Phylogenetic Diversity of Dissimilatory Sulfite Reductase Genes from Deep-sea Cold Seep Sediment

The phylogenetic diversity of dissimilatory sulfite reductase (DSR, EC 1.8.99.3) -subunit genes from a deep-sea cold seep was analyzed. Bulk genomic DNA was extracted from the cold seep sediment and used for amplification by polymerase chain reaction (PCR) of DSR -subunit gene. Two sizes of PCR products, 1.4 kb (expected) and 1.3 kb (unexpected), were amplified. Sixteen clones of the 1.4-kb amplicons and 16 clones of 1.3-kb amplicons, a total of 32 clones, were obtained and grouped into operational DSR units (ODUs) based on restriction fragment length polymorphism (RFLP) by digestion with HaeIII and MboI. A total of 14 ODUs, i.e., 5 ODUs from 1.4-kb amplicon clones and 9 ODUs from 1.3-kb amplicon clones, were recovered. About 400 bp of the 5 ends of all the clones was sequenced and validated the RFLP-based ODU grouping. All the 5-end 400-bp sequences of ODUs, even from the 1.3-kb amplicons, showed the characteristic DSR amino acid sequence motifs. The ODUs from 1.4-kb amplicons were closely related to the -Proteobacterial lineage with the DSR genes from -Proteobacterial epibionts of the hot vent worm Alvinella pompejana. The ODUs from 1.3-kb amplicons were mostly related to the unknown but possibly archaeal lineage. The diversity of the DSR genes may indicate the diversity of sulfate reducers in the seep sediment as well as the complexity of electron donors including methane.     

Phylogenetic Diversity and Distribution of Dissimilatory Sulfite Reductase Genes from Deep-Sea Sediment Cores

The diversity and distribution of sulfate-reducing prokaryotes (SRP) was investigated in the Nankai Trough sediments of off-central Japan by exploring the diversity of a functional gene, dissimilatory sulfite reductase (dsrAB). Bulk DNAs were extracted from five piston-cored samples (up to 4.5 m long) with 41 vertical sections, and full-length dsrABgene sequences (ca. 1.9 kb) were PCR amplified and cloned. A total of 382 dsrAB clones yielded eight phylogenetic groups with an indigenous group forming a unique dsrAB lineage. The deltaproteobacterial dsrAB genes were found in almost all sediment samples, especially in the surface layer. One unique dsrAB clone group was also widespread in the dsrAB profiles of the studied sediments, and the percentage of its clones was generally shown gradual increase with sediment depth.     

Denitrification, Dissimilatory Reduction of Nitrate to Ammonium, and Nitrification in a Bioturbated Estuarine Sediment as Measured with 15N and Microsensor Techniques

Nitrogen and oxygen transformations were studied in a bioturbated (reworked by animals) estuarine sediment (Norsminde Fjord, Denmark) by using a combination of ¹⁵N isotope (NO₃^-), specific inhibitor (C₂H₂), and microsensor (N₂O and O₂) techniques in a continuous-flow core system. The estuarine water was NO₃^- rich (125 to 600 μM), and NO₃^- was consistently taken up by the sediment on the four occasions studied. Total NO₃^- uptake (3.6 to 34.0 mmol of N m^-2 day^-1) corresponded closely to N₂ production (denitrification) during the experimental steady state, which indicated that dissimilatory, as well as assimilatory, NO₃^- reduction to NH₄⁺ was insignificant. When C₂H₂ was applied in the flow system, denitrification measured as N₂O production was often less (58 to 100%) than the NO₃^- uptake because of incomplete inhibition of N₂O reduction. The NO₃^- formed by nitrification and not immediately denitrified but released to the overlying water, uncoupled nitrification, was calculated both from ¹⁵NO₃^- dilution and from changes in NO₃^- uptake before and after C₂H₂ addition. These two approaches gave similar results, with rates ranging between 0 and 8.1 mmol of N m^-2 day^-1 on the four occasions. Attempts to measure total nitrification activity by the difference between NH₄⁺ fluxes before and after C₂H₂ addition failed because of non-steady-state NH₄⁺ fluxes. The vertical distribution of denitrification and oxygen consumption was studied by use of N₂O and O₂ microelectrodes. The N₂O profiles measured during the experimental steady state were often irregularly shaped, and the buildup of N₂O after C₂H₂ was added was much too fast to be described by a simple diffusion model. Only bioturbation by a dense population of infauna could explain these observations. This was corroborated by the relationship between diffusive and total fluxes, which showed that only 19 to 36 and 29 to 62% of the total O₂ uptake and denitrification, respectively, were due to diffusion-reaction processes at the regular sediment surface, excluding animal burrows.     

