarhus Bay (Denmark). Sequencing and qPCR were combined to determine the depth distributions of bacterial and archaeal taxa (16S rRNA genes) and sulfate-reducing microorganisms (SRM) (dsrB gene). Mapping of radionuclides throughout the sediment revealed a region of intense bioturbation at 0-6 cm depth. The transition from bioturbated sediment to the subsurface below (7 cm depth) was marked by a shift from dominant surface populations to common deep biosphere taxa (e.g., Chloroflexi and Atribacteria). Changes in community composition occurred in parallel to drops in microbial activity and abundance caused by reduced energy availability below the mixed sediment surface. These results offer direct evidence for the hypothesis that deep subsurface microbial communities present in Aarhus Bay mainly assemble already centimeters below the sediment surface, below the bioturbation zone.

Environmental filtering determines family-level structure of sulfate-reducing microbial communities in subsurface marine sediments.

Recent work has shown that subsurface microbial communities assemble by selective survival of surface community members during sediment burial, but it remains unclear to what extent the compositions of the subsurface communities are a product of their founding population at the sediment surface or of the changing geochemical conditions during burial. Here we investigate this question for communities of sulfate-reducing microorganisms (SRMs). We collected marine sediment samples from the upper 3-5 m at four geochemically contrasting sites in the Skagerrak and Baltic Sea and measured SRM abundance (quantitative PCR of dsrB), metabolic activity (radiotracer rate measurements), and community composition (Illumina sequencing of dsrB amplicons). These data showed that SRM abundance, richness, and phylogenetic clustering as determined by the nearest taxon index peaked below the bioturbation zone and above the depth of sulfate depletion. Minimum cell-specific rates of sulfate reduction did not vary substantially between sites. SRM communities at different sites were best distinguished based on their composition of amplicon sequence variants (ASVs), while communities in different geochemical zones were best distinguished based on their composition of SRM families. This demonstrates environmental filtering of SRM communities in sediment while a site-specific fingerprint of the founding community is retained.

Microbial Organic Matter Degradation Potential in Baltic Sea Sediments Is Influenced by Depositional Conditions and In Situ Geochemistry.

Globally, marine sediments are a vast repository of organic matter, which is degraded through various microbial pathways, including polymer hydrolysis and monomer fermentation. The sources, abundances, and quality (i.e., labile or recalcitrant) of the organic matter and the composition of the microbial assemblages vary between sediments. Here, we examine new and previously published sediment metagenomes from the Baltic Sea and the nearby Kattegat region to determine connections between geochemistry and the community potential to degrade organic carbon. Diverse organic matter hydrolysis encoding genes were present in sediments between 0.25 and 67 meters below seafloor and were in higher relative abundances in those sediments that contained more organic matter. New analysis of previously published metatranscriptomes demonstrated that many of these genes were transcribed in two organic-rich Holocene sediments. Some of the variation in deduced pathways in the metagenomes correlated with carbon content and depositional conditions. Fermentation-related genes were found in all samples and encoded multiple fermentation pathways. Notably, genes involved in alcohol metabolism were amongst the most abundant of these genes, indicating that this is an important but underappreciated aspect of sediment carbon cycling. This study is a step towards a more complete understanding of microbial food webs and the impacts of depositional facies on present sedimentary microbial communities.IMPORTANCE Sediments sequester organic matter over geologic time scales and impact global climate regulation. Microbial communities in marine sediments drive organic matter degradation, but the factors controlling their assemblages and activities, which in turn impact their role in organic matter degradation, are not well understood. Hence, determining the role of microbial communities in carbon cycling in various sediment types is necessary for predicting future sediment carbon cycling. We examined microbial communities in Baltic Sea sediments, which were deposited across various climatic and geographical regimes to determine the relationship between microbial potential for breakdown of organic matter and abiotic factors, including geochemistry and sediment lithology. The findings from this study will contribute to our understanding of carbon cycling in the deep biosphere and how microbial communities live in deeply buried environments.

Intracellular nitrate in sediments of an oxygen-deficient marine basin is linked to pelagic diatoms.

Intracellular nitrate is an important electron acceptor in oxygen-deficient aquatic environments, either for the nitrate-storing microbes themselves, or for ambient microbial communities through nitrate leakage. This study links the spatial distribution of intracellular nitrate with the abundance and identity of nitrate-storing microbes in sediments of the Bornholm Basin, an environmental showcase for severe hypoxia. Intracellular nitrate (up to 270 nmol cm-3 sediment) was detected at all 18 stations along a 35-km transect through the basin and typically extended as deep as 1.6 cm into the sediment. Intracellular nitrate contents were particularly high at stations where chlorophyll contents suggested high settling rates of pelagic primary production. The depth distribution of intracellular nitrate matched that of the diatom-specific photopigment fucoxanthin in the upper 1.6 cm and calculations support that diatoms are the major nitrate-storing microbes in these sediments. In contrast, other known nitrate-storing microbes, such as sulfide-oxidizing bacteria and foraminifers, played only a minor role, if any. Strikingly, 18S rRNA gene sequencing revealed that the majority of the diatoms in the sediment were pelagic species. We conclude that intracellular nitrate stored by pelagic diatoms is transported to the seafloor by settling phytoplankton blooms, implying a so far overlooked 'biological nitrate pump'.

