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At least 361 records · Page 20Linked to original sources

Subsurface microbial diversity in deep-granitic-fracture water in Colorado

A microbial community analysis using 16S rRNA gene sequencing was performed on borehole water and a granite rock core from Henderson Mine, a >1,000-meter-deep molybdenum mine near Empire, CO. Chemical analysis of borehole water at two separate depths (1,044 m and 1,004 m below the mine entrance) suggests that a sharp chemical gradient exists, likely from the mixing of two distinct subsurface fluids, one metal rich and one relatively dilute; this has created unique niches for microorganisms. The microbial community analyzed from filtered, oxic borehole water indicated an abundance of sequences from iron-oxidizing bacteria ( Gallionella spp.) and was compared to the community from the same borehole after 2 weeks of being plugged with an expandable packer. Statistical analyses with UniFrac revealed a significant shift in community structure following the addition of the packer. Phospholipid fatty acid (PLFA) analysis suggested that Nitrosomonadales dominated the oxic borehole, while PLFAs indicative of anaerobic bacteria were most abundant in the samples from the plugged borehole. Microbial sequences were represented primarily by Firmicutes , Proteobacteria , and a lineage of sequences which did not group with any identified bacterial division; phylogenetic analyses confirmed the presence of a novel candidate division. This “Henderson candidate division” dominated the clone libraries from the dilute anoxic fluids. Sequences obtained from the granitic rock core (1,740 m below the surface) were represented by the divisions Proteobacteria (primarily the family Ralstoniaceae ) and Firmicutes . Sequences grouping within Ralstoniaceae were also found in the clone libraries from metal-rich fluids yet were absent in more dilute fluids. Lineage-specific comparisons, combined with phylogenetic statistical analyses, show that geochemical variance has an important effect on microbial community structure in deep, subsurface systems.

Applied and Environmental Microbiology↗

Comparative assessment of a restored and natural wetland using 13C-DNA SIP reveals a higher potential for methane production in the restored wetland

Wetlands are the largest natural source of methane (CH 4 ), a potent greenhouse gas produced by methanogens. Methanogenesis rates are controlled by environmental factors such as redox potential, temperature, and carbon and electron acceptor availability and are presumably dependent on the composition of the active methanogen community. We collected intact soil cores from a restored and natural freshwater depressional wetland on Maryland’s Delmarva Peninsula (USA) to assess the effects of wetland restoration and redox shifts on microbial processes. Intact soil cores were incubated under either saturated (anoxic) or unsaturated (oxic) conditions and amended with 13 C-acetate for quantitative stable isotope probing (qSIP) of the 16S rRNA gene. Restored wetland cores supported a distinct community of methanogens compared to natural cores, and acetoclastic methanogens putatively identified in the genus Methanosarcina were among the most abundant taxa in restored anoxic and oxic cores. The active microbial communities in the restored wetland cores were also distinguished by the unique presence of facultatively anaerobic bacteria belonging to the orders Firmicutes and Bacteroidetes . In natural wetland incubations, methanogen populations were not among the most abundant taxa, and these communities were instead distinguished by the unique presence of aerobic bacteria in the phyla Acidobacteria , Actinobacteria , and class Alphaproteobacteria . Iron-reducing bacteria, in the genus Geobacter , were active across all redox conditions in both the restored and the natural cores, except the natural oxic–anoxic condition. These findings suggest an overall higher potential for methanogenesis in the restored wetland site compared to the natural wetland site, even when there is evidence of Fe reduction.

Maryland↗

Ecophysiology of "halarsenatibacter silvermanii" strain SLAS-1 T , gen. nov., sp. nov., a facultative chemoautotrophic arsenate respirer from salt-saturated Searles Lake, California

Searles Lake occupies a closed basin harboring salt-saturated, alkaline brines that have exceptionally high concentrations of arsenic oxyanions. Strain SLAS-1 T was previously isolated from Searles Lake (R. S. Oremland, T. R. Kulp, J. Switzer Blum, S. E. Hoeft, S. Baesman, L. G. Miller, and J. F. Stolz, Science 308:1305-1308, 2005). We now describe this extremophile with regard to its substrate affinities, its unusual mode of motility, sequenced arrABD gene cluster, cell envelope lipids, and its phylogenetic alignment within the order Halanaero-bacteriales, assigning it the name "Halarsenatibacter silvermanii" strain SLAS-1 T . We also report on the substrate dynamics of an anaerobic enrichment culture obtained from Searles Lake that grows under conditions of salt saturation and whose members include a novel sulfate reducer of the order Desulfovibriales, the archaeon Halorhabdus utahensis, as well as a close homolog of strain SLAS-1 T . Copyright ?? 2009, American Society for Microbiology. All Rights Reserved.

