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.

The metabolic rate of the biosphere and its components.

We assessed the relationship between rates of biological energy utilization and the biomass sustained by that energy utilization, at both the organism and biosphere level. We compiled a dataset comprising >10,000 basal, field, and maximum metabolic rate measurements made on >2,900 individual species, and, in parallel, we quantified rates of energy utilization, on a biomass-normalized basis, by the global biosphere and by its major marine and terrestrial components. The organism-level data, which are dominated by animal species, have a geometric mean among basal metabolic rates of 0.012 W (g C)-1 and an overall range of more than six orders of magnitude. The biosphere as a whole uses energy at an average rate of 0.005 W (g C)-1 but exhibits a five order of magnitude range among its components, from 0.00002 W (g C)-1 for global marine subsurface sediments to 2.3 W (g C)-1 for global marine primary producers. While the average is set primarily by plants and microorganisms, and by the impact of humanity upon those populations, the extremes reflect systems populated almost exclusively by microbes. Mass-normalized energy utilization rates correlate strongly with rates of biomass carbon turnover. Based on our estimates of energy utilization rates in the biosphere, this correlation predicts global mean biomass carbon turnover rates of ~2.3 y-1 for terrestrial soil biota, ~8.5 y-1 for marine water column biota, and ~1.0 y-1 and ~0.01 y-1 for marine sediment biota in the 0 to 0.1 m and >0.1 m depth intervals, respectively.

Metabolic responses of thermophilic endospores to sudden heat-induced perturbation in marine sediment samples.

Microbially mediated processes in a given habitat tend to be catalyzed by abundant populations that are ecologically adapted to exploit specific environmental characteristics. Typically, metabolic activities of rare populations are limited but may be stimulated in response to acute environmental stressors. Community responses to sudden changes in temperature and pressure can include suppression and activation of different populations, but these dynamics remain poorly understood. The permanently cold ocean floor hosts countless low-abundance microbes including endospores of thermophilic bacteria. Incubating sediments at high temperature resuscitates viable spores, causing the proliferation of bacterial populations. This presents a tractable system for investigating changes in a microbiome's community structure in response to dramatic environmental perturbations. Incubating permanently cold Arctic fjord sediments at 50°C for 216 h with and without volatile fatty acid amendment provoked major changes in community structure. Germination of thermophilic spores from the sediment rare biosphere was tracked using mass spectrometry-based metabolomics, radiotracer-based sulfate reduction rate measurements, and high-throughput 16S rRNA gene sequencing. Comparing community similarity at different intervals of the incubations showed distinct temporal shifts in microbial populations, depending on organic substrate amendment. Metabolite patterns indicated that amino acids and other sediment-derived organics were decomposed by fermentative Clostridia within the first 12-48 h. This fueled early and late phases of exponential increases in sulfate reduction, highlighting the cross-feeding of volatile fatty acids as electron donors for different sulfate-reducing Desulfotomaculia populations. The succession of germinated endospores triggered by sudden exposure to high temperature and controlled by nutrient availability offers a model for understanding the ecological response of dormant microbial communities following major environmental perturbations.

Growth of microaerophilic Fe(II)-oxidizing bacteria using Fe(II) produced by Fe(III) photoreduction.

