Ecophysiology and geochemistry of microbial arsenic oxidation within a high arsenic, circumneutral hot spring system of the Alvord Desert

Microbial metabolism of arsenic has gained considerable interest, due to the potential of microorganisms to drive arsenic cycling and significantly influence the geochemistry of naturally arsenic-rich or anthropogenically arsenic-polluted environments. Alvord Hot Spring in southeastern Oregon is a circumneutral hot spring with an average arsenic concentration of 4.5 mg L(-1) (60 microM). Hydrogeochemical analyses indicated significant arsenite oxidation, increased pH and decreased temperature along the stream channels flowing into Alvord Hot Spring. The dynamic range of pH and temperature over the length of three stream channels were 6.76-7.06 and 69.5-78.2 degrees C, respectively. Biofilm samples showed As(III) oxidation ex situ. 16S rRNA gene studies of sparse upstream biofilm indicated a dominance of bacteria related to Sulfurihydrogenibium, Thermus, and Thermocrinis. The lush downstream biofilm community included these same three groups but was more diverse with sequences related to uncultured OP10 bacterial phylum, uncultured Bacteroidetes, and an uncultured clade. Isolation of an arsenite oxidizer was conducted with artificial hot spring medium and yielded the isolate A03C, which is closely related to Thermus aquaticus based on 16S rRNA gene analysis. Thus, this study demonstrated the bacterial diversity along geochemical gradients of temperature, pH and As(III): As(V), and provided evidence of microbial arsenite oxidation within the Alvord Hot Spring system.     

Dissimilatory reduction of Fe(III) and other electron acceptors by a Thermus isolate

A thermophilic bacterium that can use O2, NO3-, Fe(III), and S0 as terminal electron acceptors for growth was isolated from groundwater sampled at a 3.2-km depth in a South African gold mine. This organism, designated SA-01, clustered most closely with members of the genus Thermus, as determined by 16S rRNA gene (rDNA) sequence analysis. The 16S rDNA sequence of SA-01 was >98% similar to that of Thermus strain NMX2 A.1, which was previously isolated by other investigators from a thermal spring in New Mexico. Strain NMX2 A.1 was also able to reduce Fe(III) and other electron acceptors. Neither SA-01 nor NMX2 A.1 grew fermentatively, i.e., addition of an external electron acceptor was required for anaerobic growth. Thermus strain SA-01 reduced soluble Fe(III) complexed with citrate or nitrilotriacetic acid (NTA); however, it could reduce only relatively small quantities (0.5 mM) of hydrous ferric oxide except when the humic acid analog 2,6-anthraquinone disulfonate was added as an electron shuttle, in which case 10 mM Fe(III) was reduced. Fe(III)-NTA was reduced quantitatively to Fe(II); reduction of Fe(III)-NTA was coupled to the oxidation of lactate and supported growth through three consecutive transfers. Suspensions of Thermus strain SA-01 cells also reduced Mn(IV), Co(III)-EDTA, Cr(VI), and U(VI). Mn(IV)-oxide was reduced in the presence of either lactate or H2. Both strains were also able to mineralize NTA to CO2 and to couple its oxidation to Fe(III) reduction and growth. The optimum temperature for growth and Fe(III) reduction by Thermus strains SA-01 and NMX2 A.1 is approximately 65 degrees C; their optimum pH is 6.5 to 7.0. This is the first report of a Thermus sp. being able to couple the oxidation of organic compounds to the reduction of Fe, Mn, or S.     

Molecular characterization and in situ quantification of anoxic arsenite-oxidizing denitrifying enrichment cultures

