Dissimilatory arsenate reduction with sulfide as electron donor: experiments with mono lake water and Isolation of strain MLMS-1, a chemoautotrophic arsenate respirer

Anoxic bottom water from Mono Lake, California, can biologically reduce added arsenate without any addition of electron donors. Of the possible in situ inorganic electron donors present, only sulfide was sufficiently abundant to drive this reaction. We tested the ability of sulfide to serve as an electron donor for arsenate reduction in experiments with lake water. Reduction of arsenate to arsenite occurred simultaneously with the removal of sulfide. No loss of sulfide occurred in controls without arsenate or in sterilized samples containing both arsenate and sulfide. The rate of arsenate reduction in lake water was dependent on the amount of available arsenate. We enriched for a bacterium that could achieve growth with sulfide and arsenate in a defined, mineral medium and purified it by serial dilution. The isolate, strain MLMS-1, is a gram-negative, motile curved rod that grows by oxidizing sulfide to sulfate while reducing arsenate to arsenite. Chemoautotrophy was confirmed by the incorporation of H(14)CO(3)(-) into dark-incubated cells, but preliminary gene probing tests with primers for ribulose-1,5-biphosphate carboxylase/oxygenase did not yield PCR-amplified products. Alignment of 16S rRNA sequences indicated that strain MLMS-1 was in the delta-Proteobacteria, located near sulfate reducers like Desulfobulbus sp. (88 to 90% similarity) but more closely related (97%) to unidentified sequences amplified previously from Mono Lake. However, strain MLMS-1 does not grow with sulfate as its electron acceptor.     

Sulfide Oxidation Coupled to Arsenate Reduction by a Diverse Microbial Community in a Soda Lake

We characterized the arsenate-reducing, sulfide-oxidizing population of Mono Lake, California, by analyzing the distribution and diversity of rrnA, cbbL, and dissimilatory arsenate reductase (arrA) genes in environmental DNA, arsenate-plus sulfide-amended lake water, mixed cultures, and isolates. The arsenate-reducing community was diverse. An organism represented by an rrnA sequence previously retrieved from Mono Lake and affiliated with the Desulfobulbaceae (Deltaproteobacteria) appears to be an important member of the arsenate-reducing, sulfide-oxidizing community. Sulfide oxidation coupled with arsenate reduction appears to proceed via a two-electron transfer, resulting in the production of arsenite and an intermediate S compound that is subsequently disproportionated. A realgar-like As/S mineral was formed in some experiments.     

Effects of Imposed Salinity Gradients on Dissimilatory Arsenate Reduction, Sulfate Reduction, and Other Microbial Processes in Sediments from Two California Soda Lakes

Salinity effects on microbial community structure and on potential rates of arsenate reduction, arsenite oxidation, sulfate reduction, denitrification, and methanogenesis were examined in sediment slurries from two California soda lakes. We conducted experiments with Mono Lake and Searles Lake sediments over a wide range of salt concentrations (25 to 346 g liter⁻¹). With the exception of sulfate reduction, rates of all processes demonstrated an inverse relationship to total salinity. However, each of these processes persisted at low but detectable rates at salt saturation. Denaturing gradient gel electrophoresis analysis of partial 16S rRNA genes amplified from As(V) reduction slurries revealed that distinct microbial populations grew at low (25 to 50 g liter⁻¹), intermediate (100 to 200 g liter⁻¹), and high (>300 g liter⁻¹) salinity. At intermediate and high salinities, a close relative of a cultivated As-respiring halophile was present. These results suggest that organisms adapted to more dilute conditions can remain viable at high salinity and rapidly repopulate the lake during periods of rising lake level. In contrast to As reduction, sulfate reduction in Mono Lake slurries was undetectable at salt saturation. Furthermore, sulfate reduction was excluded from Searles Lake sediments at any salinity despite the presence of abundant sulfate. Sulfate reduction occurred in Searles Lake sediment slurries only following inoculation with Mono Lake sediment, indicating the absence of sulfate-reducing flora. Experiments with borate-amended Mono Lake slurries suggest that the notably high (0.46 molal) concentration of borate in the Searles Lake brine was responsible for the exclusion of sulfate reducers from that ecosystem.     

