Rapid and simple method for the most-probable-number estimation of arsenic-reducing bacteria.

A rapid and simple most-probable-number (MPN) procedure for the enumeration of dissimilatory arsenic-reducing bacteria (DARB) is presented. The method is based on the specific detection of arsenite, the end product of anaerobic arsenate respiration, by a precipitation reaction with sulfide. After 4 weeks of incubation, the medium for the MPN method is acidified to pH 6 and sulfide is added to a final concentration of about 1 mM. The brightly yellow arsenic trisulfide precipitates immediately and can easily be scored at arsenite concentrations as low as 0.05 mM. Abiotic reduction of arsenate upon sulfide addition, which could yield false positives, apparently produces a soluble As-S intermediate, which does not precipitate until about 1 h after sulfide addition. Using the new MPN method, population estimates of pure cultures of DARB were similar to direct cell counts. MPNs of environmental water and sediment samples yielded DARB numbers between 10(1) and 10(5) cells per ml or gram (dry weight), respectively. Poisoned and sterilized controls showed that potential abiotic reductants in environmental samples did not interfere with the MPN estimates. A major advantage is that the assay can be easily scaled to a microtiter plate format, enabling analysis of large numbers of samples by use of multichannel pipettors. Overall, the MPN method provides a rapid and simple means for estimating population sizes of DARB, a diverse group of organisms for which no comprehensive molecular markers have been developed yet.     

A rapid colony screening method for the detection of arsenate-reducing bacteria

A rapid and simple method has been developed for the detection of arsenate reducing bacteria based on the presence of arsenite [As (III)], the end product of anaerobic arsenate [As (V)] respiration. Confirmation of As (III) product is made by the reduction of starch-iodine complex. The method can be used over a large pH range (5.5–9.0) and can easily be determined at arsenite concentration as low as 0.025 mM. Major advantages of this technique are that a large number of samples can be analyzed easily at a time.     

Molecular methods to detect and monitor dissimilatory arsenate-respiring bacteria (DARB) in sediments

Dissimilatory arsenate-respiring bacteria (DARB) reduce arsenate to arsenite and may play a significant role in arsenic mobilization in aquifers and anoxic sediments. Many studies have been conducted with pure cultures of DARB to understand their involvement in arsenic contamination. However, few studies have examined uncultured DARB in the environment. In order to investigate uncultured DARB in anoxic sediments, genes encoding arsenate respiratory reductases ( arr ) were targeted as a genetic marker. Degenerate primers for the α-subunit of arr genes were designed and used with PCR amplification to detect uncultured DARB in the sediments collected from three stations (upper, mid and lower bay) in the Chesapeake Bay. Phylogenetic analysis of putative arrA genes revealed the diversity of DARB with distinct community structures at each of the three stations. Arsenate reduction in sediment communities was confirmed using enrichment cultures established with sediment samples from the upper bay. In addition, terminal restriction fragment length polymorphism analysis of the putative arrA genes showed changes in the community structure of DARB in the enrichment cultures while reducing arsenate. This was also confirmed by cloning and sequence analysis of the arrA genes obtained from the enrichment cultures. Thus, we were able to detect diverse uncultured DARB in sediments, as well as to describe changes in DARB community structure during arsenic reduction in anoxic environments.     

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 H¹⁴CO₃⁻ 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 δ-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.     

Molecular Characterization of the Total Bacteria and Dissimilatory Arsenate-Reducing Bacteria in Core Sediments of the Jianghan Plain,Central China

Arsenic contamination in groundwater has been reported in the Jianghan Plain of China since 2005, yet little is known about the microbial communities involved in As mobilization in this area, especially the dissimilatory arsenate-reducing bacteria (DARB) communities. Here, we conducted a cultivation-independent investigation on core sediments collected from a region with arsenic-contaminated groundwater in the Jianghan Plain to reveal the total bacteria and DARB community structures. Highly diverse As-resistant bacteria communities were found from sediment samples via high-throughput sequencing of 16S rRNA genes. Notably, we identified 27 unique arrA gene (encoding the alpha subunit of dissimilatory arsenate reductase) phylotypes, none of which was related to any previously described arrA gene sequence. This suggests a novel and unique DARB community in the sediments of the Jianghan Plain and expands our knowledge about the distribution and diversity of this group of bacteria in natural environments. Moreover, RDA and CCA demonstrated that total bacterial communities and specific functional groups are controlled by different environmental factors. Specifically, sediment pH, NH₄⁺, total nitrogen, total Fe, total organic carbon and total phosphorus were the key factors driving total bacterial community compositions, while As significantly shaped DARB community structures. This report is the first to describe DARB communities and their correlation with environmental factors in Jianghan Plain sediments, which could give us clues about the origin of the arsenic contamination of groundwater in this region.     