Linkage of High Rates of Sulfate Reduction in Yellowstone Hot Springs to Unique Sequence Types in the Dissimilatory Sulfate Respiration Pathway

Diversity, habitat range, and activities of sulfate-reducing prokaryotes within hot springs in Yellowstone National Park were characterized using endogenous activity measurements, molecular characterization, and enrichment. Five major phylogenetic groups were identified using PCR amplification of the dissimilatory sulfite reductase genes (dsrAB) from springs demonstrating significant sulfate reduction rates, including a warm, acidic (pH 2.5) stream and several nearly neutral hot springs with temperatures reaching 89°C. Three of these sequence groups were unrelated to named lineages, suggesting that the diversity and habitat range of sulfate-reducing prokaryotes exceeds that now represented in culture.     

Dissimilatory Oxidation and Reduction of Elemental Sulfur in Thermophilic Archaea

The oxidation and reduction of elemental sulfur and reduced inorganic sulfur species are some of the most important energy-yielding reactions for microorganisms living in volcanic hot springs, solfataras, and submarine hydrothermal vents, including both heterotrophic, mixotrophic, and chemolithoautotrophic, carbon dioxide-fixing species. Elemental sulfur is the electron donor in aerobic archaea like Acidianus and Sulfolobus. It is oxidized via sulfite and thiosulfate in a pathway involving both soluble and membrane-bound enzymes. This pathway was recently found to be coupled to the aerobic respiratory chain, eliciting a link between sulfur oxidation and oxygen reduction at the level of the respiratory heme copper oxidase. In contrast, elemental sulfur is the electron acceptor in a short electron transport chain consisting of a membrane-bound hydrogenase and a sulfur reductase in (facultatively) anaerobic chemolithotrophic archaea Acidianus and Pyrodictium species. It is also the electron acceptor in organoheterotrophic anaerobic species like Pyrococcus and Thermococcus, however, an electron transport chain has not been described as yet. The current knowledge on the composition and properties of the aerobic and anaerobic pathways of dissimilatory elemental sulfur metabolism in thermophilic archaea is summarized in this contribution.     

Abiotic Reduction of 4-Chloronitrobenzene to 4-Chloroaniline in a Dissimilatory Iron-Reducing Enrichment Culture

4-chloronitrobenzene (4-Cl-NB) was rapidly reduced to 4-chloroaniline with half-lives of minutes in a dissimilatory Fe(III)-reducing enrichment culture. The initial pseudo-first-order rate constants at 25°C ranged from 0.11 to 0.19 per minute. The linear Arrhenius correlation in a temperature range of 6 to 85°C and the unchanged reactivity after pasteurization indicated that the nitroreduction occurred abiotically. A fine-grained black solid which was identified as poorly crystalline magnetite (Fe₃O₄) by X-ray diffraction accumulated in the enrichments. Magnetite produced by the Fe(III)-reducing bacterium Geobacter metallireducens GS-15 and synthetic magnetite also reduced 4-Cl-NB. These results suggest that the reduction of 4-Cl-NB by the enrichment material was a surface-mediated reaction by dissimilatory formed Fe(II) associated with magnetite.     