Applied and Environmental Microbiology↗

Large fractions of CO 2 -fixing microorganisms in pristine limestone aquifers appear to be involved in the oxidation of reduced sulfur and nitrogen compounds

The traditional view of the dependency of subsurface environments on surface-derived allochthonous carbon inputs is challenged by increasing evidence for the role of lithoautotrophy in aquifer carbon flow. We linked information on autotrophy (Calvin-Benson-Bassham cycle) with that from total microbial community analysis in groundwater at two superimposed—upper and lower—limestone groundwater reservoirs (aquifers). Quantitative PCR revealed that up to 17% of the microbial population had the genetic potential to fix CO 2 via the Calvin cycle, with abundances of cbbM and cbbL genes, encoding RubisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase) forms I and II, ranging from 1.14 × 10 3 to 6 × 10 6 genes liter −1 over a 2-year period. The structure of the active microbial communities based on 16S rRNA transcripts differed between the two aquifers, with a larger fraction of heterotrophic, facultative anaerobic, soil-related groups in the oxygen-deficient upper aquifer. Most identified CO 2 -assimilating phylogenetic groups appeared to be involved in the oxidation of sulfur or nitrogen compounds and harbored both RubisCO forms I and II, allowing efficient CO 2 fixation in environments with strong oxygen and CO 2 fluctuations. The genera Sulfuricella and Nitrosomonas were represented by read fractions of up to 78 and 33%, respectively, within the cbbM and cbbL transcript pool and accounted for 5.6 and 3.8% of 16S rRNA sequence reads, respectively, in the lower aquifer. Our results indicate that a large fraction of bacteria in pristine limestone aquifers has the genetic potential for autotrophic CO 2 fixation, with energy most likely provided by the oxidation of reduced sulfur and nitrogen compounds.

Applied and Environmental Microbiology↗

Microbial formation of ethane in anoxic estuarine sediments

Estuarine sediment slurries produced methane and traces of ethane when incubated under hydrogen. Formation of methane occurred over a broad temperature range with an optimum above 65°C. Ethane formation had a temperature optimum at 40°C. Formation of these two gases was inhibited by air, autoclaving, incubation at 4 and 80°C, and by the methanogenic inhibitor, 2-bromoethanesulfonic acid. Ethane production was stimulated by addition of ethylthioethanesulfonic acid, and production from ethylthioethanesulfonic acid was blocked by 2-bromoethanesulfonic acid. A highly purified enrichment culture of a methanogenic bacterium obtained from sediments produced traces of ethane from ethylthioethanesulfonic acid. These results indicate that the small quantities of ethane found in anaerobic sediments can be formed by certain methanogenic bacteria.

Applied and Environmental Microbiology↗

Formation of methane and carbon dioxide from dimethylselenide in anoxic sediments and by a methanogenic bacterium

Anaerobic San Francisco Bay salt marsh sediments rapidly metabolized [ 14 C]dimethylselenide (DMSe) to 14 CH 4 and 14 CO 2 . Addition of selective inhibitors (2-bromoethanesulfonic acid or molybdate) to these sediments indicated that both methanogenic and sulfate-respiring bacteria could degrade DMSe to gaseous products. However, sediments taken from the selenium-contaminated Kesterson Wildlife Refuge produced only 14 CO 2 from [ 14 C]DMSe, implying that methanogens were not important in the Kesterson samples. A pure culture of a dimethylsulfide (DMS)-grown methylotrophic methanogen converted [ 14 C]DMSe to 14 CH 4 and 14 CO 2 . However, the organism could not grow on DMSe. Addition of DMS to either sediments or the pure culture retarded the metabolism of DMSe. This effect appeared to be caused by competitive inhibition, thereby indicating a common enzyme system for DMS and DMSe metabolism. DMSe appears to be degraded as part of the DMS pool present in anoxic environments. These results suggest that methylotrophic methanogens may demethylate methylated forms of other metals and metalloids found in nature.