Biological characterization of Bacillus flexus strain SSAI1 transforming highly toxic arsenite to less toxic arsenate mediated by periplasmic arsenite oxidase enzyme encoded by aioAB genes

Bacillus flexus strain SSAI1 isolated from agro-industry waste, Tuem, Goa, India displayed high arsenite resistance as minimal inhibitory concentration was 25 mM in mineral salts medium. This bacterial strain exposed to 10 mM arsenite demonstrated rapid arsenite oxidation and internalization of 7 mM arsenate within 24 h. The Fourier transformed infrared (FTIR) spectroscopy of cells exposed to arsenite revealed important functional groups on the cell surface interacting with arsenite. Furthermore, scanning electron microscopy combined with electron dispersive X-ray spectroscopy (SEM-EDAX) of cells exposed to arsenite revealed clumping of cells with no surface adsorption of arsenite. Transmission electron microscopy coupled with electron dispersive X-ray spectroscopic (TEM-EDAX) analysis of arsenite exposed cells clearly demonstrated ultra-structural changes and intracellular accumulation of arsenic. Whole-genome sequence analysis of this bacterial strain interestingly revealed the presence of large number of metal(loid) resistance genes, including aioAB genes encoding arsenite oxidase responsible for the oxidation of highly toxic arsenite to less toxic arsenate. Enzyme assay further confirmed that arsenite oxidase is a periplasmic enzyme. The genome of strain SSAI1 also carried glpF, aioS and aioE genes conferring resistance to arsenite. Therefore, multi-metal(loid) resistant arsenite oxidizing Bacillus flexus strain SSAI1 has potential to bioremediate arsenite contaminated environmental sites and is the first report of its kind.

     

An indirect fluorescent antibody staining technique for determining population levels ofThiobacillus ferrooxidans in acid mine drainage waters

Thiobacillus ferrooxidans is believed to be responsible for the oxidation of ferrous ion at low pH, the rate-limiting step in the oxidation of pyrite ores and subsequent formation of acid mine drainage (AMD). It has been suggested that efforts to control this environmental problem include procedures that would inhibit this bacterium. At present, a most probable number (MPN) procedure requiring a minimum of 10 days is used to enumerate this microorganism in natural waters. If control of AMD through inhibition ofT. ferrooxidans is to be feasible, it will be necessary to develop a more rapid method to determine population levels to facilitate application of control measures.An indirect fluorescent antibody (FA) staining technique was developed for this purpose which provided reliable estimates within a few hours. Artificial samples containing approximated numbers ofT. ferrooxidans were analyzed using the FA and MPN procedures, and the FA technique more closely approximated expected numbers of cells. The MPN method was excessively conservative, detecting only 3% to 21% of the cells enumerated by the FA procedure.     

Uptake and toxicity of arsenite and arsenate in cultured brain astrocytes

Inorganic arsenicals are environmental toxins that have been connected with neuropathies and impaired cognitive functions. To investigate whether such substances accumulate in brain astrocytes and affect their viability and glutathione metabolism, we have exposed cultured primary astrocytes to arsenite or arsenate. Both arsenicals compromised the cell viability of astrocytes in a time- and concentration-dependent manner. However, the early onset of cell toxicity in arsenite-treated astrocytes revealed the higher toxic potential of arsenite compared with arsenate. The concentrations of arsenite and arsenate that caused within 24 h half-maximal release of the cytosolic enzyme lactate dehydrogenase were around 0.3 mM and 10 mM, respectively. The cellular arsenic contents of astrocytes increased rapidly upon exposure to arsenite or arsenate and reached after 4 h of incubation almost constant steady state levels. These levels were about 3-times higher in astrocytes that had been exposed to a given concentration of arsenite compared with the respective arsenate condition. Analysis of the intracellular arsenic species revealed that almost exclusively arsenite was present in viable astrocytes that had been exposed to either arsenate or arsenite. The emerging toxicity of arsenite 4 h after exposure was accompanied by a loss in cellular total glutathione and by an increase in the cellular glutathione disulfide content. These data suggest that the high arsenite content of astrocytes that had been exposed to inorganic arsenicals causes an increase in the ratio of glutathione disulfide to glutathione which contributes to the toxic potential of these substances.     