Iron(II) (Fe(II)) can be formed by abiotic Fe(III) photoreduction, particularly when Fe(III) is organically complexed. Light-influenced environments often overlap or even coincide with oxic or microoxic geochemical conditions, for example, in sediments. So far, it is unknown whether microaerophilic Fe(II)-oxidizing bacteria are able to use the Fe(II) produced by Fe(III) photoreduction as electron donor. Here, we present an adaption of the established agar-stabilized gradient tube approach in comparison with liquid cultures for the cultivation of microaerophilic Fe(II)-oxidizing microorganisms by using a ferrihydrite-citrate mixture undergoing Fe(III) photoreduction as Fe(II) source. We quantified oxygen and Fe(II) gradients with amperometric and voltammetric microelectrodes and evaluated microbial growth by qPCR of 16S rRNA genes. We showed that gradients of dissolved Fe(II) (maximum Fe(II) concentration of 1.25 mM) formed in the gradient tubes when incubated in blue or UV light (400-530 nm or 350-400 nm). Various microaerophilic Fe(II)-oxidizing bacteria (Curvibacter sp. and Gallionella sp.) grew by oxidizing Fe(II) that was produced in situ by Fe(III) photoreduction. Best growth for these species, based on highest gene copy numbers, was observed in incubations using UV light in both liquid culture and gradient tubes containing 8 mM ferrihydrite-citrate mixtures (1:1), due to continuous light-induced Fe(II) formation. Microaerophilic Fe(II)-oxidizing bacteria contributed up to 40% to the overall Fe(II) oxidation within 24 h of incubation in UV light. Our results highlight the potential importance of Fe(III) photoreduction as a source of Fe(II) for Fe(II)-oxidizing bacteria by providing Fe(II) in illuminated environments, even under microoxic conditions.

Influence of Fe(III) source, light quality, photon flux and presence of oxygen on photoreduction of Fe(III)-organic complexes - Implications for light-influenced coastal freshwater and marine sediments.

Iron(III) photoreduction is an important source of Fe(II) in illuminated aquatic and sedimentary environments. Under oxic conditions, the Fe(II) can be re-oxidized by oxygen (O2) forming reactive O-species such as hydrogen peroxide (H2O2) which further react with Fe(II) thus enhancing Fe(II) oxidation rates. However, it is unknown by aquatic sediments how the parameters wavelength of radiation, photon flux, origin of Fe(III) source and presence or absence of O2 influence the extent of Fe(II) and H2O2 turnover. We studied this using batch experiments with different Fe(III)-organic complexes mimicking sedimentary conditions. We found that wavelengths <500 nm are necessary to initiate Fe(III) photoreduction and that the photon flux, wavelength and identity of Fe(III)-complexing organic acids control the kinetics of Fe(III) photoreduction. The formation of photo-susceptible Fe(III)-organic complexes did not depend on whether the Fe(III) source was biogenically produced, poorly-crystalline Fe(III) oxyhydroxides or chemically synthesized ferrihydrite. Oxic conditions caused chemical re-oxidation of Fe(II) and accumulation of H2O2. The photon flux, wavelength and availability of Fe(III)-complexing organic molecules are critical for the balance between concurrent Fe(III) photoreduction and abiotic Fe(II) oxidation and may even lead to a steady-state concentration of Fe(II) in the micromolar range. These results help understand and predict Fe(III) photoreduction dynamics and in-situ formation of Fe(II) in oxic or anoxic, illuminated and organic-rich environments.

Response to substrate limitation by a marine sulfate-reducing bacterium.

Sulfate-reducing microorganisms (SRM) in subsurface sediments live under constant substrate and energy limitation, yet little is known about how they adapt to this mode of life. We combined controlled chemostat cultivation and transcriptomics to examine how the marine sulfate reducer, Desulfobacterium autotrophicum, copes with substrate (sulfate or lactate) limitation. The half-saturation uptake constant (Km) for lactate was 1.2 µM, which is the first value reported for a marine SRM, while the Km for sulfate was 3 µM. The measured residual lactate concentration in our experiments matched values observed in situ in marine sediments, supporting a key role of SRM in the control of lactate concentrations. Lactate limitation resulted in complete lactate oxidation via the Wood-Ljungdahl pathway and differential overexpression of genes involved in uptake and metabolism of amino acids as an alternative carbon source. D. autotrophicum switched to incomplete lactate oxidation, rerouting carbon metabolism in response to sulfate limitation. The estimated free energy was significantly lower during sulfate limitation (-28 to -33 kJ mol-1 sulfate), suggesting that the observed metabolic switch is under thermodynamic control. Furthermore, we detected the upregulation of putative sulfate transporters involved in either high or low affinity uptake in response to low or high sulfate concentration.