Selenate-dependent anaerobic arsenite oxidation by a bacterium from Mono Lake, California

Arsenate was produced when anoxic Mono Lake water samples were amended with arsenite and either selenate or nitrate. Arsenite oxidation did not occur in killed control samples or live samples with no added terminal electron acceptor. Potential rates of anaerobic arsenite oxidation with selenate were comparable to those with nitrate ( approximately 12 to 15 mumol.liter(-1) h(-1)). A pure culture capable of selenate-dependent anaerobic arsenite oxidation (strain ML-SRAO) was isolated from Mono Lake water into a defined salts medium with selenate, arsenite, and yeast extract. This strain does not grow chemoautotrophically, but it catalyzes the oxidation of arsenite during growth on an organic carbon source with selenate. No arsenate was produced in pure cultures amended with arsenite and nitrate or oxygen, indicating that the process is selenate dependent. Experiments with washed cells in mineral medium demonstrated that the oxidation of arsenite is tightly coupled to the reduction of selenate. Strain ML-SRAO grows optimally on lactate with selenate or arsenate as the electron acceptor. The amino acid sequences deduced from the respiratory arsenate reductase gene (arrA) from strain ML-SRAO are highly similar (89 to 94%) to those from two previously isolated Mono Lake arsenate reducers. The 16S rRNA gene sequence of strain ML-SRAO places it within the Bacillus RNA group 6 of gram-positive bacteria having low G+C content.     

Dissimilatory Arsenate and Sulfate Reduction in Sediments of Two Hypersaline, Arsenic-Rich Soda Lakes: Mono and Searles Lakes, California

A radioisotope method was devised to study bacterial respiratory reduction of arsenate in sediments. The following two arsenic-rich soda lakes in California were chosen for comparison on the basis of their different salinities: Mono Lake (~90 g/liter) and Searles Lake (~340 g/liter). Profiles of arsenate reduction and sulfate reduction were constructed for both lakes. Reduction of [⁷³As]arsenate occurred at all depth intervals in the cores from Mono Lake (rate constant [k] = 0.103 to 0.04 h⁻¹) and Searles Lake (k = 0.012 to 0.002 h⁻¹), and the highest activities occurred in the top sections of each core. In contrast, [³⁵S]sulfate reduction was measurable in Mono Lake (k = 7.6 ×10⁴ to 3.2 × 10⁻⁶ h⁻¹) but not in Searles Lake. Sediment DNA was extracted, PCR amplified, and separated by denaturing gradient gel electrophoresis (DGGE) to obtain phylogenetic markers (i.e., 16S rRNA genes) and a partial functional gene for dissimilatory arsenate reduction (arrA). The amplified arrA gene product showed a similar trend in both lakes; the signal was strongest in surface sediments and decreased to undetectable levels deeper in the sediments. More arrA gene signal was observed in Mono Lake and was detectable at a greater depth, despite the higher arsenate reduction activity observed in Searles Lake. A partial sequence (about 900 bp) was obtained for a clone (SLAS-3) that matched the dominant DGGE band found in deeper parts of the Searles Lake sample (below 3 cm), and this clone was found to be closely related to SLAS-1, a novel extremophilic arsenate respirer previously cultivated from Searles Lake.     

Anaerobic Oxidation of Arsenite in Mono Lake Water and by a Facultative, Arsenite-Oxidizing Chemoautotroph, Strain MLHE-1

Arsenite [As(III)]-enriched anoxic bottom water from Mono Lake, California, produced arsenate [As(V)] during incubation with either nitrate or nitrite. No such oxidation occurred in killed controls or in live samples incubated without added nitrate or nitrite. A small amount of biological As(III) oxidation was observed in samples amended with Fe(III) chelated with nitrolotriacetic acid, although some chemical oxidation was also evident in killed controls. A pure culture, strain MLHE-1, that was capable of growth with As(III) as its electron donor and nitrate as its electron acceptor was isolated in a defined mineral salts medium. Cells were also able to grow in nitrate-mineral salts medium by using H₂ or sulfide as their electron donor in lieu of As(III). Arsenite-grown cells demonstrated dark ¹⁴CO₂ fixation, and PCR was used to indicate the presence of a gene encoding ribulose-1,5-biphosphate carboxylase/oxygenase. Strain MLHE-1 is a facultative chemoautotroph, able to grow with these inorganic electron donors and nitrate as its electron acceptor, but heterotrophic growth on acetate was also observed under both aerobic and anaerobic (nitrate) conditions. Phylogenetic analysis of its 16S ribosomal DNA sequence placed strain MLHE-1 within the haloalkaliphilic Ectothiorhodospira of the γ-Proteobacteria. Arsenite oxidation has never been reported for any members of this subgroup of the Proteobacteria.     