Oxidative Transformation of Trithioarsenate Along Alkaline Geothermal Drainages—Abiotic versus Microbially Mediated Processes

Trithioarsenate is the predominant arsenic species at the source of alkaline, sulfidic geothermal springs in Yellowstone National Park. Kinetic studies along seven drainage channels showed that upon discharge the major initial reaction is rapid transformation to arsenite. When aerating a trithioarsenate solution in the laboratory, 10 to 20% of trithioarsenate dissociates abiotically before reaching a steady state with arsenite and thiosulfate. In the geothermal springs, trithioarsenate is completely converted to arsenite and rate constants of 0.2 to 1.9 min?1 are 40 to 500 times higher than in the laboratory, indicating microbial catalysis. Abiotic transformation of trithioarsenate to arsenate requires the presence of a strong oxidizing agent in the laboratory and no evidence was found for direct transformation of thioarsenates to arsenate in the geothermal drainage channels. The simultaneous increase of arsenite and arsenate observed upon trithioarsenate dissociation in some hot springs confirms that the main reaction is thioarsenate transformation to arsenite before microbially catalyzed oxidation to arsenate. In contrast to previous investigations in acidic hot springs, microbially catalyzed arsenate production in near-neutral to alkaline hot springs is not inhibited by the presence of sulfide. Phylogenetic analysis showed that arsenate production coincides with the temperature-dependent occurrence of organisms closely related to Thermocrinis ruber, a sulfur-oxidizing bacterium.     

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.     

Most probable number methodology for quantifying dilute concentrations and fluxes of Salmonella in surface waters

Aims: To better understand and manage the fate and transport of Salmonella in agricultural watersheds, we developed a culture‐based, five tube–four dilution most probable number (MPN) method for enumerating dilute densities of Salmonella in environmental waters. Methods and Results: The MPN method was a combination of a filtration technique for large sample volumes of environmental water, standard selective media for Salmonella and a TaqMan confirmation step. This method has determined the density of Salmonella in 20‐l samples of pond inflow and outflow streams as low as 0·1 MPN l^?1 and a low 95% confidence level 0·015 MPN l^?1. Salmonella densities ranged from not detectable to 0·55 MPN l^?1 for pond inflow samples and from not detectable to 3·4 MPN l^?1 for pond outflow samples. Salmonella densities of pond inflow samples were associated with densities of Escherichia coli and faecal enterococci that indicated stream contamination with faeces and with nondetectable pond outflow densities of the faecal indicator bacteria. The MPN methodology was extended to flux determinations by integrating with volumetric measurements of pond inflow (mean flux of 2·5 l s^?1) and outflow (mean flux of 5·6 l s^?1). Fluxes of Salmonella ranged from 100 to greater than 10⁴ MPN h^?1. Conclusions: This is a culture‐based method that can detect small numbers of Salmonella in environmental waters of watersheds containing animal husbandry and wildlife. Significance and Impact of the Study: Applying this method to environmental waters will improve our understanding of the transport and fate of Salmonella in agricultural watersheds, and can be the basis of valuable collections of environmental Salmonella.     

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.     

Preparation and purification of As-labeled arsenate and arsenite for use in biological experiments

Inorganic arsenic may occur in biological systems as arsenite or arsenate, these two forms of arsenic differing markedly in both their chemical and biological properties (1). Preparations of arsenic-74 sometimes contain arsenic in both oxidation states. Lunde (2) reported that a sample of ⁷⁴As-labeled sodium arsenate contained 60% of the arsenic-74 as arsenite, while Chan et al. (3) found that labeled arsenate samples contained 0.1 to 1% of an impurity which did not migrate with authentic arsenate during paper electrophoresis.     