California↗

Microbial hydroxylation of quinoline in contaminated groundwater: evidence for incorporation of the oxygen atom of water.

Studies conducted in an aquifer contaminated by creosote suggest that quinoline is converted to 2(1H)quinolinone by an indigenous consortium of microorganisms. Laboratory microbial experiments using H 2 18 O indicate that water is the source of the oxygen atom for this hydroxylation reaction under aerobic and anaerobic conditions.

Applied and Environmental Microbiology↗

Rates of microbial metabolism in deep coastal plain aquifers

Rates of microbial metabolism in deep anaerobic aquifers of the Atlantic coastal plain of South Carolina were investigated by both microbiological and geochemical techniques. Rates of [2- 14 C]acetate and [U- 14 C]glucose oxidation as well as geochemical evidence indicated that metabolic rates were faster in the sandy sediments composing the aquifers than in the clayey sediments of the confining layers. In the sandy aquifer sediments, estimates of the rates of CO 2 production (millimoles of CO 2 per liter per year) based on the oxidation of [2- 14 C] acetate were 9.4 × 10 −3 to 2.4 × 10 −1 for the Black Creek aquifer, 1.1 × 10 −2 for the Middendorf aquifer, and <7 × 10 −5 for the Cape Fear aquifer. These estimates were at least 2 orders of magnitude lower than previously published estimates that were based on the accumulation of CO 2 in laboratory incubations of similar deep subsurface sediments. In contrast, geochemical modeling of groundwater chemistry changes along aquifer flowpaths gave rate estimates that ranged from 10 −4 to 10 −6 mmol of CO 2 per liter per year. The age of these sediments (ca. 80 million years) and their organic carbon content suggest that average rates of CO 2 production could have been no more than 10 −4 mmol per liter per year. Thus, laboratory incubations may greatly overestimate the in situ rates of microbial metabolism in deep subsurface environments. This has important implications for the use of laboratory incubations in attempts to estimate biorestoration capacities of deep aquifers. The rate estimates from geochemical modeling indicate that deep aquifers are among the most oligotrophic aquatic environments in which there is ongoing microbial metabolism.

South Carolina↗

Electron transport in the dissimilatory iron reducer, GS-15

Mechanisms for electron transport to Fe(III) were investigated in GS-15, a novel anaerobic microorganism which can obtain energy for growth hy coupling the complete oxidation of organic acids or aromatic compounds to the reduction of Fe(III) to Fe(II). The results indicate that Fe(III) reduction proceeds through a type b cytochrome and a membrane-bound Fe(III) reductase which is distinct from the nitrate reductase.

Applied and Environmental Microbiology↗

Oxidation of polycyclic aromatic hydrocarbons under sulfate-reducing conditions

[ 14 C]naphthalene and phenanthrene were oxidized to 14 CO 2 without a detectable lag under strict anaerobic conditions in sediments from San Diego Bay, San Diego, Calif., that were heavily contaminated with polycyclic aromatic hydrocarbons (PAHs) but not in less contaminated sediments. Sulfate reduction was necessary for PAH oxidation. These results suggest that the self-purification capacity of PAH-contaminated sulfate-reducing environments may be greater than previously recognized.

California↗

Isolation of Geobacter species from diverse sedimentary environments

In an attempt to better understand the microorganisms responsible for Fe(III) reduction in sedimentary environments, Fe(III)-reducing microorganisms were enriched for and isolated from freshwater aquatic sediments, a pristine deep aquifer, and a petroleum-contaminated shallow aquifer. Enrichments were initiated with acetate or toluene as the electron donor and Fe(III) as the electron acceptor. Isolations were made with acetate or benzoate. Five new strains which could obtain energy for growth by dissimilatory Fe(III) reduction were isolated. All five isolates are gram- negative strict anaerobes which grow with acetate as the electron donor and Fe(III) as the electron acceptor. Analysis of the 16S rRNA sequence of the isolated organisms demonstrated that they all belonged to the genus Geobacter in the delta subdivision of the Proteobacteria . Unlike the type strain, Geobacter metallireducens , three of the five isolates could use H 2 as an electron donor for Fe(III) reduction. The deep subsurface isolate is the first Fe(III) reducer shown to completely oxidize lactate to carbon dioxide, while one of the freshwater sediment isolates is only the second Fe(III) reducer known that can oxidize toluene. The isolation of these organisms demonstrates that Geobacter species are widely distributed in a diversity of sedimentary environments in which Fe(III) reduction is an important process.