To explore the bacteria involved in the oxidation of arsenite (As(III)) under denitrifying conditions, three enrichment cultures (ECs) and one mixed culture (MC) were characterized that originated from anaerobic environmental samples. The oxidation of As(III) (0.5 mM) was dependent on NO₃⁻ addition and N₂ formation was dependent on As(III) addition. The ratio of N₂–N formed to As(III) fed approximated the expected stoichiometry of 2.5. A 16S rRNA gene clone library analysis revealed three predominant phylotypes. The first, related to the genus Azoarcus from the division Betaproteobacteria , was found in the three ECs. The other two predominant phylotypes were closely related to the genera Acidovorax and Diaphorobacter within the Comamonadaceae family of Betaproteobacteria , and one of these was present in all of the cultures examined. FISH confirmed that Azoarcus accounted for a large fraction of bacteria present in the ECs. The Azoarcus clones had 96% sequence homology with Azoarcus sp. strain DAO1, an isolate previously reported to oxidize As(III) with nitrate. FISH analysis also confirmed that Comamonadaceae were present in all cultures. Pure cultures of Azoarcus and Diaphorobacter were isolated and shown to be responsible for nitrate-dependent As(III) oxidation. These results, taken as a whole, suggest that bacteria within the genus Azoarcus and the family Comamonadaceae are involved in the observed anoxic oxidation of As(III).     

Manganese and Arsenic Oxidation Performance of Bacterium-Yunotaki 86 (BY86) from Hokkaido,Japan, and the Bacterium's Phylogeny

We examined the Mn(II) oxidation performance of a bacterium, BY86, collected at Yunotaki Falls Hokkaido, Japan. The bacterium showed rapid oxidation of Mn(II), and brown precipitates containing Mn formed within a few days of incubation. The presence of higher oxidation states of Mn than Mn(II) was ascertained by the UV-vis and XANES sutdy. This bacterium did not oxidize As(III) to As(V) in the absence of Mn. In the presence of Mn, however, As(III) was rapidly oxidized to As(V) on the cell surfaces. These findings indicate that BY86 does not have the ability to directly oxidize As(III) to As(V) within a short period of contact, but indirectly oxidizes it by the Mn oxides generated on the cell surfaces. A phylogenetical study disclosed that BY86 was most closely related to Bacillus cereus with an identity of 99.90%. It is expected that our findings in this study will contribute to the study of Mn(II)-oxidizing bacteria, which play an important role in the biogeochemical cycling of Mn as well as other trace elements including As.     

Redox cycling of arsenic by the hydrothermal marine bacterium Marinobacter santoriniensis

Marinobacter santoriniensis NKSG1^T is a mesophilic, dissimilatory arsenate-reducing and arsenite-oxidizing bacterium isolated from an arsenate-reducing enrichment culture. The inoculum was obtained from arsenic-rich shallow marine hydrothermal sediment from Santorini, Greece, with evidence of arsenic redox cycling. Growth studies demonstrated M. santoriniensis NKSG1^T is capable of conserving energy from the reduction of arsenate [As(V)] with acetate or lactate as the electron donor, and of oxidizing arsenite [As(III)] heterotrophically with oxygen as the electron acceptor. The oxidation of As(III) coincided with the expression of the aoxB gene encoding for the catalytic molybdopterin subunit of the heterodimeric arsenite oxidase operon, indicating the reaction is enzymatically controlled, and M. santoriniensis NKSG1^T is a heterotrophic As(III)-oxidizing bacterium. Although it is clear that this organism also performs dissimilatory As(V) reduction, no amplification of the arrA arsenate reductase gene was attained using a range of primers and PCR conditions. Marinobacter santoriniensis NKSG1^T belongs to a genus of bacteria widely occurring in marine environments, including hydrothermal sediments, and is among the first marine bacteria shown to be capable of either anaerobic As(V) respiration or aerobic As(III) oxidation.     

Interaction of the isolated domain II/III of Thermus thermophilus elongation factor Tu with the nucleotide exchange factor EF-Ts.

The middle and C-terminal domain (domain II/III) of elongation factor Tu from Thermus thermophilus lacking the GTP/GDP binding domain have been prepared by treating nucleotide-free protein with Staphylococcus aureus V8 protease. The isolated domain II/III of EF-Tu has a compact structure and high resistance against tryptic treatment and thermal denaturation. As demonstrated by circular dichroism spectroscopy, the isolated domain II/III does not contain any alpha-helical structure. Nucleotide exchange factor, EF-Ts, was found to interact with domain II/III, whereas the binding of aminoacyl-tRNA, GDP and GTP to this EF-Tu fragment could not be detected.     

