Characterizing the distribution and rates of microbial sulfate reduction at Middle Valley hydrothermal vents

Few studies have directly measured sulfate reduction at hydrothermal vents, and relatively little is known about how environmental or ecological factors influence rates of sulfate reduction in vent environments. A better understanding of microbially mediated sulfate reduction in hydrothermal vent ecosystems may be achieved by integrating ecological and geochemical data with metabolic rate measurements. Here we present rates of microbially mediated sulfate reduction from three distinct hydrothermal vents in the Middle Valley vent field along the Juan de Fuca Ridge, as well as assessments of bacterial and archaeal diversity, estimates of total biomass and the abundance of functional genes related to sulfate reduction, and in situ geochemistry. Maximum rates of sulfate reduction occurred at 90 °C in all three deposits. Pyrosequencing and functional gene abundance data revealed differences in both biomass and community composition among sites, including differences in the abundance of known sulfate-reducing bacteria. The abundance of sequences for Thermodesulfovibro-like organisms and higher sulfate reduction rates at elevated temperatures suggests that Thermodesulfovibro-like organisms may have a role in sulfate reduction in warmer environments. The rates of sulfate reduction presented here suggest that—within anaerobic niches of hydrothermal deposits—heterotrophic sulfate reduction may be quite common and might contribute substantially to secondary productivity, underscoring the potential role of this process in both sulfur and carbon cycling at vents.     

Bacterial Populations (First Record) at Two Shallow Hydrothermal Vents of the Mexican Pacific West Coast

Thermophilic and metal-oxidizing bacteria were identified in shallow hydrothermal vents on the western Mexican coast. The role of these bacteria in biomineralization processes observed in the vents is explained, and the effect of the vents on biodiversity of prokaryotes is discussed. Research was done at two shallow hydrothermal vent sites: Bahía Concepción (BC) in the Baja California Peninsula and Punta Mita (PM), on the central Pacific coast. Temperature at the sediments proximal to the vents was similar, but the redox potentials (0.5 V in BC and ?0.3 V in PM) and pH (6.2 in BC and as low as 4.5 in PM) differed. The composition of the discharged water ranged from nearly seawater to lower-salinity fluids, and the gas phase was mainly CO₂ at BC and N₂ and CH₄ at PM. The study focuses on the biogeochemical processes related to the different species of bacteria present in the studied sites, which are involved in the anaerobic oxidation of methane (AOM), seawater sulfate reduction, and metal oxidation. The detected bacterial lineages represented typical deep vent species, which disproves a previous hypothesis that proposed that different consortia were populating deep and shallow hydrothermal vents. The results obtained here show that the main parameter affecting the bacterial groups present in shallow vents was the redox potential: gamma-, delta-, and epsilon proteobacteria as well as Bacteriodetes are present under the oxidizing conditions of BC, and Thermotogae, Aquificae, and Planctomycetes are present in PM. Sunlight abundance favored the prevalence of halophilic and Chlorofleaxae bacteria in both areas.     

Primary Formation Path of Formaldehyde in Hydrothermal Vents

Formaldehyde is abundant in the universe and one of the fundamental molecules for life. Hydrothermal vents produce a substantial amount of hydrogen molecules by serpentinization and promote reductive reactions of single carbon compounds. The abundance of formaldehyde is expected to be low due to the high Gibbs free energy in hydrothermal vents. We consider two competing formation pathways of formaldehyde: (1) the reduction of CO by H₂ and (2) the reduction of HCOOH by H₂ to form a methanediol, followed by the dehydration of the methanediol. We performed a number of quantum chemical simulations to examine the formation of formaldehyde in the gas phase as well as in aqueous solution. The energy barrier is significantly reduced by the catalytic effect of water molecules in aqueous solution and becomes lowest when a water cluster consisted of 5 water molecules catalyzes the reduction. The energy barrier to form a methanediol by the reduction of HCOOH is lower by 17.5 kcal/mol than that to form a formaldehyde by the reduction of CO. Considering the low energy barrier to dehydrate methanediol, the primary pathway to form formaldehyde in hydrothermal vents is concluded to be the reduction of HCOOH by H₂, followed by the dehydration of methanediol.     