Applied and Environmental Microbiology↗

Microbial degradation of hydrochlorofluorocarbons (CHCl2F and CHCl2CF3) in soils and sediments

The ability of microorganisms to degrade trace levels of the hydrochlorofluorocarbons HCFC-21 and HCFC-123 was investigated. Methanotroph- linked oxidation of HCFC-21 was observed in aerobic soils, and anaerobic degradation of HCFC-21 occurred in freshwater and salt marsh sediments. Microbial degradation of HCFC-123 was observed in anoxic freshwater and salt marsh sediments, and the recovery of 1,1,1-trifluoro-2-chloroethane indicated the involvement of reductive dechlorination. No degradation of HCFC-123 was observed in aerobic soils. In same experiments, HCFCs were degraded at low (parts per billion) concentrations, raising the possibility that bacteria in nature remove HCFCs from the atmosphere.

Applied and Environmental Microbiology↗

Microbial diversity in a hydrocarbon- and chlorinated-solvent- contaminated aquifer undergoing intrinsic bioremediation

A culture-independent molecular phylogenetic approach was used to survey constituents of microbial communities associated with an aquifer contaminated with hydrocarbons (mainly jet fuel) and chlorinated solvents undergoing intrinsic bioremediation. Samples were obtained from three redox zones: methanogenic, methanogenic-sulfate reducing, and iron or sulfate reducing. Small-subunit rRNA genes were amplified directly from aquifer material DNA by PCR with universally conserved or Bacteria - or Archaea -specific primers and were cloned. A total of 812 clones were screened by restriction fragment length polymorphisms (RFLP), approximately 50% of which were unique. All RFLP types that occurred more than once in the libraries, as well as many of the unique types, were sequenced. A total of 104 (94 bacterial and 10 archaeal) sequence types were determined. Of the 94 bacterial sequence types, 10 have no phylogenetic association with known taxonomic divisions and are phylogenetically grouped in six novel division level groups (candidate divisions WS1 to WS6); 21 belong to four recently described candidate divisions with no cultivated representatives (OP5, OP8, OP10, and OP11); and 63 are phylogenetically associated with 10 well-recognized divisions. The physiology of two particularly abundant sequence types obtained from the methanogenic zone could be inferred from their phylogenetic association with groups of microorganisms with a consistent phenotype. One of these sequence types is associated with the genus Syntrophus ; Syntrophus spp. produce energy from the anaerobic oxidation of organic acids, with the production of acetate and hydrogen. The organism represented by the other sequence type is closely related to Methanosaeta spp., which are known to be capable of energy generation only through aceticlastic methanogenesis. We hypothesize, therefore, that the terminal step of hydrocarbon degradation in the methanogenic zone of the aquifer is aceticlastic methanogenesis and that the microorganisms represented by these two sequence types occur in syntrophic association.

Applied and Environmental Microbiology↗

Estimation of methanogen biomass via quantitation of coenzyme M

Determination of the role of methanogenic bacteria in an anaerobic ecosystem often requires quantitation of the organisms. Because of the extreme oxygen sensitivity of these organisms and the inherent limitations of cultural techniques, an accurate biomass value is very difficult to obtain. We standardized a simple method for estimating methanogen biomass in a variety of environmental matrices. In this procedure we used the thiol biomarker coenzyme M (CoM) (2-mercaptoethanesulfonic acid), which is known to be present in all methanogenic bacteria. A high-performance liquid chromatography-based method for detecting thiols in pore water (A. Vairavamurthy and M. Mopper, Anal. Chim. Acta 78:363–370, 1990) was modified in order to quantify CoM in pure cultures, sediments, and sewage water samples. The identity of the CoM derivative was verified by using liquid chromatography-mass spectroscopy. The assay was linear for CoM amounts ranging from 2 to 2,000 pmol, and the detection limit was 2 pmol of CoM/ml of sample. CoM was not adsorbed to sediments. The methanogens tested contained an average of 19.5 nmol of CoM/mg of protein and 0.39 ± 0.07 fmol of CoM/cell. Environmental samples contained an average of 0.41 ± 0.17 fmol/cell based on most-probable-number estimates. CoM was extracted by using 1% tri-( N )-butylphosphine in isopropanol. More than 90% of the CoM was recovered from pure cultures and environmental samples. We observed no interference from sediments in the CoM recovery process, and the method could be completed aerobically within 3 h. Freezing sediment samples resulted in 46 to 83% decreases in the amounts of detectable CoM, whereas freezing had no effect on the amounts of CoM determined in pure cultures. The method described here provides a quick and relatively simple way to estimate methanogenic biomass.