Growth of Strain SES-3 with Arsenate and Other Diverse Electron Acceptors

The selenate-respiring bacterial strain SES-3 was able to use a variety of inorganic electron acceptors to sustain growth. SES-3 grew with the reduction of arsenate to arsenite, Fe(III) to Fe(II), or thiosulfate to sulfide. It also grew in medium in which elemental sulfur, Mn(IV), nitrite, trimethylamine N-oxide, or fumarate was provided as an electron acceptor. Growth on oxygen was microaerophilic. There was no growth with arsenite or chromate. Washed suspensions of cells grown on selenate or nitrate had a constitutive ability to reduce arsenate but were unable to reduce arsenite. These results suggest that strain SES-3 may occupy a niche as an environmental opportunist by being able to take advantage of a diversity of electron acceptors.     

Effects of imposed salinity gradients on dissimilatory arsenate reduction, sulfate reduction, and other microbial processes in sediments from two California soda lakes

Salinity effects on microbial community structure and on potential rates of arsenate reduction, arsenite oxidation, sulfate reduction, denitrification, and methanogenesis were examined in sediment slurries from two California soda lakes. We conducted experiments with Mono Lake and Searles Lake sediments over a wide range of salt concentrations (25 to 346 g liter(-1)). With the exception of sulfate reduction, rates of all processes demonstrated an inverse relationship to total salinity. However, each of these processes persisted at low but detectable rates at salt saturation. Denaturing gradient gel electrophoresis analysis of partial 16S rRNA genes amplified from As(V) reduction slurries revealed that distinct microbial populations grew at low (25 to 50 g liter(-1)), intermediate (100 to 200 g liter(-1)), and high (>300 g liter(-1)) salinity. At intermediate and high salinities, a close relative of a cultivated As-respiring halophile was present. These results suggest that organisms adapted to more dilute conditions can remain viable at high salinity and rapidly repopulate the lake during periods of rising lake level. In contrast to As reduction, sulfate reduction in Mono Lake slurries was undetectable at salt saturation. Furthermore, sulfate reduction was excluded from Searles Lake sediments at any salinity despite the presence of abundant sulfate. Sulfate reduction occurred in Searles Lake sediment slurries only following inoculation with Mono Lake sediment, indicating the absence of sulfate-reducing flora. Experiments with borate-amended Mono Lake slurries suggest that the notably high (0.46 molal) concentration of borate in the Searles Lake brine was responsible for the exclusion of sulfate reducers from that ecosystem.     

Characterization of Microbial Arsenate Reduction in the Anoxic Bottom Waters of Mono Lake,California

Dissimilatory reduction of arsenate (DAsR) occurs in the arsenic-rich, anoxic water column of Mono Lake, California, yet the microorganisms responsible for this observed in situ activity have not been identified. To gain insight as to which microorganisms mediate this phenomenon, as well as to some of the biogeochemical constraints on this activity, we conducted incubations of arsenate-enriched bottom water coupled with inhibition/amendment studies and Denaturing Gradient Gel Electrophoresis (DGGE) characterization techniques. DAsR was totally inhibited by filter-sterilization and by nitrate, partially inhibited (~50%) by selenate, but only slightly (~25%) inhibited by oxyanions that block sulfate-reduction (molybdate and tungstate). The apparent inhibition by nitrate, however, was not due to action as a preferred electron acceptor to arsenate. Rather, nitrate addition caused a rapid, microbial re-oxidation of arsenite to arsenate, which gave the overall appearance of no arsenate loss. A similar microbial oxidation of As(III) was also found with Fe(III), a fact that has implications for the recycling of As(V) in Mono Lake's anoxic bottom waters. DAsR could be slightly (10%) stimulated by substrate amendments of lactate, succinate, malate, or glucose, but not by acetate, suggesting that the DAsR microflora is not electron donor limited. DGGE analysis of amplified 16S rDNA gene fragments from incubated arsenate-enriched bottom waters revealed the presence of two bands that were not present in controls without added arsenate. The resolved sequences of these excised bands indicated the presence of members of the epsilon ( Sulfurospirillum ) and delta ( Desulfovibrio ) subgroups of the Proteobacteria , both of which have representative species that are capable of anaerobic growth using arsenate as their electron acceptor.     