Rapid Microbial Production of Filamentous Sulfur Mats at Hydrothermal Vents

During recent oceanographic cruises to Pacific hydrothermal vent sites (9°N and the Guaymas Basin), the rapid microbial formation of filamentous sulfur mats by a new chemoautotrophic, hydrogen sulfide-oxidizing bacterium was documented in both in situ and shipboard experiments. Observations suggest that formation of these sulfur mats may be a factor in the initial colonization of hydrothermal surfaces by macrofaunal Alvinella worms. This novel metabolic capability, previously shown to be carried out by a coastal strain in H₂S continuous-flow reactors, may be an important, heretofore unconsidered, source of microbial organic matter production at deep-sea hydrothermal vents.     

Substrate Induced Denitrification over or under Estimates Shifts in Soil N2/N2O Ratios

The increase in atmospheric nitrous oxide (N₂O), a potent greenhouse and ozone depleting gas, is of serious global concern. Soils are large contributors to this increase through microbial processes that are enhanced in agricultural land due to nitrogenous fertilizer applications. Denitrification, a respiratory process using nitrogen oxides as electron acceptors in the absence of oxygen, is the main source of N₂O. The end product of denitrification is benign dinitrogen (N₂) and understanding what regulates the shift in ratio of N₂O and N₂ emission is crucial for mitigation strategies. The role of organic carbon in controlling N₂O reduction is poorly understood, and mostly based on application of glucose. Here we investigated how a range of carbon compounds (succinate, butyrate, malic acid, acetate, glucose, sucrose and cysteine) affect denitrifier N₂/N₂O production stoichiometry under laboratory conditions. The results show that a soil''s capability in efficiently reducing N₂O to N₂ is C substrate dependent and most compounds tested were different in regards to this efficiency compared to glucose. We challenge the concept of using glucose as a model soil C compound in furthering our understanding of denitrification and specifically the efficiency in the N₂O reductase enzyme. Organic acids, commonly exuded by roots, increased N₂/N₂O ratios compared to glucose, and therefore mitigated net N₂O release and we suggest provides better insights into soil regulatory aspects of N₂O reduction. The widespread use of glucose in soil laboratory studies could lead to misleading knowledge on the functioning of denitrification in soils with regards to N₂O reduction.     

The Geoglobus acetivorans Genome: Fe(III) Reduction,Acetate Utilization,Autotrophic Growth,and Degradation of Aromatic Compounds in a Hyperthermophilic Archaeon

Geoglobus acetivorans is a hyperthermophilic anaerobic euryarchaeon of the order Archaeoglobales isolated from deep-sea hydrothermal vents. A unique physiological feature of the members of the genus Geoglobus is their obligate dependence on Fe(III) reduction, which plays an important role in the geochemistry of hydrothermal systems. The features of this organism and its complete 1,860,815-bp genome sequence are described in this report. Genome analysis revealed pathways enabling oxidation of molecular hydrogen, proteinaceous substrates, fatty acids, aromatic compounds, n-alkanes, and organic acids, including acetate, through anaerobic respiration linked to Fe(III) reduction. Consistent with the inability of G. acetivorans to grow on carbohydrates, the modified Embden-Meyerhof pathway encoded by the genome is incomplete. Autotrophic CO₂ fixation is enabled by the Wood-Ljungdahl pathway. Reduction of insoluble poorly crystalline Fe(III) oxide depends on the transfer of electrons from the quinone pool to multiheme c-type cytochromes exposed on the cell surface. Direct contact of the cells and Fe(III) oxide particles could be facilitated by pilus-like appendages. Genome analysis indicated the presence of metabolic pathways for anaerobic degradation of aromatic compounds and n-alkanes, although an ability of G. acetivorans to grow on these substrates was not observed in laboratory experiments. Overall, our results suggest that Geoglobus species could play an important role in microbial communities of deep-sea hydrothermal vents as lithoautotrophic producers. An additional role as decomposers would close the biogeochemical cycle of carbon through complete mineralization of various organic compounds via Fe(III) respiration.     