Anaerobic arsenite oxidation by novel denitrifying isolates

Autotrophic microorganisms have been isolated that are able to derive energy from the oxidation of arsenite [As(III)] to arsenate [As(V)] under aerobic conditions. Based on chemical energetics, microbial oxidation of As(III) can occur in the absence of oxygen, and may be relevant in some environments. Enrichment cultures were established from an arsenic contaminated industrial soil amended with As(III) as the electron donor, inorganic C as the carbon source and nitrate as the electron acceptor. In the active enrichment cultures, oxidation of As(III) was stoichiometrically coupled to the reduction of NO(3) (-). Two autotrophic As(III)-oxidizing strains were isolated that completely oxidized 5 mM As(III) within 7 days under denitrifying conditions. Based on 16S rRNA gene sequencing results, strain DAO1 was 99% related to Azoarcus and strain DAO10 was most closely related to a Sinorhizobium. The nitrous oxide reductase (nosZ) and the RuBisCO Type II (cbbM) genes were successfully amplified from both isolates underscoring their ability to denitrify and fix CO(2) while coupled to As(III) oxidation. Although limited work has been done to examine the diversity of anaerobic autotrophic oxidizers of As(III), this process may be an important component in the biological cycling of arsenic within the environment.     

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 [14C]glucose to anoxic samples resulted in the production of 14CO2, 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 approximately 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 (approximately 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.     

Dynamics and mechanisms of arsenite oxidation by freshwater lake sediments

There has been increasing concern over As in freshwater environments from sources such as arsenical pesticides, smelters, coal-fired power plants, and erosion caused by intensive land use. Arsenic in the reduced state, As (III) (arsenite), is much more toxic, more soluble and mobile, than when in the oxidized state, As (V) (arsenate). This paper summarizes the dynamics and mechanisms involved in the oxidation of As (III) to As (V) by freshwater lake sediments. Sediments from selected freshwater lakes in southern Saskatchewan oxidize As (III) to As (V) predominantly through an abiotic process. Solution analysis of As (III) and As (V) by colorimetry, and examination of the oxidation state of surface-sorbed As species by X-ray photoelectron spectroscopy, indicate that Mn present in the sediment is the primary electron acceptor in the oxidation of As (III). The transformation of As (III) to As (V) by carbonate and silicate minerals, common in sediments, is not evident. The heat of activation, ΔH_a, for the depletion (oxidation plus sorption) of As (III) by the sediments, varies from 3.3 to 8.5 kcal mole⁻¹, indicating that the process is predominantly diffusion-controlled. The Mn present in a series of particle size fractions ( < 2– > 20 μm) of the sediments may potentially detoxify As (III) in aquatic systems, by converting it to As (V).     

Inhibition of iron(II) oxidation by arsenic(III,V) in Thiobacillus ferrooxidans: Effects on arsenopyrite bioleaching

Summary The more complex inhibitory effect of As(III) than that of As(V) on Fe(II) oxidation in a non-growing Thiobacillus ferrooxidans suspension was demonstrated. The yield of arsenic bioextraction from a chalcopyrite concentrate was not affected by arsenic inhibition due to the low sensitivity of the strain to arsenic ions, supported by a spontaneous conversion of As(III) to As(V).     

Microbial arsenite oxidation with oxygen,nitrate, or an electrode as the sole electron acceptor

The purpose of this study was to identify bacteria that can perform As(III) oxidation for environmental bioremediation. Two bacterial strains, named JHS3 and JHW3, which can autotrophically oxidize As(III)–As(V) with oxygen as an electron acceptor, were isolated from soil and water samples collected in the vicinity of an arsenic-contaminated site. According to 16S ribosomal RNA sequence analysis, both strains belong to the ?-Proteobacteria class and share 99% sequence identity with previously described strains. JHS3 appears to be a new strain of the Acinetobacter genus, whereas JHW3 is likely to be a novel strain of the Klebsiella genus. Both strains possess the aioA gene encoding an arsenite oxidase and are capable of chemolithoautotrophic growth in the presence of As(III) up to 10 mM as a primary electron donor. Cell growth and As(III) oxidation rate of both strains were significantly enhanced during cultivation under heterotrophic conditions. Under anaerobic conditions, only strain JHW3 oxidized As(III) using nitrate or a solid-state electrode of a bioelectrochemical system as a terminal electron acceptor. Kinetic studies of As(III) oxidation under aerobic condition demonstrated a higher V _max and K _m from strain JHW3 than strain JHS3. This study indicated the potential application of strain JHW3 for remediation of subsurface environments contaminated with arsenic.     