Sulfide oxidation coupled to arsenate reduction by a diverse microbial community in a soda lake

We characterized the arsenate-reducing, sulfide-oxidizing population of Mono Lake, California, by analyzing the distribution and diversity of rrnA, cbbL, and dissimilatory arsenate reductase (arrA) genes in environmental DNA, arsenate-plus sulfide-amended lake water, mixed cultures, and isolates. The arsenate-reducing community was diverse. An organism represented by an rrnA sequence previously retrieved from Mono Lake and affiliated with the Desulfobulbaceae (Deltaproteobacteria) appears to be an important member of the arsenate-reducing, sulfide-oxidizing community. Sulfide oxidation coupled with arsenate reduction appears to proceed via a two-electron transfer, resulting in the production of arsenite and an intermediate S compound that is subsequently disproportionated. A realgar-like As/S mineral was formed in some experiments.     

ArxA, a new clade of arsenite oxidase within the DMSO reductase family of molybdenum oxidoreductases

Arsenotrophy, growth coupled to autotrophic arsenite oxidation or arsenate respiratory reduction, occurs only in the prokaryotic domain of life. The enzymes responsible for arsenotrophy belong to distinct clades within the DMSO reductase family of molybdenum-containing oxidoreductases: specifically arsenate respiratory reductase, ArrA, and arsenite oxidase, AioA (formerly referred to as AroA and AoxB). A new arsenite oxidase clade, ArxA, represented by the haloalkaliphilic bacterium Alkalilimnicola ehrlichii strain MLHE-1 was also identified in the photosynthetic purple sulfur bacterium Ectothiorhodospira sp. strain PHS-1. A draft genome sequence of PHS-1 was completed and an arx operon similar to MLHE-1 was identified. Gene expression studies showed that arxA was strongly induced with arsenite. Microbial ecology investigation led to the identification of additional arxA-like sequences in Mono Lake and Hot Creek sediments, both arsenic-rich environments in California. Phylogenetic analyses placed these sequences as distinct members of the ArxA clade of arsenite oxidases. ArxA-like sequences were also identified in metagenome sequences of several alkaline microbial mat environments of Yellowstone National Park hot springs. These results suggest that ArxA-type arsenite oxidases appear to be widely distributed in the environment presenting an opportunity for further investigations of the contribution of Arx-dependent arsenotrophy to the arsenic biogeochemical cycle.     

Rapid Biotransformation of Arsenate into Oxo-Arsenosugars by a Freshwater Unicellular Green Alga,Chlamydomonas reinhardtii

We examined the short-term metabolic processes of arsenate for 24 h in a freshwater unicellular green alga, Chlamydomonas reinhardtii wild-type strain CC-125. The arsenic species in the algal extracts were identified by high-performance liquid chromatography/inductively coupled plasma mass spectrometry after water extraction using a sonicator. Speciation analyses of arsenic showed that the levels of arsenite, arsenate, and methylarsonic acid in the cells rapidly increased for 30 min to 1 h, and those of dimethylarsinic acid and oxo-arsenosugar-glycerol also tended to increase continuously for 24 h, while that of oxo-arsenosugar-phosphate was quite low and fluctuated throughout the experiment. These results indicate that this alga can rapidly biotransform arsenate into oxo-arsenosugar-glycerol for at least 10 min and then oxo-arsenosugar-phosphate through both reduction of incorporated arsenate to arsenite and methylation of arsenite and/or arsenate retained in the cells to dimethylarsinic acid via methylarsonic acid as an possible intermediate.     