The characteristics and comparative analysis of methanotrophs reveal genomic insights into Methylomicrobium sp. enriched from marine sediments

Methanotrophic bacteria are widespread and use methane as a sole carbon and energy source. They also play a crucial role in marine ecosystems by preventing the escape of methane into the atmosphere from diverse methane sources, such as methane seeps and hydrothermal vents. Despite their importance for methane carbon cycling, relatively few marine methanotrophic bacteria have been isolated and studied at the genomic level. Herein, we report the genome of a marine methanotrophic member of the genus Methylomicrobium, metagenome-assembled genome (MAG) wino1, which was obtained through enrichment using methane as the sole carbon source. Phylogenetic analysis based on 16S rRNA sequences and comparison of pmoA genes supported the close relationship of MAG-wino1 to the genus Methylomicrobium and it possessed a genome of 5.06 Mb encoding many specialized methanotrophic genes. A comparison of MAG-wino1 with the genomes of other strains (Methylomicrobium alcaliphilum 20Z^T and Methylomicrobium buryatense 5G) showed that genes (e.g. ectABC, ask, and mscLS) involved in the accumulation of compatible solutes required for survival in marine environments might be conserved. Methane utilization genes, including methanol dehydrogenase, and key enzymes related to ribulose monophosphate (RuMP) metabolism were identified. The wino1 genome harbored nitrogen fixation, urease, urea and nitrate transporter genes involved in the exploitation of nitrogen sources. Poly-β-hydroxybutyrate degradation and glycogen synthesis-related genes may facilitate survival under nutrient-limiting conditions. Additionally, genome analysis revealed three dominant taxa in the enrichment culture, methanotroph Methylomicrobium sp., methylotroph Methyloceanibacter sp., and non-methylotroph Labrenzia sp., which provided insights into microbial associations in marine sediments.     

Symbiotic relationships between soil fungi and plants reduce N2O emissions from soil

N₂O is a potent greenhouse gas involved in the destruction of the protective ozone layer in the stratosphere and contributing to global warming. The ecological processes regulating its emissions from soil are still poorly understood. Here, we show that the presence of arbuscular mycorrhizal fungi (AMF), a dominant group of soil fungi, which form symbiotic associations with the majority of land plants and which influence a range of important ecosystem functions, can induce a reduction in N₂O emissions from soil. To test for a functional relationship between AMF and N₂O emissions, we manipulated the abundance of AMF in two independent greenhouse experiments using two different approaches (sterilized and re-inoculated soil and non-mycorrhizal tomato mutants) and two different soils. N₂O emissions were increased by 42 and 33% in microcosms with reduced AMF abundance compared to microcosms with a well-established AMF community, suggesting that AMF regulate N₂O emissions. This could partly be explained by increased N immobilization into microbial or plant biomass, reduced concentrations of mineral soil N as a substrate for N₂O emission and altered water relations. Moreover, the abundance of key genes responsible for N₂O production (nirK) was negatively and for N₂O consumption (nosZ) positively correlated to AMF abundance, indicating that the regulation of N₂O emissions is transmitted by AMF-induced changes in the soil microbial community. Our results suggest that the disruption of the AMF symbiosis through intensification of agricultural practices may further contribute to increased N₂O emissions.     

Nitrous oxide respiring bacteria in biogas digestates for reduced agricultural emissions

Inoculating agricultural soils with nitrous oxide respiring bacteria (NRB) can reduce N₂O-emission, but would be impractical as a standalone operation. Here we demonstrate that digestates obtained after biogas production are suitable substrates and vectors for NRB. We show that indigenous NRB in digestates grew to high abundance during anaerobic enrichment under N₂O. Gas-kinetics and meta-omic analyses showed that these NRB’s, recovered as metagenome-assembled genomes (MAGs), grew by harvesting fermentation intermediates of the methanogenic consortium. Three NRB’s were isolated, one of which matched the recovered MAG of a Dechloromonas, deemed by proteomics to be the dominant producer of N₂O-reductase in the enrichment. While the isolates harbored genes required for a full denitrification pathway and could thus both produce and sequester N₂O, their regulatory traits predicted that they act as N₂O sinks in soil, which was confirmed experimentally. The isolates were grown by aerobic respiration in digestates, and fertilization with these NRB-enriched digestates reduced N₂O emissions from soil. Our use of digestates for low-cost and large-scale inoculation with NRB in soil can be taken as a blueprint for future applications of this powerful instrument to engineer the soil microbiome, be it for enhancing plant growth, bioremediation, or any other desirable function.Subject terms: Environmental sciences, Environmental microbiology     

Evaluation of enrichments of sulfate reducing bacteria from pristine hydrothermal vents sediments as potential inoculum for reducing trichloroethylene