Reduction of Selenate and Selenite to Elemental Selenium by a Pseudomonas stutzeri Isolate

A Pseudomonas stutzeri isolate rapidly reduced both selenite and selenate ions to elemental selenium at initial concentrations of both anions of up to 48.1 mM. Optimal selenium reduction occurred under aerobic conditions between pH 7.0 and 9.0 and at temperatures of 25 to 35°C. Reduction of both selenite and selenate was unaffected by a number of anions except for sulfite, chromate, and tungstate ions, which inhibited both growth and reduction.     

Dissimilatory Nitrate Reduction in Anaerobic Sediments Leading to River Nitrite Accumulation

Recent studies on Northern Ireland rivers have shown that summer nitrite (NO(inf2)(sup-)) concentrations greatly exceed the European Union guideline of 3 (mu)g of N liter(sup-1) for rivers supporting salmonid fisheries. In fast-flowing aerobic small streams, NO(inf2)(sup-) is thought to originate from nitrification, due to the retardation of Nitrobacter strains by the presence of free ammonia. Multiple regression analyses of NO(inf2)(sup-) concentrations against water quality variables of the six major rivers of the Lough Neagh catchment in Northern Ireland, however, suggested that the high NO(inf2)(sup-) concentrations found in the summer under warm, slow-flow conditions may result from the reduction of NO(inf3)(sup-). This hypothesis was supported by field observations of weekly changes in N species. Here, reduction of NO(inf3)(sup-) was observed to occur simultaneously with elevation of NO(inf2)(sup-) levels and subsequently NH(inf4)(sup+) levels, indicating that dissimilatory NO(inf3)(sup-) reduction to NH(inf4)(sup+) (DNRA) performed by fermentative bacteria (e.g., Aeromonas and Vibrio spp.) is responsible for NO(inf2)(sup-) accumulation in these large rivers. Mechanistic studies in which (sup15)N-labelled NO(inf3)(sup-) in sediment extracts was used provided further support for this hypothesis. Maximal concentrations of NO(inf2)(sup-) accumulation (up to 1.4 mg of N liter(sup-1)) were found in sediments deeper than 6 cm associated with a high concentration of metabolizable carbon and anaerobic conditions. The (sup15)N enrichment of the NO(inf2)(sup-) was comparable to that of the NO(inf3)(sup-) pool, indicating that the NO(inf2)(sup-) was predominantly NO(inf3)(sup-) derived. There is evidence which suggests that the high NO(inf2)(sup-) concentrations observed arose from the inhibition of the DNRA NO(inf2)(sup-) reductase system by NO(inf3)(sup-).     

Regulation of Dissimilatory Fe(III) Reduction Activity in Shewanella putrefaciens

Under anaerobic conditions, Shewanella putrefaciens is capable of respiratory-chain-linked, high-rate dissimilatory iron reduction via both a constitutive and inducible Fe(III)-reducing system. In the presence of low levels of dissolved oxygen, however, iron reduction by this microorganism is extremely slow. Fe(II)-trapping experiments in which Fe(III) and O₂ were presented simultaneously to batch cultures of S. putrefaciens indicated that autoxidation of Fe(II) was not responsible for the absence of Fe(III) reduction. Inhibition of cytochrome oxidase with CN⁻ resulted in a high rate of Fe(III) reduction in the presence of dissolved O₂, which suggested that respiratory control mechanisms did not involve inhibition of Fe(III) reductase activities or Fe(III) transport by molecular oxygen. Decreasing the intracellular ATP concentrations by using an uncoupler, 2,4-dinitrophenol, did not increase Fe(III) reduction, indicating that the reduction rate was not controlled by the energy status of the cell. Control of electron transport at branch points could account for the observed pattern of respiration in the presence of the competing electron acceptors Fe(III) and O₂.     