Facile reduction of arsenate in methanogenic sludge

Due to the recent enactment of a stricter drinking water standard for arsenate, large quantities of arsenate-laden drinking water residuals will be disposed in municipal landfills. The objective of this study was to determine the role of methanogenic consortia on the conversion of arsenate. Methanogenic conditions commonly occur in mature municipal solid waste landfills. The results indicate the rapid and facile reduction of arsenate to arsenite in methanogenic sludge. Endogenous substrates in the sludge were sufficient to support the reductive biotransformation. However the rates of arsenate reduction were stimulated by the addition of exogenous electron donating substrates, such as H2, lactate or a mixture of volatile fatty acids. A selective methanogenic inhibitor stimulated arsenate reduction in microcosms supplied with H2, suggesting that methanogens competed with arsenate reducers for the electron donor. Rates of arsenate reduction increased with arsenate concentration up to 2 mM, higher concentrations were inhibitory. The electron shuttle, anthraquinone-2,6-disulfonate, used as a model of humic quinone moieties, was shown to significantly increase rates of arsenate reduction at substoichiometric concentrations. The presence of sulfur compounds, sulfate and sulfide, did not affect the rate of arsenate transformation but lowered the yield of soluble arsenite, due to the precipitation of arsenite with sulfides. The results taken as a whole suggest that arsenate disposed into anaerobic environments may readily be converted to arsenite increasing the mobility of arsenic. The extent of the increased mobility will depend on the concentration of sulfides generated from sulfate reduction.     

Genomic Responses to Arsenic in the Cyanobacterium Synechocystis sp. PCC 6803

Arsenic is a ubiquitous contaminant and a toxic metalloid which presents two main redox states in nature: arsenite [As^III] and arsenate [As^V]. Arsenic resistance in Synechocystis sp. strain PCC 6803 is mediated by the arsBHC operon and two additional arsenate reductases encoded by the arsI1 and arsI2 genes. Here we describe the genome-wide responses to the presence of arsenate and arsenite in wild type and mutants in the arsenic resistance system. Both forms of arsenic produced similar responses in the wild type strain, including induction of several stress related genes and repression of energy generation processes. These responses were transient in the wild type strain but maintained in time in an arsB mutant strain, which lacks the arsenite transporter. In contrast, the responses observed in a strain lacking all arsenate reductases were somewhat different and included lower induction of genes involved in metal homeostasis and Fe-S cluster biogenesis, suggesting that these two processes are targeted by arsenite in the wild type strain. Finally, analysis of the arsR mutant strain revealed that ArsR seems to only control 5 genes in the genome. Furthermore, the arsR mutant strain exhibited hypersentivity to nickel, copper and cadmium and this phenotype was suppressed by mutation in arsB but not in arsC gene suggesting that overexpression of arsB is detrimental in the presence of these metals in the media.     

Sulfur-Oxidizing Bacteria in Soap Lake (Washington State), a Meromictic, Haloalkaline Lake with an Unprecedented High Sulfide Content

Culture-dependent and -independent techniques were used to study the diversity of chemolithoautotrophic sulfur-oxidizing bacteria in Soap Lake (Washington State), a meromictic, haloalkaline lake containing an unprecedentedly high sulfide concentration in the anoxic monimolimnion. Both approaches revealed the dominance of bacteria belonging to the genus Thioalkalimicrobium, which are common inhabitants of soda lakes. A dense population of Thioalkalimicrobium (up to 10⁷ cells/ml) was found at the chemocline, which is characterized by a steep oxygen-sulfide gradient. Twelve Thioalkalimicrobium strains exhibiting three different phenotypes were isolated in pure culture from various locations in Soap Lake. The isolates fell into two groups according to 16S rRNA gene sequence analysis. One of the groups was closely related to T. cyclicum, which was isolated from Mono Lake (California), a transiently meromictic, haloalkaline lake. The second group, consisting of four isolates, was phylogenetically and phenotypically distinct from known Thioalkalimicrobium species and unique to Soap Lake. It represented a new species, for which we suggest the name Thioalkalimicrobium microaerophilum sp. nov.     