The evaluation of enrichments from pristine hydrothermal vents sediments on its capability of reducing trichloroethylene (TCE) under sulfate reducing conditions with lactate and volatile fatty acids (VFAs) as substrates was performed. Effect of the possible TCE biodegradation intermediates cis and trans 1,2 dichloroethenes on sulfate reduction (SR) was also evaluated. The influence of cyanocobalamin (CNB₁₂) and riboflavin (RF) on the SR and biodegradation of TCE was also determined. Sediments from the vents were incubated at 37°C and supplemented with 4 g l⁻¹ SO₄ ²⁻, lactate or VFAs and amended in the corresponding treatments with either CNB₁₂ or RF in separated experiments. A percentage of TCE removal of 86 (150 μmol l⁻¹ initial concentration) was attained coupled to 48% sulfate depletion with lactate as substrate. Up to 93% removal of TCE (300 μmol l⁻¹ initial concentration) and 40% of sulfate was reached for VFAs as electron donor. A combination of lactate and CNB₁₂ yielded the best SR. The overall results suggest a syntrophic association in this microbial community in which sulfate reducers, dehalogenating, and probably halorespiring bacteria may be interacting and taking advantage of the fermentation of substrates differently, but without interruption of SR in spite of the fact that TCE was always present. It was also clear that sulfate reduction must be established in the cultures before any degradation can occur. The microbial community present in these hydrothermal vents sediments could be a new source of inoculum for bioreactors designed for dechlorination purposes.     

The uptake and excretion of partially oxidized sulfur expands the repertoire of energy resources metabolized by hydrothermal vent symbioses

Symbiotic associations between animals and chemoautotrophic bacteria crowd around hydrothermal vents. In these associations, symbiotic bacteria use chemical reductants from venting fluid for the energy to support autotrophy, providing primary nutrition for the host. At vents along the Eastern Lau Spreading Center, the partially oxidized sulfur compounds (POSCs) thiosulfate and polysulfide have been detected in and around animal communities but away from venting fluid. The use of POSCs for autotrophy, as an alternative to the chemical substrates in venting fluid, could mitigate competition in these communities. To determine whether ESLC symbioses could use thiosulfate to support carbon fixation or produce POSCs during sulfide oxidation, we used high-pressure, flow-through incubations to assess the productivity of three symbiotic mollusc genera—the snails Alviniconcha spp. and Ifremeria nautilei, and the mussel Bathymodiolus brevior—when oxidizing sulfide and thiosulfate. Via the incorporation of isotopically labelled inorganic carbon, we found that the symbionts of all three genera supported autotrophy while oxidizing both sulfide and thiosulfate, though at different rates. Additionally, by concurrently measuring their effect on sulfur compounds in the aquaria with voltammetric microelectrodes, we showed that these symbioses excreted POSCs under highly sulfidic conditions, illustrating that these symbioses could represent a source for POSCs in their habitat. Furthermore, we revealed spatial disparity in the rates of carbon fixation among the animals in our incubations, which might have implications for the variability of productivity in situ. Together, these results re-shape our thinking about sulfur cycling and productivity by vent symbioses, demonstrating that thiosulfate may be an ecologically important energy source for vent symbioses and that they also likely impact the local geochemical regime through the excretion of POSCs.     

A novel inhibitory interaction between dimethylsulfoniopropionate (DMSP) and the denitrification pathway

Dimethylsulfoniopropionate (DMSP) is an abundant organic sulfur compound in marine algae and denitrification influences nitrogen availability to primary producers, the key regulators of coastal eutrophication. In this study, we tested the effect of DMSP on the nitrous oxide (N₂O) reduction step of denitrification in sediments and biofilms from the Douro and Ave estuaries (NW Portugal) and in pure cultures of a denitrifying bacterium, Ruegeria pomeroyi. N₂O accumulation rates were monitored in sediment slurries and bacterial cell suspensions amended with DMSP concentrations ranging from 0 to 5 mM. In these treatments N₂O accumulation rates increased linearly with DMSP concentration (R ² from 0.89 to 0.99, p < 0.001), suggesting an inhibitory effect of DMSP on the nitrous oxide reductase activity. The addition of DMSP to sediments and bacterial culture resulted in accumulation of dimethylsulfide (DMS) as well as N₂O. However, no direct inhibition on N₂O reductase activity by DMS was observed. Natural concentrations of DMSP in the different estuarine sites were found to be linearly correlated to natural N₂O effluxes (R ² = 0.64, p < 0.001), suggesting that DMSP may negatively affect N₂O reductase in situ. This newly identified interaction between DMSP and N₂O emissions may have a significant ecological role as the inhibition of the nitrous oxide reduction enhances nitrogen loss via N₂O. Since N₂O is a powerful greenhouse gas, the results from our study may be important for evaluating climate change scenarios.     