Interactions between temperature and energy supply drive microbial communities in hydrothermal sediment.

Temperature and bioavailable energy control the distribution of life on Earth, and interact with each other due to the dependency of biological energy requirements on temperature. Here we analyze how temperature-energy interactions structure sediment microbial communities in two hydrothermally active areas of Guaymas Basin. Sites from one area experience advective input of thermogenically produced electron donors by seepage from deeper layers, whereas sites from the other area are diffusion-dominated and electron donor-depleted. In both locations, Archaea dominate at temperatures >45 °C and Bacteria at temperatures <10 °C. Yet, at the phylum level and below, there are clear differences. Hot seep sites have high proportions of typical hydrothermal vent and hot spring taxa. By contrast, high-temperature sites without seepage harbor mainly novel taxa belonging to phyla that are widespread in cold subseafloor sediment. Our results suggest that in hydrothermal sediments temperature determines domain-level dominance, whereas temperature-energy interactions structure microbial communities at the phylum-level and below.

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.

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.

The effect of temperature on sulfur and oxygen isotope fractionation by sulfate reducing bacteria (Desulfococcus multivorans).

Temperature influences microbiological growth and catabolic rates. Between 15 and 35 °C the growth rate and cell specific sulfate reduction rate of the sulfate reducing bacterium Desulfococcus multivorans increased with temperature. Sulfur isotope fractionation during sulfate reduction decreased with increasing temperature from 27.2 ‰ at 15 °C to 18.8 ‰ at 35 °C which is consistent with a decreasing reversibility of the metabolic pathway as the catabolic rate increases. Oxygen isotope fractionation, in contrast, decreased between 15 and 25 °C and then increased again between 25 and 35 °C, suggesting increasing reversibility in the first steps of the sulfate reducing pathway at higher temperatures. This points to a decoupling in the reversibility of sulfate reduction between the steps from the uptake of sulfate into the cell to the formation of sulfite, relative to the whole pathway from sulfate to sulfide. This observation is consistent with observations of increasing sulfur isotope fractionation when sulfate reducing bacteria are living near their upper temperature limit. The oxygen isotope decoupling may be a first signal of changing physiology as the bacteria cope with higher temperatures.

Sub-seafloor biogeochemical processes and microbial life in the Baltic Sea.

The post-glacial Baltic Sea has experienced extreme changes that are archived today in the deep sediments. IODP Expedition 347 retrieved cores down to 100 m depth and studied the climate history and the deep biosphere. We here review the biogeochemical and microbiological highlights and integrate these with other studies from the Baltic seabed. Cell numbers, endospore abundance and organic matter mineralization rates are extremely high. A 100-fold drop in cell numbers with depth results from a small difference between growth and mortality in the ageing sediment. Evidence for growth derives from a D:L amino acid racemization model, while evidence for mortality derives from the abundance and potential activity of lytic viruses. The deep communities assemble at the bottom of the bioturbated zone from the founding surface community by selection of organisms suited for life under deep sediment conditions. The mean catabolic per-cell rate of microorganisms drops steeply with depth to a life in slow-motion, typical for the deep biosphere. The subsurface life under extreme energy limitation is facilitated by exploitation of recalcitrant substrates, by biochemical protection of nucleic acids and proteins and by repair mechanisms for random mismatches in DNA or damaged amino acids in proteins.

Photochemistry of iron in aquatic environments.