Simultaneous Reduction of Nitrate and Selenate by Cell Suspensions of Selenium-Respiring Bacteria

Washed-cell suspensions of Sulfurospirillum barnesii reduced selenate [Se(VI)] when cells were cultured with nitrate, thiosulfate, arsenate, or fumarate as the electron acceptor. When the concentration of the electron donor was limiting, Se(VI) reduction in whole cells was approximately fourfold greater in Se(VI)-grown cells than was observed in nitrate-grown cells; correspondingly, nitrate reduction was ~11-fold higher in nitrate-grown cells than in Se(VI)-grown cells. However, a simultaneous reduction of nitrate and Se(VI) was observed in both cases. At nonlimiting electron donor concentrations, nitrate-grown cells suspended with equimolar nitrate and selenate achieved a complete reductive removal of nitrogen and selenium oxyanions, with the bulk of nitrate reduction preceding that of selenate reduction. Chloramphenicol did not inhibit these reductions. The Se(VI)-respiring haloalkaliphile Bacillus arsenicoselenatis gave similar results, but its Se(VI) reductase was not constitutive in nitrate-grown cells. No reduction of Se(VI) was noted for Bacillus selenitireducens, which respires selenite. The results of kinetic experiments with cell membrane preparations of S. barnesii suggest the presence of constitutive selenate and nitrate reduction, as well as an inducible, high-affinity nitrate reductase in nitrate-grown cells which also has a low affinity for selenate. The simultaneous reduction of micromolar Se(VI) in the presence of millimolar nitrate indicates that these organisms may have a functional use in bioremediating nitrate-rich, seleniferous agricultural wastewaters. Results with ⁷⁵Se-selenate tracer show that these organisms can lower ambient Se(VI) concentrations to levels in compliance with new regulations proposed for release of selenium oxyanions into the environment.     

The Influence of Carbon Source on the Products of Dissimilatory Iron Reduction

We have examined the influence of carbon source on both the rate of iron reduction and the mineralogy of the reduction products with Shewanella putrefaciens strain W3-18-1. When pyruvate was the carbon source, the secondary products were spherules composed of siderite. When uridine was used as the carbon source, the products were hexagonal plate-like structures identified as iron carbonate hydroxide hydrate, also known as carbonate green rust, a precursor to fougerite. When lactate was used as the carbon source, products were a mixture of iron carbonate hydroxide and magnetite. In terms of reaction stoichiometry, there were differences in the amount of acetate produced depending on the starting organic carbon source. Incubation with pyruvate produced a relatively large amount of acetate compared to incubation with uridine and lactate. There were also differences in the final pH of the cultures. While the pH for incubations with lactate started at 8.6 and ended between 8.0–8.3, the pH of cultures incubated with uridine was found to be almost a full unit lower at the conclusion of the experiment (～7.4). Solubility diagrams based on the chemistry found in our experiments predict that the production of Fe²⁺ _(aq) should always lead to the formation of magnetite. However, strain W3-18-1 produced different minerals depending on the carbon source utilized as the electron acceptor.     

Diversity of Dissimilatory Sulfite Reductase Genes (dsrAB) in a Salt Marsh Impacted by Long-Term Acid Mine Drainage