As2O3 oxidation by vitamin C: cell culture studies

The ability of As₂O₃ to induce apoptosis in various malignant cell lines has made it a potential treatment agent for several malignancies. In this study the chemical stability of As₂O₃ (As(III)) in cell-free growth media with various compositions was studied (MEM with different amount of amino acids and DMEM). Special attention was given to evaluate the influence of serum (FBS; fetal bovine serum) absence and vitamin C addition on the oxidation of As(III) to As(V) in cell-free growth media. FBS is an important source of antioxidants and vitamin C (ascorbic acid) is acting as a prooxidant in millimolar concentrations. Media were incubated with As(III) (0.6, 2 and 7 μmol l⁻¹) up to 72 h. Experiments were performed at 37°C in light or/and in the dark, with or without added serum (10%) or vitamin C (1.4, 0.14 mM). Metabolites were followed with high-performance liquid chromatography directly coupled to a hydride generation-atomic fluorescence spectrometry system. After 72 h up to 30% of As(III) was transformed into As(V) in MEMs and up to 35% in DMEM when exposed in dark. Light had no influence on transformations in MEMs, but changed the situation dramatically in DMEM where almost all As(III) was oxidized to As(V) after 72 h when exposed to light. Except for some faster oxidation rate the absence of FBS had little effect on the transformation rate in all media. The most visible impact on As(III) oxidation was observed by addition of vitamin C. Addition of vitamin C (1.4 mM) transformed almost all As(III) to As(V) within 72 h. In lower concentrations (0.14 mM) a pro-oxidative effect was still observed reaching approximately 60% oxidation of As(III) during 72 h. All oxidation processes could be explained by pseudo first order reaction kinetics, yielding reaction rates increasing with initial As(III) concentration and vitamin C concentration whereas the FBS content additionally increased the As(III) oxidation rate in the DMEM (light). The temporal oxidation of As(III) to As(V) in various cell-free growth media necessitates routine checking of the valence state of arsenic during cell culture experiments and the results of biological effects attributed to As(III) should be interpreted with caution. Special attention is needed particularly in cases with vitamin C which was acting pro-oxidatively in all conditions examined.     

Chemical and biological pathways in the bacterial oxidation of arsenopyrite

Abstract: A moderately thermophilic mixed culture of bacteria catalysed the oxidative solubilization of arsenopyrite to give Fe(III), S(VI) and As(V). Toxic effects were observed in a few experiments due to teh build-up of As(III). The bacterial oxidation of arsenopyrite involved direct attack of the bacteria on the mineral to give AS(III). Subsequent oxidation of AS(III) to AS(V) occurred reaction with FE(III), but only in the presence of pyrite, which provide a catalytic surface. Arsenopyrite was unable to act as a catalyst. The pyrite- catalysed oxidation of As(III) to AS(V) by FE(III) usually only went to completion in the presence of bacteria, possibly due to their role in the provision of clean catalytic surfaces. Thus, toxic concentrations of As(III) may accumulate in reactors during the bacterial oxidation of arsenopyrite due to the absence of pyrite or a clean pyrite surface or to low concentrations of the effective oxidizing agent, Fe(III).     

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 [¹⁴C]glucose to anoxic samples resulted in the production of ¹⁴CO₂, 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 ~10⁶ 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 (~10² 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.     