Possible use of N2O in MPN tubes for enumeration of denitrifiers

N₂O when used in most probable number tubes as the only electron acceptor was not consumed at dilutions, down to 10^–3 per gram. Additions of fresh carbon source and NO₃ after growth did not stimulate N₂O reduction. Since pure cultures of denitrifiers grew well under the same conditions this result was unexpected, the explanation has not been found. This approach, if successful, has the advantage that only denitrifiers can reduce N₂O to N₂. Further work in designing a method to use N₂O as an electron acceptor in MPN tubes is suggested.     

Strain-level genomic variation in natural populations of Lebetimonas from an erupting deep-sea volcano

Chemolithoautotrophic Epsilonproteobacteria are ubiquitous in sulfidic, oxygen-poor habitats, including hydrothermal vents, marine oxygen minimum zones, marine sediments and sulfidic caves and have a significant role in cycling carbon, hydrogen, nitrogen and sulfur in these environments. The isolation of diverse strains of Epsilonproteobacteria and the sequencing of their genomes have revealed that this group has the metabolic potential to occupy a wide range of niches, particularly at dynamic deep-sea hydrothermal vents. We expand on this body of work by examining the population genomics of six strains of Lebetimonas, a vent-endemic, thermophilic, hydrogen-oxidizing Epsilonproteobacterium, from a single seamount in the Mariana Arc. Using Lebetimonas as a model for anaerobic, moderately thermophilic organisms in the warm, anoxic subseafloor environment, we show that genomic content is highly conserved and that recombination is limited between closely related strains. The Lebetimonas genomes are shaped by mobile genetic elements and gene loss as well as the acquisition of novel functional genes by horizontal gene transfer, which provide the potential for adaptation and microbial speciation in the deep sea. In addition, these Lebetimonas genomes contain two operons of nitrogenase genes with different evolutionary origins. Lebetimonas expressed nifH during growth with nitrogen gas as the sole nitrogen source, thus providing the first evidence of nitrogen fixation in any Epsilonproteobacteria from deep-sea hydrothermal vents. In this study, we provide a comparative overview of the genomic potential within the Nautiliaceae as well as among more distantly related hydrothermal vent Epsilonproteobacteria to broaden our understanding of microbial adaptation and diversity in the deep sea.     

Adaptations to Submarine Hydrothermal Environments Exemplified by the Genome of Nautilia profundicola

Submarine hydrothermal vents are model systems for the Archaean Earth environment, and some sites maintain conditions that may have favored the formation and evolution of cellular life. Vents are typified by rapid fluctuations in temperature and redox potential that impose a strong selective pressure on resident microbial communities. Nautilia profundicola strain Am-H is a moderately thermophilic, deeply-branching Epsilonproteobacterium found free-living at hydrothermal vents and is a member of the microbial mass on the dorsal surface of vent polychaete, Alvinella pompejana. Analysis of the 1.7-Mbp genome of N. profundicola uncovered adaptations to the vent environment—some unique and some shared with other Epsilonproteobacterial genomes. The major findings included: (1) a diverse suite of hydrogenases coupled to a relatively simple electron transport chain, (2) numerous stress response systems, (3) a novel predicted nitrate assimilation pathway with hydroxylamine as a key intermediate, and (4) a gene (rgy) encoding the hallmark protein for hyperthermophilic growth, reverse gyrase. Additional experiments indicated that expression of rgy in strain Am-H was induced over 100-fold with a 20°C increase above the optimal growth temperature of this bacterium and that closely related rgy genes are present and expressed in bacterial communities residing in geographically distinct thermophilic environments. N. profundicola, therefore, is a model Epsilonproteobacterium that contains all the genes necessary for life in the extreme conditions widely believed to reflect those in the Archaean biosphere—anaerobic, sulfur, H₂- and CO₂-rich, with fluctuating redox potentials and temperatures. In addition, reverse gyrase appears to be an important and common adaptation for mesophiles and moderate thermophiles that inhabit ecological niches characterized by rapid and frequent temperature fluctuations and, as such, can no longer be considered a unique feature of hyperthermophiles.     