Desulfurispira natronophila gen. nov. sp. nov.: an obligately anaerobic dissimilatory sulfur-reducing bacterium from soda lakes

Anaerobic enrichment cultures with elemental sulfur as electron acceptor and either acetate or propionate as electron donor and carbon source at pH 10 and moderate salinity inoculated with sediments from soda lakes in Kulunda Steppe (Altai, Russia) resulted in the isolation of two novel members of the bacterial phylum Chrysiogenetes. The isolates, AHT11 and AHT19, represent the first specialized obligate anaerobic dissimilatory sulfur respirers from soda lakes. They use either elemental sulfur/polysulfide or arsenate as electron acceptor and a few simple organic compounds as electron donor and carbon source. Elemental sulfur is reduced to sulfide through intermediate polysulfide, while arsenate is reduced to arsenite. The bacteria belong to the obligate haloalkaliphiles, with a pH growth optimum from 10 to 10.2 and a salt range from 0.2 to 3.0 M Na⁺ (optimum 0.4–0.6 M). According to the phylogenetic analysis, the two strains were close to each other, but distinct from the nearest relative, the haloalkaliphilic sulfur-reducing bacterium Desulfurispirillum alkaliphilum, which was isolated from a bioreactor. On the basis of distinct phenotype and phylogeny, the soda lake isolates are proposed as a new genus and species, Desulfurispira natronophila (type strain AHT11^T = DSM22071^T = UNIQEM U758^T).     

Arsenic-resistant bacteria associated with roots of the wild Cirsium arvense (L.) plant from an arsenic polluted soil,and screening of potential plant growth-promoting characteristics

A rhizobacterial community, associated with the roots of wild thistle Cirsium arvense (L.) growing in an arsenic polluted soil, was studied by fluorescence in situ hybridization (FISH) analysis in conjunction with cultivation-based methods. In the bulk, rhizosphere, and rhizoplane fractions of the soil, the qualitative picture obtained by FISH analysis of the main phylogenetic bacterial groups was similar and was predominantly comprised of Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria. The arsenic-resistant isolates belonged to 13 genera, the most abundant being those of Bacillus, Achromobacter, Brevundimonas, Microbacterium, and Ochrobactrum. Most bacteria grew in the presence of high arsenic concentrations (over 100 mM arsenate and 10 mM arsenite). Most strains possessed the ArsC, ArsB and ACR3 genes homologous to arsenate reductase and to the two classes of arsenite efflux pumps, respectively, peculiar to the ars operon of the arsenic detoxification system. ArsB and ACR3 were present simultaneously in highly resistant strains. An inconsistency between 16S rRNA phylogenetic affiliations and the arsenate reductase sequences of the strains was observed, indicating possible horizontal transfer of arsenic resistance genes in the soil bacterial community. Several isolates were able to reduce arsenate and to oxidise arsenite. In particular, Ancylobacter dichloromethanicum strain As3-1b possessed both characteristics, and arsenite oxidation occurred in the strain also under chemoautotrophic conditions. Some rhizobacteria produced siderophores, indole acetic acid and 1-amino-cyclopropane-1-carboxylic acid deaminase, thus possessing potential plant growth-promoting traits.     

Distribution of RuBisCO Genotypes along a Redox Gradient in Mono Lake, California

Partial sequences of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) (EC 4.1.1.39) genes were retrieved from samples taken along a redox gradient in alkaline, hypersaline Mono Lake, Calif. The form I gene (cbbL) was found in all samples, whereas form II (cbbM) was not retrieved from any of the samples. None of the RuBisCO sequences we obtained were closely related (nucleotide similarity, <90%) to sequences in the database. Some could be attributed to organisms isolated from the lake (Cyanobium) or appearing in enrichment cultures. Most (52%) of the sequences fell into in one clade, containing sequences that were identical to sequences retrieved from an enrichment culture grown with nitrate and sulfide, and another clade contained sequences identical to those retrieved from an arsenate-reducing, sulfide-oxidizing enrichment.     

Analysis of Methane Monooxygenase Genes in Mono Lake Suggests That Increased Methane Oxidation Activity May Correlate with a Change in Methanotroph Community Structure

Mono Lake is an alkaline hypersaline lake that supports high methane oxidation rates. Retrieved pmoA sequences showed a broad diversity of aerobic methane oxidizers including the type I methanotrophs Methylobacter (the dominant genus), Methylomicrobium, and Methylothermus, and the type II methanotroph Methylocystis. Stratification of Mono Lake resulted in variation of aerobic methane oxidation rates with depth. Methanotroph diversity as determined by analysis of pmoA using new denaturing gradient gel electrophoresis primers suggested that variations in methane oxidation activity may correlate with changes in methanotroph community composition.