Deep-biosphere methane production stimulated by geofluids in the Nankai accretionary complex.

Microbial life inhabiting subseafloor sediments plays an important role in Earth's carbon cycle. However, the impact of geodynamic processes on the distributions and carbon-cycling activities of subseafloor life remains poorly constrained. We explore a submarine mud volcano of the Nankai accretionary complex by drilling down to 200 m below the summit. Stable isotopic compositions of water and carbon compounds, including clumped methane isotopologues, suggest that ~90% of methane is microbially produced at 16° to 30°C and 300 to 900 m below seafloor, corresponding to the basin bottom, where fluids in the accretionary prism are supplied via megasplay faults. Radiotracer experiments showed that relatively small microbial populations in deep mud volcano sediments (102 to 103 cells cm-3) include highly active hydrogenotrophic methanogens and acetogens. Our findings indicate that subduction-associated fluid migration has stimulated microbial activity in the mud reservoir and that mud volcanoes may contribute more substantially to the methane budget than previously estimated.

Sulfate Transporters in Dissimilatory Sulfate Reducing Microorganisms: A Comparative Genomics Analysis.

The first step in the sulfate reduction pathway is the transport of sulfate across the cell membrane. This uptake has a major effect on sulfate reduction rates. Much of the information available on sulfate transport was obtained by studies on assimilatory sulfate reduction, where sulfate transporters were identified among several types of protein families. Despite our growing knowledge on the physiology of dissimilatory sulfate-reducing microorganisms (SRM) there are no studies identifying the proteins involved in sulfate uptake in members of this ecologically important group of anaerobes. We surveyed the complete genomes of 44 sulfate-reducing bacteria and archaea across six phyla and identified putative sulfate transporter encoding genes from four out of the five surveyed protein families based on homology. We did not find evidence that ABC-type transporters (SulT) are involved in the uptake of sulfate in SRM. We speculate that members of the CysP sulfate transporters could play a key role in the uptake of sulfate in thermophilic SRM. Putative CysZ-type sulfate transporters were present in all genomes examined suggesting that this overlooked group of sulfate transporters might play a role in sulfate transport in dissimilatory sulfate reducers alongside SulP. Our in silico analysis highlights several targets for further molecular studies in order to understand this key step in the metabolism of SRMs.

Microbial turnover times in the deep seabed studied by amino acid racemization modelling.

The study of active microbial populations in deep, energy-limited marine sediments has extended our knowledge of the limits of life on Earth. Typically, microbial activity in the deep biosphere is calculated by transport-reaction modelling of pore water solutes or from experimental measurements involving radiotracers. Here we modelled microbial activity from the degree of D:L-aspartic acid racemization in microbial necromass (remains of dead microbial biomass) in sediments up to ten million years old. This recently developed approach (D:L-amino acid modelling) does not require incubation experiments and is highly sensitive in stable, low-activity environments. We applied for the first time newly established constraints on several important input parameters of the D:L-amino acid model, such as a higher aspartic acid racemization rate constant and a lower cell-specific carbon content of sub-seafloor microorganisms. Our model results show that the pool of necromass amino acids is turned over by microbial activity every few thousand years, while the turnover times of vegetative cells are in the order of years to decades. Notably, microbial turnover times in million-year-old sediment from the Peru Margin are up to 100-fold shorter than previous estimates, highlighting the influence of microbial activities on element cycling over geologic time scales.

Identity, Abundance, and Reactivation Kinetics of Thermophilic Fermentative Endospores in Cold Marine Sediment and Seawater.

Cold marine sediments harbor endospores of fermentative and sulfate-reducing, thermophilic bacteria. These dormant populations of endospores are believed to accumulate in the seabed via passive dispersal by ocean currents followed by sedimentation from the water column. However, the magnitude of this process is poorly understood because the endospores present in seawater were so far not identified, and only the abundance of thermophilic sulfate-reducing endospores in the seabed has been quantified. We investigated the distribution of thermophilic fermentative endospores (TFEs) in water column and sediment of Aarhus Bay, Denmark, to test the role of suspended dispersal and determine the rate of endospore deposition and the endospore abundance in the sediment. We furthermore aimed to determine the time course of reactivation of the germinating TFEs. TFEs were induced to germinate and grow by incubating pasteurized sediment and water samples anaerobically at 50°C. We observed a sudden release of the endospore component dipicolinic acid immediately upon incubation suggesting fast endospore reactivation in response to heating. Volatile fatty acids (VFAs) and H2 began to accumulate exponentially after 3.5 h of incubation showing that reactivation was followed by a short phase of outgrowth before germinated cells began to divide. Thermophilic fermenters were mainly present in the sediment as endospores because the rate of VFA accumulation was identical in pasteurized and non-pasteurized samples. Germinating TFEs were identified taxonomically by reverse transcription, PCR amplification and sequencing of 16S rRNA. The water column and sediment shared the same phylotypes, thereby confirming the potential for seawater dispersal. The abundance of TFEs was estimated by most probable number enumeration, rates of VFA production, and released amounts of dipicolinic acid during germination. The surface sediment contained ∼105-106 inducible TFEs cm-3. TFEs thus outnumber thermophilic sulfate-reducing endospores by an order of magnitude. The abundance of cultivable TFEs decreased exponentially with sediment depth with a half-life of 350 years. We estimate that 6 × 109 anaerobic thermophilic endospores are deposited on the seafloor per m2 per year in Aarhus Bay, and that these thermophiles represent >10% of the total endospore community in the surface sediment.