Light energy is a driver for many biogeochemical element cycles in aquatic systems. The sunlight-induced photochemical reduction of ferric iron (Fe(iii) photoreduction) to ferrous iron (Fe(ii)) by either direct ligand-to-metal charge transfer or by photochemically produced radicals can be an important source of dissolved Feaq2+ in aqueous and sedimentary environments. Reactive oxygen species (ROS) are formed by a variety of light-dependent reactions. Those ROS can oxidize Fe(ii) or reduce Fe(iii), and due to their high reactivity they are key oxidants in aquatic systems where they influence many other biogeochemical cycles. In oxic waters with circumneutral pH, the produced Fe(ii) reaches nanomolar concentrations and serves as a nutrient, whereas in acidic waters, freshwater and marine sediments, which are rich in Fe(ii), the photochemically formed Fe(ii) can reach concentrations of up to 100 micromolar and be used as additional electron donor for acidophilic aerobic, microaerophilic, phototrophic and, if nitrate is present, for nitrate-reducing Fe(ii)-oxidizing bacteria. Therefore, Fe(iii) photoreduction may not only control the primary productivity in the oceans but has a tremendous impact on Fe cycling in the littoral zone of freshwater and marine environments. In this review, we summarize photochemical reactions involving Fe, discuss the role of ROS in Fe cycling, and highlight the importance of photoreductive processes in the environment.

Fe(III) Photoreduction Producing Feaq2+ in Oxic Freshwater Sediment.

Iron(III) (Fe(III)) photoreduction plays an important role in Fe cycling and Fe(II) bioavailability in the upper ocean. Although well described for water columns, it is currently unknown to what extent light impacts the production of dissolved Fe(II) (Fe2+) in aquatic sediments. We performed high-resolution voltammetric microsensor measurements and demonstrated light-induced Fe2+ release in freshwater sediments from Lake Constance. Fe2+ concentrations increased up to 40 µM in the top 3 mm of the sediment during illumination in the visible range of light (400-700 nm), even in the presence of oxygen (100-280 µM). The Fe2+ release was strongly dependent on the organic matter content of the sediment. A lack of photoreduced Fe2+ was measured in sediments with concentrations of organic carbon <6 mg L-1. The simultaneous presence of sedimentary Fe(III) photoreduction besides microbial and abiotic Fe2+ oxidation by oxygen suggests an active Fe redox cycling in the oxic and photic zone of aquatic sediments. Here, we provide evidence for a relevant contribution of Fe(III) photoreduction to the bio-geochemical Fe redox cycle in aquatic freshwater sediments.

Origin of Short-Chain Organic Acids in Serpentinite Mud Volcanoes of the Mariana Convergent Margin.

Serpentinitic systems are potential habitats for microbial life due to frequently high concentrations of microbial energy substrates, such as hydrogen (H2), methane (CH4), and short-chain organic acids (SCOAs). Yet, many serpentinitic systems are also physiologically challenging environments due to highly alkaline conditions (pH > 10) and elevated temperatures (>80°C). To elucidate the possibility of microbial life in deep serpentinitic crustal environments, International Ocean Discovery Program (IODP) Expedition 366 drilled into the Yinazao, Fantangisña, and Asùt Tesoru serpentinite mud volcanoes on the Mariana Forearc. These mud volcanoes differ in temperature (80, 150, 250°C, respectively) of the underlying subducting slab, and in the porewater pH (11.0, 11.2, 12.5, respectively) of the serpentinite mud. Increases in formate and acetate concentrations across the three mud volcanoes, which are positively correlated with temperature in the subducting slab and coincide with strong increases in H2 concentrations, indicate a serpentinization-related origin. Thermodynamic calculations suggest that formate is produced by equilibrium reactions with dissolved inorganic carbon (DIC) + H2, and that equilibration continues during fluid ascent at temperatures below 80°C. By contrast, the mechanism(s) of acetate production are not clear. Besides formate, acetate, and H2 data, we present concentrations of other SCOAs, methane, carbon monoxide, and sulfate, δ13C-data on bulk carbon pools, and microbial cell counts. Even though calculations indicate a wide range of microbial catabolic reactions to be thermodynamically favorable, concentration profiles of potential energy substrates, and very low cell numbers suggest that microbial life is scarce or absent. We discuss the potential roles of temperature, pH, pressure, and dispersal in limiting the occurrence of microbial life in deep serpentinitic environments.