Chemolithotrophic Sulfur-Oxidizing Bacteria from the Galapagos Rift Hydrothermal Vents

Three distinct physiological types of sulfur-oxidizing bacteria were enriched and isolated from samples collected at several deep-sea hydrothermal vents (2,550 m) of the Galapagos Rift ocean floor spreading center. Twelve strains of the obligately chemolithotrophic genus Thiomicrospira were obtained from venting water and from microbial mats covering surfaces in the immediate vicinity of the vents. From these and other sources two types of obligately heterotrophic sulfur oxidizers were repeatedly isolated that presumably oxidized thiosulfate either to sulfate (acid producing; 9 strains) or to polythionates (base producing; 74 strains). The former were thiobacilli-like, exhibiting a thiosulfate-stimulated increase in growth and CO₂ incorporation, whereas the latter were similar to previously encountered pseudomonad-like heterotrophs. The presence of chemolithotrophic sulfur-oxidizing bacteria in the sulfide-containing hydrothermal water supports the hypothesis that chemosynthesis provides a substantial primary food source for the rich populations of invertebrates found in the immediate vicinity of the vents.     

Prebiotic Synthesis of Glycine from Ethanolamine in Simulated Archean Alkaline Hydrothermal Vents

Submarine hydrothermal vents are generally considered as the likely habitats for the origin and evolution of early life on Earth. In recent years, a novel hydrothermal system in Archean subseafloor has been proposed. In this model, highly alkaline and high temperature hydrothermal fluids were generated in basalt-hosted hydrothermal vents, where H₂ and CO₂ could be abundantly provided. These extreme conditions could have played an irreplaceable role in the early evolution of life. Nevertheless, sufficient information has not yet been obtained for the abiotic synthesis of amino acids, which are indispensable components of life, at high temperature and alkaline condition. This study aims to propose a new method for the synthesis of glycine in simulated Archean submarine alkaline vent systems. We investigated the formation of glycine from ethanolamine under conditions of high temperature (80–160 °C) and highly alkaline solutions (pH = 9.70). Experiments were performed in an anaerobic environment under mild pressure (0.1–8.0 MPa) at the same time. The results suggested that the formation of glycine from ethanolamine occurred rapidly and efficiently in the presence of metal powders, and was favored by high temperatures and high pressures. The experiment provides a new pathway for prebiotic glycine formation and points out the phenomenal influence of high-temperature alkaline hydrothermal vents in origin of life in the early ocean.     

Deep-sea hydrothermal vent Epsilonproteobacteria encode a conserved and widespread nitrate reduction pathway (Nap)

Despite the frequent isolation of nitrate-respiring Epsilonproteobacteria from deep-sea hydrothermal vents, the genes coding for the nitrate reduction pathway in these organisms have not been investigated in depth. In this study we have shown that the gene cluster coding for the periplasmic nitrate reductase complex (nap) is highly conserved in chemolithoautotrophic, nitrate-reducing Epsilonproteobacteria from deep-sea hydrothermal vents. Furthermore, we have shown that the napA gene is expressed in pure cultures of vent Epsilonproteobacteria and it is highly conserved in microbial communities collected from deep-sea vents characterized by different temperature and redox regimes. The diversity of nitrate-reducing Epsilonproteobacteria was found to be higher in moderate temperature, diffuse flow vents than in high temperature black smokers or in low temperatures, substrate-associated communities. As NapA has a high affinity for nitrate compared with the membrane-bound enzyme, its occurrence in vent Epsilonproteobacteria may represent an adaptation of these organisms to the low nitrate concentrations typically found in vent fluids. Taken together, our findings indicate that nitrate reduction is widespread in vent Epsilonproteobacteria and provide insight on alternative energy metabolism in vent microorganisms. The occurrence of the nap cluster in vent, commensal and pathogenic Epsilonproteobacteria suggests that the ability of these bacteria to respire nitrate is important in habitats as different as the deep-sea vents and the human body.