Coupled Arsenotrophy in a Hot Spring Photosynthetic Biofilm at Mono Lake,California

Red-pigmented biofilms grow on rock and cobble surfaces present in anoxic hot springs located on Paoha Island in Mono Lake. The bacterial community was dominated (∼ 85% of 16S rRNA gene clones) by sequences from the photosynthetic Ectothiorhodospira genus. Scraped biofilm materials incubated under anoxic conditions rapidly oxidized As(III) to As(V) in the light via anoxygenic photosynthesis but could also readily reduce As(V) to As(III) in the dark at comparable rates. Back-labeling experiments with ⁷³As(V) demonstrated that reduction to ⁷³As(III) also occurred in the light, thereby illustrating the cooccurrence of these two anaerobic processes as an example of closely coupled arsenotrophy. Oxic biofilms also oxidized As(III) to As(V). Biofilms incubated with [¹⁴C]acetate oxidized the radiolabel to ¹⁴CO₂ in the light but not the dark, indicating a capacity for photoheterotrophy but not chemoheterotrophy. Anoxic, dark-incubated samples demonstrated As(V) reduction linked to additions of hydrogen or sulfide but not acetate. Chemoautotrophy linked to As(V) as measured by dark fixation of [¹⁴C]bicarbonate into cell material was stimulated by either H₂ or HS⁻. Functional genes for the arsenate respiratory reductase (arrA) and arsenic resistance (arsB) were detected in sequenced amplicons of extracted DNA, with about half of the arrA sequences closely related (∼98% translated amino acid identity) to those from the family Ectothiorhodospiraceae. Surprisingly, no authentic PCR products for arsenite oxidase (aoxB) were obtained, despite observing aerobic arsenite oxidation activity. Collectively, these results demonstrate close linkages of these arsenic redox processes occurring within these biofilms.Oxyanions of the group 15 element arsenic, arsenate [As(V)] and arsenite [As(III)], have been known for millennia to be potent poisons. Despite its well-established toxicity to life, the phenomenon of arsenic resistance was discovered whereby some microorganisms maintain an otherwise “normal” existence in the presence of high concentrations of As(V) or As(III) (17, 29, 31). More recently it has become recognized that certain representatives from the bacterial and archaeal domains can actually exploit the electrochemical potential of the As(V)/As(III) redox couple (+130 mV) to gain energy for growth. This can be achieved either by employing As(III) as an autotrophic electron donor or by using As(V) as a respiratory electron acceptor (18, 21, 34). The latter phenomenon, although most commonly associated with chemoheterotrophy, can also employ inorganic substances like sulfide or H₂. Indeed, As(V)-respiring anaerobes displaying a capacity for chemoautotrophy with these electron donors have been isolated and described (5, 7, 16). We recently reported that photoautotrophy is supported by As(III) in anoxic biofilms located in hot springs on Paoha Island in Mono Lake, CA (15). This process represented a novel means of As(III) oxidation achieved via anoxygenic photosynthesis occurring in certain photosynthetic bacteria (i.e., Ectothiorhodospira) and possibly within some cyanobacteria as well (e.g., “Oscillatoria”).Whether or not a microbial habitat is overtly oxic or anoxic, or temporally shifts between these two states over a diel cycle, critical energy linkages between aerobes and anaerobes have long been known for the biogeochemical cycles of key elements, such as sulfur, iron, and nitrogen. Most prominently studied is the case of nitrogen, whereby an ecological coupling exists between the processes of nitrification and denitrification (9, 10, 28). The former process provides energy to aerobic nitrifiers, while the latter process consumes the nitrate produced by this reaction, thereby meeting the energy needs of the denitrifiers.For arsenic, the detection of both As(III) oxidation and As(V) reduction in oxic and anoxic incubations of freshly collected periphyton suggested that an analogous coupled process may also occur for this element (12). Similarly, several uncontaminated soils in Japan displayed a capacity for either As(V) reduction or As(III) oxidation upon arsenic oxyanion amendment and whether they were incubated under oxic or anoxic conditions (39). A defined coculture consisting of an aerobic As(III) oxidizer (strain OL1) and an anaerobic As(V) respirer (strain Y5) was shown to function in this fashion under manipulated laboratory conditions of oxygen tension (26). We pursued the phenomenon of coupled arsenic metabolism further by using materials collected from the hot spring biofilms in Mono Lake, but we focused on examination of the cycling of arsenic under anoxic conditions.In this paper we report results obtained by manipulated incubations of red-pigmented biofilms found in the hot springs of Paoha Island. Preliminary community characterizations of these biofilms show that they are dominated by Bacteria from the genus Ectothiorhodospira but also harbor an assemblage of Archaea related to the Halobacteriacaea. Incubation results have demonstrated the presence of the following arsenic metabolic activities: respiratory As(V) reduction, photosynthetic anaerobic As(III) oxidation, and aerobic As(III) oxidation, along with the ecophysiological conditions under which they occur. Surprisingly, we were unable to obtain authentic PCR products for arsenite oxidase genes (aoxB), despite observing aerobic As(III) oxidation activity. These biofilms serve as a model system for how anaerobic cycling of arsenic can be sustained with oxidation of As(III) by anoxygenic photosynthesis coupled to regeneration of this electron donor via dissimilatory As(V) reduction. The significance that such a light-driven anaerobic ecosystem may have played in the Archean Earth is discussed.     