Applied and Environmental Microbiology↗

Structural and spectral features of selenium nanospheres produced by Se-respiring bacteria

Certain anaerobic bacteria respire toxic selenium oxyanions and in doing so produce extracellular accumulations of elemental selenium [Se(0)]. We examined three physiologically and phylogenetically diverse species of selenate- and selenite-respiring bacteria, Sulfurospirillum barnesii, Bacillus selenitireducens, and Selenihalanaerobacter shriftii, for the occurrence of this phenomenon. When grown with selenium oxyanions as the electron acceptor, all of these organisms formed extracellular granules consisting of stable, uniform nanospheres (diameter, ???300 nm) of Se(0) having monoclinic crystalline structures. Intracellular packets of Se(0) were also noted. The number of intracellular Se(0) packets could be reduced by first growing cells with nitrate as the electron acceptor and then adding selenite ions to washed suspensions of the nitrate-grown cells. This resulted in the formation of primarily extracellular Se nanospheres. After harvesting and cleansing of cellular debris, we observed large differences in the optical properties (UV-visible absorption and Raman spectra) of purified extracellular nanospheres produced in this manner by the three different bacterial species. The spectral properties in turn differed substantially from those of amorphous Se(0) formed by chemical oxidation of H2Se and of black, vitreous Se(0) formed chemically by reduction of selenite with ascorbate. The microbial synthesis of Se(0) nanospheres results in unique, complex, compacted nanostructural arrangements of Se atoms. These arrangements probably reflect a diversity of enzymes involved in the dissimilatory reduction that are subtly different in different microbes. Remarkably, these conditions cannot be achieved by current methods of chemical synthesis.

Applied and Environmental Microbiology↗

Redox transformations of arsenic oxyanions in periphyton communities

Periphyton ( Cladophora sp.) samples from a suburban stream lacking detectable dissolved As were able to reduce added As(V) to As(III) when incubated under anoxic conditions and, conversely, oxidized added As(III) to As(V) with aerobic incubation. Both types of activity were abolished in autoclaved controls, thereby demonstrating its biological nature. The reduction of As(V) was inhibited by chloramphenicol, indicating that it required the synthesis of new protein. Nitrate also inhibited As(V) reduction, primarily because it served as a preferred electron acceptor to which the periphyton community was already adapted. However, part of the inhibition was also caused by microbial reoxidation of As(III) linked to nitrate. Addition of [ 14 C]glucose to anoxic samples resulted in the production of 14 CO 2 , suggesting that the observed As(V) reduction was a respiratory process coupled to the oxidation of organic matter. The population density of As(V)-reducing bacteria within the periphyton increased with time and with the amount of As(V) added, reaching values as high as ∼10 6 cells ml −1 at the end of the incubation. This indicated that dissimilatory As(V) reduction in these populations was linked to growth. However, As(V)-respiring bacteria were found to be present, albeit at lower numbers (∼10 2 ml −1 ), in freshly sampled periphyton. These results demonstrate the presence of a bacterial population within the periphyton communities that is capable of two key arsenic redox transformations that were previously studied in As-contaminated environments, which suggests that these processes are widely distributed in nature. This assumption was reinforced by experiments with estuarine samples of Cladophora sericea in which we detected a similar capacity for anaerobic As(V) reduction and aerobic As(III) oxidation.

Applied and Environmental Microbiology↗

Chloroethene biodegradation in sediments at 4°C

Microbial reductive dechlorination of [1,2- 14 C]trichloroethene to [ 14 C] cis -dichloroethene and [ 14 C]vinyl chloride was observed at 4&deg;C in anoxic microcosms prepared with cold temperature-adapted aquifer and river sediments from Alaska. Microbial anaerobic oxidation of [1,2- 14 C] cis -dichloroethene and [1,2- 14 C]vinyl chloride to 14 CO 2 also was observed under these conditions.