Large sulfur isotope fractionation by bacterial sulfide oxidation.

A sulfide-oxidizing microorganism, Desulfurivibrio alkaliphilus (DA), generates a consistent enrichment of sulfur-34 (34 S) in the produced sulfate of +12.5 per mil or greater. This observation challenges the general consensus that the microbial oxidation of sulfide does not result in large 34 S enrichments and suggests that sedimentary sulfides and sulfates may be influenced by metabolic activity associated with sulfide oxidation. Since the DA-type sulfide oxidation pathway is ubiquitous in sediments, in the modern environment, and throughout Earth history, the enrichments and depletions in 34 S in sediments may be the combined result of three microbial metabolisms: microbial sulfate reduction, the disproportionation of external sulfur intermediates, and microbial sulfide oxidation.

Unravelling the sulphur cycle of marine sediments.

In this invited paper, I give a personal description of my start as a young scientist studying the sulphur cycle of marine sediments. The initial objective was to quantify the processes experimentally using 35 S as a tracer. The results showed an unexpected large contribution of sulphate reduction to the oxidation of sediment organic matter. This implied that sulphate reducing bacteria can perform complete oxidation of their substrates to CO2 . A quantification of sulphate reducing bacteria compared to measured rates of sulphate reduction revealed that the viable counting techniques available at the time vastly underestimated the true cell numbers. Through our introduction of microsensors for oxygen, sulphide and pH, the microbial oxidation of H2 S could be analysed at high spatial resolution. Studies of the large, filamentous sulphur bacteria revealed surprising adaptations to sulphide oxidation based on anaerobic respiration with nitrate stored in vacuoles.

Microbial biomass turnover times and clues to cellular protein repair in energy-limited deep Baltic Sea sediments.

The discovery of active microbial life deeply buried beneath the seafloor has opened important questions: how do microorganisms cope with extreme energy limitation, what is their metabolic activity, and how do they repair damages to essential biomolecules? We used a D:L-amino acid model to calculate microbial biomass turnover times. We used a metagenome and metatranscriptome analysis to investigate the distribution of the gene that encodes Protein-L-iso aspartate(D-aspartate) O-methyltransferase (PCMT), an enzyme which recognizes damaged L-isoapartyl and D-aspartyl residues in proteins and catalyzes their repair. Sediment was retrieved during the Integrated Ocean Drilling Program (IODP) Expedition 347 from Landsort Deep and the Little Belt in the Baltic Sea. The study covers the period from the Baltic Ice Lake ca. 13 000 years ago to the present. Our results provide new knowledge on microbial biomass turnover times and protein repair in relation to different regimes of organic matter input. For the first time, we show that the PCMT gene was widely distributed and expressed among phylogenetically diverse groups of microorganisms. Our findings suggest that microbial communities are capable of repairing D-amino acids within proteins using energy obtained from the degradation of a mixture of labile compounds in microbial necromass and more recalcitrant organic matter.

The Biogeochemical Sulfur Cycle of Marine Sediments.

Microbial dissimilatory sulfate reduction to sulfide is a predominant terminal pathway of organic matter mineralization in the anoxic seabed. Chemical or microbial oxidation of the produced sulfide establishes a complex network of pathways in the sulfur cycle, leading to intermediate sulfur species and partly back to sulfate. The intermediates include elemental sulfur, polysulfides, thiosulfate, and sulfite, which are all substrates for further microbial oxidation, reduction or disproportionation. New microbiological discoveries, such as long-distance electron transfer through sulfide oxidizing cable bacteria, add to the complexity. Isotope exchange reactions play an important role for the stable isotope geochemistry and for the experimental study of sulfur transformations using radiotracers. Microbially catalyzed processes are partly reversible whereby the back-reaction affects our interpretation of radiotracer experiments and provides a mechanism for isotope fractionation. We here review the progress and current status in our understanding of the sulfur cycle in the seabed with respect to its microbial ecology, biogeochemistry, and isotope geochemistry.