Sulfate-reducing bacteria (SRB) play a major role in the coupled biogeochemical cycling of sulfur and chalcophilic metal(loid)s. By implication, they can exert a strong influence on the speciation and mobility of multiple metal(loid) contaminants. In this study, we combined DsrAB gene sequencing and sulfur isotopic profiling to identify the phylogeny and distribution of SRB and to assess their metabolic activity in salt marsh sediments exposed to acid mine drainage (AMD) for over 100 years. Recovered dsrAB sequences from three sites sampled along an AMD flow path indicated the dominance of a single Desulfovibrio species. Other major sequence clades were related most closely to Desulfosarcina, Desulfococcus, Desulfobulbus, and Desulfosporosinus species. The presence of metal sulfides with low δ³⁴S values relative to δ³⁴S values of pore water sulfate showed that sediment SRB populations were actively reducing sulfate under ambient conditions (pH of ∼2), although possibly within less acidic microenvironments. Interestingly, δ³⁴S values for pore water sulfate were lower than those for sulfate delivered during tidal inundation of marsh sediments. 16S rRNA gene sequence data from sediments and sulfur isotope data confirmed that sulfur-oxidizing bacteria drove the reoxidation of biogenic sulfide coupled to oxygen or nitrate reduction over a timescale of hours. Collectively, these findings imply a highly dynamic microbially mediated cycling of sulfate and sulfide, and thus the speciation and mobility of chalcophilic contaminant metal(loid)s, in AMD-impacted marsh sediments.Salt marshes exhibit high primary production rates (1, 101) and form biogeochemical “transition zones” for nutrient production, transport, and cycling between terrestrial and coastal marine environments (41, 66, 100). These zones also serve to reduce the flux of potentially toxic metals in contaminated groundwater to estuaries (12, 99, 106). Both functions depend strongly on microbial activity, especially that of sulfate-reducing bacteria (SRB) (42, 62, 67). SRB recycle much of the sedimentary organic carbon pool in marsh sediments (42-44) and indirectly inhibit production of the greenhouse gas methane (37, 71). They can restrict the mobility of dissolved contaminant metals by inducing precipitation of poorly soluble metal sulfides, and studies have examined their use in constructed wetlands to bioremediate acid mine drainage (AMD) and other metalliferous waste streams (11, 35, 40, 46, 50, 76, 90, 94, 104). However, the high acidity and metal concentrations inherent to AMD can inhibit SRB growth (15, 88, 98), and preferential growth of iron- and sulfur-oxidizing bacteria over SRB has been observed in some treatment wetlands (39).For natural salt marshes, 16S ribosomal nucleic acid- and phospholipid fatty acid (PLFA)-based analyses have shown that SRB commonly comprise a significant fraction of the microbial community (13, 24, 31, 34, 51, 58). Studies of salt marsh dissimilatory sulfite reductase genes (dsrAB), a highly conserved functional phylogenetic marker of prokaryotic sulfate reducers (49, 57, 102, 103, 107), have revealed both novel and deeply branching clades (3). Studies of mining-impacted sites at pH 2.0 to 7.8 (5, 7, 39, 70, 72, 77, 84), of soils and geothermal settings at a pH of ∼4 (55, 68), of metal-contaminated estuaries at pH 6.8 to 7.2 (65), and of hypersaline lakes at pH 7.5 (56) further outline the distribution and tolerance of specific groups and species of SRB under geochemically stringent conditions. Other findings point toward the existence of deltaproteobacteria in environments at a pH of ∼1 (10), although it is unknown if these include SRB. SRB diversity in salt marshes under long-term contamination by AMD has not been well investigated. Such studies may provide useful information for bioremediation projects in estuarine environments, as well as general insights into relationships between SRB physiology and the geochemistry of AMD.We studied the diversity of SRB, based on phylogenetic analysis of recovered DsrAB gene sequences (∼1.9 kb), in natural salt marsh sediments of the San Francisco Bay impacted by AMD for over 100 years. Sulfur isotope ratio and concentration measurements of pore water sulfate and metal sulfide minerals provided information about the spatial and temporal extent of active bacterial sulfate reduction (BSR) in sediment cores taken from specific sites along an AMD flow path. Collectively, the results revealed a tidal marsh system characterized by rapidly cycling bacterial sulfate reduction and sulfide reoxidation associated with oscillating tidal inundation and groundwater infiltration.     