The marine sulfate reducer Desulfobacterium autotrophicum HRM2 can switch between low and high apparent half-saturation constants for dissimilatory sulfate reduction.

Studies of the kinetics of dissimilatory sulfate reduction in marine sediment have shown that a mixture of marine sulfate-reducing bacteria (SRB) can reduce sulfate with both a high and low apparent sulfate half-saturation constant (Km). However, all marine pure cultures investigated have shown only low-sulfate affinity sulfate reduction kinetics. It remains unknown whether marine high sulfate-affinity sulfate reduction is catalyzed by unknown SRB or whether known SRB possess unrecognized high-affinity sulfate reduction systems. We used 35S-sulfate incubation experiments to show that cultures of Desulfobacterium autotrophicum HMR2 will switch from low-affinity to high-affinity sulfate reduction when sulfate concentrations fall below 500 µM. The mean Km was 150 µM at high sulfate concentrations and 8 µM at low sulfate concentrations. The high-affinity Km value is comparable to values found in SRB inhabiting freshwater sediments and D. autotrophicum cultures could deplete sulfate to below our detection limit of 25 nM. The switch in Km value was accompanied by a change in the expression of genes encoding membrane-bound transport proteins putatively involved in sulfate uptake in D. autotrophicum. Our results demonstrate that a marine sulfate reducer can efficiently reduce sulfate at both high and low sulfate concentrations, possibly by activation of different sulfate transporters in the membrane.

Evidence for the Existence of Autotrophic Nitrate-Reducing Fe(II)-Oxidizing Bacteria in Marine Coastal Sediment.

Nitrate-reducing Fe(II)-oxidizing microorganisms were described for the first time ca. 20 years ago. Most pure cultures of nitrate-reducing Fe(II) oxidizers can oxidize Fe(II) only under mixotrophic conditions, i.e., when an organic cosubstrate is provided. A small number of nitrate-reducing Fe(II)-oxidizing cultures have been proposed to grow autotrophically, but unambiguous evidence for autotrophy has not always been provided. Thus, it is still unclear whether or to what extent Fe(II) oxidation coupled to nitrate reduction is an enzymatically catalyzed and energy-yielding autotrophic process or whether Fe(II) is abiotically oxidized by nitrite from heterotrophic nitrate reduction. The aim of the present study was to find evidence for the existence of autotrophic nitrate-reducing Fe(II) oxidizers in coastal marine sediments. Microcosm incubations showed that with increasing incubation times, the stoichiometric ratio of reduced nitrate/oxidized Fe(II) [NO3-reduced/Fe(II)oxidized] decreased, indicating a decreasing contribution of heterotrophic denitrification and/or an increasing contribution of autotrophic nitrate-reducing Fe(II) oxidation over time. After incubations of sediment slurries for >10 weeks, nitrate-reducing activity ceased, although nitrate was still present. This suggests that heterotrophic nitrate reduction had ceased due to the depletion of readily available organic carbon. However, after the addition of Fe(II) to these batch incubation mixtures, the nitrate-reducing activity resumed, and Fe(II) was oxidized, indicating the activity of autotrophic nitrate-reducing Fe(II) oxidizers. The concurrent reduction of 14C-labeled bicarbonate concentrations unambiguously proved that autotrophic C fixation occurred during Fe(II) oxidation and nitrate reduction. Our results clearly demonstrated that autotrophic nitrate-reducing Fe(II)-oxidizing bacteria were present in the investigated coastal marine sediments. IMPORTANCE: Twenty years after the discovery of nitrate-reducing Fe(II) oxidizers, it is still controversially discussed whether autotrophic nitrate-reducing Fe(II)-oxidizing microorganisms exist and to what extent Fe(II) oxidation in this reduction/oxidation process is enzymatically catalyzed or which role abiotic side reactions of Fe(II) with reactive N species play. Most pure cultures of nitrate-reducing Fe(II) oxidizers are mixotrophic; i.e., they need an organic cosubstrate to maintain their activity over several cultural transfers. For the few existing autotrophic isolates and enrichment cultures, either the mechanism of nitrate-reducing Fe(II) oxidation is not known or evidence for their autotrophic lifestyle is controversial. In the present study, we provide evidence for the existence of autotrophic nitrate-reducing Fe(II) oxidizers in coastal marine sediments. The evidence is based on stoichiometries of nitrate reduction and Fe(II) oxidation determined in microcosm incubations and the incorporation of carbon from CO2 under conditions that favor the activity of nitrate-reducing Fe(II) oxidizers.