Applied and Environmental Microbiology↗

Identification of a novel arsenite oxidase gene, arxA, in the haloalkaliphilic, arsenite-oxidizing bacterium alkalilimnicola ehrlichii strain MLHE-1

Although arsenic is highly toxic to most organisms, certain prokaryotes are known to grow on and respire toxic metalloids of arsenic (i.e., arsenate and arsenite). Two enzymes are known to be required for this arsenic-based metabolism: (i) the arsenate respiratory reductase (ArrA) and (ii) arsenite oxidase (AoxB). Both catalytic enzymes contain molybdopterin cofactors and form distinct phylogenetic clades (ArrA and AoxB) within the dimethyl sulfoxide (DMSO) reductase family of enzymes. Here we report on the genetic identification of a “new” type of arsenite oxidase that fills a phylogenetic gap between the ArrA and AoxB clades of arsenic metabolic enzymes. This “new” arsenite oxidase is referred to as ArxA and was identified in the genome sequence of the Mono Lake isolate Alkalilimnicola ehrlichii MLHE-1, a chemolithoautotroph that can couple arsenite oxidation to nitrate reduction. A genetic system was developed for MLHE-1 and used to show that arxA (gene locus ID mlg _ 0216 ) was required for chemoautotrophic arsenite oxidation. Transcription analysis also showed that mlg _ 0216 was only expressed under anaerobic conditions in the presence of arsenite. The mlg _ 0216 gene is referred to as arxA because of its greater homology to arrA relative to aoxB and previous reports that implicated Mlg_0216 (ArxA) of MLHE-1 in reversible arsenite oxidation and arsenate reduction in vitro . Our results and past observations support the position that ArxA is a distinct clade within the DMSO reductase family of proteins. These results raise further questions about the evolutionary relationships between arsenite oxidases (AoxB) and arsenate respiratory reductases (ArrA). Arsenic is toxic to most organisms and is known to cause cancer in humans. However, bacteria have adapted several biotransformation pathways that function to either couple the reduction or oxidation of arsenicals to energy conservation and growth (1). The enzymologies of these two pathways have several features in common. The arsenate respiratory reductase (ArrAB) and arsenite oxidase (AoxAB) enzymes are usually composed of at least two subunits, a small iron-sulfur cluster-containing subunit (ArrB and AoxA) and a larger molybdopterin-containing catalytic subunit (ArrA and AoxB). Although they catalyze arsenic redox chemistry, ArrA and AoxB form distinct phylogenetic clades within the dimethyl sulfoxide (DMSO) reductase family of molybdenum-containing enzymes (16, 24). Culture-dependent approaches have resulted in the isolation of a variety of diverse bacteria that metabolize arsenic (reviewed in reference 26). Many of these isolates have had their genomes sequenced, which has been insightful for understanding the composition and diversity of arr and aox gene clusters. In the arsenite-oxidizing nitrate reducer Alkalilimnicola ehrlichii strain MLHE-1 (a haloalkaliphile isolated from Mono Lake [CA]) (10, 15), bioinformatic analysis of its genome revealed the absence of genes homologous to the arsenite oxidase genes of the aoxB type. Instead, two genes ( mlg _ 0216 and mlg _ 2426 ) were identified that better resembled the catalytic subunit of the arsenate respiratory reductase (20); however, MLHE-1 has not been shown to respire (or reduce) arsenate (15). Recent work by Richey et al. (20) showed that the Mlg_0216 protein (and not Mlg_2426) was expressed under chemolithoautotrophic (10 mM arsenite and 10 mM nitrate) growth conditions. Moreover, it was shown that Mlg_0216 exhibits both arsenate reductase and arsenite oxidase activities in vitro . These observations raised the question, is the mlg _ 0216 gene required for arsenite oxidation in vivo ? In this report, we addressed this question by developing a genetic system in MLHE-1, generating strains with mutations in mlg _ 0216 and mlg _ 2426 , and physiologically characterizing the resulting strains. Our results implicate mlg _ 0216 in chemolithoautotrophic arsenite oxidation coupled to nitrate respiration.

Journal of Bacteriology↗