Oxidation of arsenite by two β-proteobacteria isolated from soil

Two heterotrophic As(III)-oxidizing bacteria, SPB-24 and SPB-31 were isolated from garden soil. Based on 16S rRNA gene sequence analysis, strain SPB-24 was closely related to genus Bordetella, and strain SPB-31 was most closely related to genus Achromobacter. Both strains exhibited high As(III) (15 mM for SPB-24 and 40 mM for SPB-31) and As(V) (>300 mM for both strains) resistance. Both strains oxidized 5 mM As(III) in minimal medium with oxidation rate of 554 and 558 μM h⁻¹ for SPB-24 and SPB-31, respectively. Washed cells of both strains oxidized As(III) over broad pH and temperature range with optimum pH 6 and temperature 42°C for both strains. The As(III) oxidation kinetic by washed cells showed K _m and V _max values of 41.7 μM and 1,166 μM h⁻¹ for SPB-24, 52 μM and 1,186 μM h⁻¹ for SPB-31. In the presence of minimal amount of carbon source, the strains showed high As(III) oxidation rate and high specific arsenite oxidase activity. The ability of strains to resist high concentration of arsenic and oxidize As(III) with highest rates reported so far makes them potential candidates for bioremediation of arsenic-contaminated environment.     

Isolation and characterization of a novel As(V)-reducing bacterium: implications for arsenic mobilization and the genus Desulfitobacterium.

Dissimilatory arsenate-reducing bacteria have been implicated in the mobilization of arsenic from arsenic-enriched sediments. An As(V)-reducing bacterium, designated strain GBFH, was isolated from arsenic-contaminated sediments of Lake Coeur d'Alene, Idaho. Strain GBFH couples the oxidation of formate to the reduction of As(V) when formate is supplied as the sole carbon source and electron donor. Additionally, strain GBFH is capable of reducing As(V), Fe(III), Se(VI), Mn(IV) and a variety of oxidized sulfur species. 16S ribosomal DNA sequence comparisons reveal that strain GBFH is closely related to Desulfitobacterium hafniense DCB-2(T) and Desulfitobacterium frappieri PCP-1(T). Comparative physiology demonstrates that D. hafniense and D. frappieri, known for reductively dechlorinating chlorophenols, are also capable of toxic metal or metalloid respiration. DNA-DNA hybridization and comparative physiological studies suggest that D. hafniense, D. frappieri, and strain GBFH should be united into one species. The isolation of an Fe(III)- and As(V)-reducing bacterium from Lake Coeur d'Alene suggests a mechanism for arsenic mobilization in these contaminated sediments while the discovery of metal or metalloid respiration in the genus Desulfitobacterium has implications for environments cocontaminated with arsenious and chlorophenolic compounds.     