Dissimilatory Iron Reduction and Odor Indicator Abatement by Biofilm Communities in Swine Manure Microcosms

Animal waste odors arising from products of anaerobic microbial metabolism create community relations problems for livestock producers. We investigated a novel approach to swine waste odor reduction: the addition of FeCl₃, a commonly used coagulant in municipal wastewater treatment, to stimulate degradation of odorous compounds by dissimilatory iron-reducing bacteria (DIRB). Two hypotheses were tested: (i) FeCl₃ is an effective source of redox-active ferric iron (Fe³⁺) for dissimilatory reduction by bacteria indigenous to swine manure, and (ii) dissimilatory iron reduction results in significant degradation of odorous compounds within 7 days. Our results demonstrated that Fe³⁺ from FeCl₃ was reduced biologically as well as chemically in laboratory microcosms prepared with prefiltered swine manure slurry and limestone gravel, which provided pH buffering and a substrate for microbial biofilm development. Addition of a 1-g liter⁻¹ equivalent concentration of Fe³⁺ from FeCl₃, but not from presynthesized ferrihydrite, caused initial, rapid solids flocculation, chemical Fe³⁺ reduction, and E_h increase, followed by a 2-day lag period. Between 2 and 6 days of incubation, increases in Fe²⁺ concentrations were accompanied by significant reductions in concentrations of volatile fatty acids used as odor indicators. Increases in Fe²⁺ concentrations between 2 and 6 days did not occur in FeCl₃-treated microcosms that were sterilized by gamma irradiation or amended with NaN₃, a respiratory inhibitor. DNA sequences obtained from rRNA gene amplicons of bacterial communities in FeCl₃-treated microcosms were closely related to Desulfitobacterium spp., which are known representatives of DIRB. Use of iron respiration to abate wastewater odors warrants further investigation.     

Bacterial Dissimilatory Reduction of Arsenic(V) to Arsenic(III) in Anoxic Sediments

Incubation of anoxic salt marsh sediment slurries with 10 mM As(V) resulted in the disappearance over time of the As(V) in conjunction with its recovery as As(III). No As(V) reduction to As(III) occurred in heat-sterilized or formalin-killed controls or in live sediments incubated in air. The rate of As(V) reduction in slurries was enhanced by addition of the electron donor lactate, H(inf2), or glucose, whereas the respiratory inhibitor/uncoupler dinitrophenol, rotenone, or 2-heptyl-4-hydroxyquinoline N-oxide blocked As(V) reduction. As(V) reduction was also inhibited by tungstate but not by molybdate, sulfate, or phosphate. Nitrate inhibited As(V) reduction by its action as a preferred respiratory electron acceptor rather than as a structural analog of As(V). Nitrate-respiring sediments could reduce As(V) to As(III) once all the nitrate was removed. Chloramphenicol blocked the reduction of As(V) to As(III) in nitrate-respiring sediments, suggesting that nitrate and arsenate were reduced by separate enzyme systems. Oxidation of [2-(sup14)C]acetate to (sup14)CO(inf2) by salt marsh and freshwater sediments was coupled to As(V). Collectively, these results show that reduction of As(V) in sediments proceeds by a dissimilatory process. Bacterial sulfate reduction was completely inhibited by As(V) as well as by As(III).     

Capacity for Denitrification and Reduction of Nitrate to Ammonia in a Coastal Marine Sediment

The capacity for dissimilatory reduction of NO₃⁻ to N₂ (N₂O) and NH₄⁺ was measured in ¹⁵NO₃⁻-amended marine sediment. Incubation with acetylene (7 × 10⁻³ atmospheres [normal]) caused accumulation of N₂O in the sediment. The rate of N₂O production equaled the rate of N₂ production in samples without acetylene. Complete inhibition of the reduction of N₂O to N₂ suggests that the “acetylene blockage technique” is applicable to assays for denitrification in marine sediments. The capacity for reduction of NO₃⁻ by denitrification decreased rapidly with depth in the sediment, whereas the capacity for reduction of NO₃⁻ to NH₄⁺ was significant also in deeper layers. The data suggested that the latter process may be equally as significant as denitrification in the turnover of NO₃⁻ in marine sediments.