An arsenic(III)-oxidizing bacterial population: selection,characterization, and performance in reactors

AIMS: To select an autotrophic arsenic(III)-oxidizing population, named CASO1, and to evaluate the performance of the selected bacteria in reactors. METHODS AND RESULTS: An As(III)-containing medium without organic substrate was used to select CASO1 from a mining environment. As(III) oxidation was studied under batch and continuous conditions. The main organisms present in CASO1 were identified with molecular biology tools. CASO1 exhibited significant As(III)-oxidizing activity between pH 3 and 8. The optimum temperature was 25 degrees C. As(III) oxidation was still observed in the presence of 1000 mg l(-1) As(III). In continuous culture mode, the As(III) oxidation rate reached 160 mg l(-1) h(-1). The CASO1 consortium contains at least two organisms - strain b3, which is phylogenetically close to Ralstonia picketii, and strain b6, which is related to the genus Thiomonas. The divergence in 16S rDNA sequences between b6 and the closest related organism was 5.9%, suggesting that b6 may be a new species. CONCLUSIONS: High As(III)-oxidizing activity can be obtained without organic nutrient supply, using a bacterial population from a mining environment. SIGNIFICANCE AND IMPACT OF THE STUDY: The biological oxidation of arsenite by the CASO1 population is of particular interest for decontamination of arsenic-contaminated waste or groundwater.     

A Microbial Arsenic Cycle in Sediments of an Acidic Mine Impoundment: Herman Pit,Clear Lake,California

The involvement of prokaryotes in the redox reactions of arsenic occurring between its +5 [arsenate; As(V)] and +3 [arsenite; As(III)] oxidation states has been well established. Most research to date has focused upon circum-neutral pH environments (e.g., freshwater or estuarine sediments) or arsenic-rich “extreme” environments like hot springs and soda lakes. In contrast, relatively little work has been conducted in acidic environments. With this in mind we conducted experiments with sediments taken from the Herman Pit, an acid mine drainage impoundment of a former mercury (cinnabar) mine. Due to the large adsorptive capacity of the abundant Fe(III)-rich minerals, we were unable to initially detect in solution either As(V) or As(III) added to the aqueous phase of live sediment slurries or autoclaved controls, although the former consumed added electron donors (i.e., lactate, acetate, hydrogen), while the latter did not. This prompted us to conduct further experiments with diluted slurries using the live materials from the first incubation as inoculum. In these experiments we observed reduction of As(V) to As(III) under anoxic conditions and reduction rates were enhanced by addition of electron donors. We also observed oxidation of As(III) to As(V) in oxic slurries as well as in anoxic slurries amended with nitrate. We noted an acid-tolerant trend for sediment slurries in the cases of As(III) oxidation (aerobic and anaerobic) as well as for anaerobic As(V) reduction. These observations indicate the presence of a viable microbial arsenic redox cycle in the sediments of this extreme environment, a result reinforced by the successful amplification of arsenic functional genes (aioA, and arrA) from these materials.     

Inhibition of microbial arsenate reduction by phosphate

The ratio of arsenite (As(III)) to arsenate (As(V)) in soils and natural waters is often controlled by the activity of As-transforming microorganisms. Phosphate is a chemical analog to As(V) and, consequently, may competitively inhibit microbial uptake and enzymatic binding of As(V), thus preventing its reduction to the more toxic, mobile, and bioavailable form - As(III). Five As-transforming bacteria isolated either from As-treated soil columns or from As-impacted soils were used to evaluate the effects of phosphate on As(V) reduction and As(III) oxidation. Cultures were initially spiked with various P:As ratios, incubated for approximately 48 h, and analyzed periodically for As(V) and As(III) concentration. Arsenate reduction was inhibited at high P:As ratios and completely suppressed at elevated levels of phosphate (500 and 1,000 μM; P inhibition constant (K(i))～20-100 μM). While high P:As ratios effectively shut down microbial As(V) reduction, the expression of the arsenate reductase gene (arsC) was not inhibited under these conditions in the As(V)-reducing isolate, Agrobacterium tumefaciens str. 5B. Further, high phosphate ameliorated As(V)-induced cell growth inhibition caused by high (1mM) As pressure. These results indicate that phosphate may inhibit As(V) reduction by impeding As(V) uptake by the cell via phosphate transport systems or by competitively binding to the active site of ArsC.