Influence of sulphur on arsenic accumulation and metabolism in rice seedlings

The influence of sulphur on the accumulation and metabolism of arsenic in rice was investigated. Rice seedlings were grown in nutrient solutions with low sulphate (1.8 μM SO₄²⁻) or high sulphate (0.7 mM SO₄²⁻) for 12 or 14 d, before being exposed to 10 μM arsenite or arsenate for 2 or 1 d, respectively. In the arsenite exposure treatment, low sulphate-pretreated rice accumulated less arsenite than high sulphate pretreated plants, but the arsenite concentrations in shoots of low sulphate pretreated rice were higher than those of high sulphate pretreated. In the arsenate exposure treatment, the low sulphate pre-treatments also resulted in less arsenite accumulation in rice roots. Sulphur deprivation in nutrient solution decreased the concentrations of non-protein thiols in rice roots exposed to either arsenite or arsenate. The low sulphate-pretreated plants had a higher arsenic transfer factor than the high sulphate-pretreated plants. The results suggest that rice sulphate nutrition plays an important role in regulating arsenic translocation from roots to shoots, possibly through the complexation of arsenite-phytochelatins.     

Relative toxicity of arsenite and arsenate on germination and early seedling growth of rice (Oryza sativa L.)

Elevated soil arsenic levels resulting from long-term use of arsenic contaminated ground for irrigation in Bangladesh may inhibit seed germination and seedling establishment of rice, the country's main food crop. A germination study on rice seeds and a short-term toxicity experiment with different concentrations of arsenite and arsenate on rice seedlings were conducted. Percent germination over control decreased significantly with increasing concentrations of arsenite and arsenate. Arsenite was found to be more toxic than arsenate for rice seed germination. There were varietal differences among the test varieties in response to arsenite and arsenate exposure. The performance of the dry season variety Purbachi was the best among the varieties. Germination of Purbachi was not inhibited at all up to 4 mg l^–1 arsenite and 8 mg l^–1 arsenate treatment. Root tolerance index (RTI) and relative shoot height (RSH) for rice seedlings decreased with increasing concentrations of arsenite and arsenate. Reduction of RTI caused by arsenate was higher than that of arsenite. In general, dry season varieties have more tolerance to arsenite or arsenate than the wet season varieties.     

The effect of arsenate and arsenite on photosynthesis in Scenedesmus obliquus. A Potentiometric study in a closed CO2-system

Abstract

A Potentiometric titration method was used to study the adverse effect of arsenate (As(V)) and arsenite (As(III)) on inorganic carbon uptake in suspensions of the green alga Scenedesmus obliquus. The measurements were performed in a closed CO₂-system with diluted synthetic seawater (1‰ salinity) as ionic medium. Usually, the algal chlorophyll concentration was 0.4 mg dm^?3, while the arsenate- and arsenite-concentrations were varied within the limits 0.1 to 200 μmol dm^?3. In some experiments arsenate toxicity was studied in the presence of 1 to 100 μmol dm^?3 of phosphate (P(V)).

With concentrations of arsenate or arsenite less than 0.1 μmol dm^?3 no toxic effects were observed. However, at As-concentrations of 200 μmol dm^?3, the algal carbon uptake was reduced by 41% with arsenate and 29% with arsenite, i.e., arsenate is more toxic to Scenedesmus obliquus than arsenite. The toxicity of arsenate was negligible in the presence of a ten fold excess of phosphate. This is probably due to chemical similarities between arsenate and phosphate causing competition between the ions for the binding sites.

The importance of taking the speciation as well as the buffer capacity of the algal system into account, when calculating the carbon uptake, is also discussed.     

Oxidation of arsenite to arsenate by a bacterium isolated from an aquatic environment

Arsenic is ubiquitous in the biosphere and frequently reported to be an environmental pollutant. Global cycling of arsenic is affected by microorganisms. This paper describes a new bacterial strain which is able to efficiently oxidize arsenite (As[III]) into arsenate (As[V]) in liquid medium. The rate of the transformation depends on the cell density. Arsenic species were separated by high performance liquid chromatography (HPLC) and quantified by inductively coupled plasma-atomic emission spectrometry (ICP-AES). The strain also exhibits high minimum inhibitory concentrations (MICs) for As[III] (6.65 mM (500 mg L^-1)) and other heavy metals, such as cadmium (1.42 mM (160 mg L^-1)) or lead (1.20 mM (250 mg L^-1)). Partial identification of the strain revealed a chemoorganotrophic, Gram-negative and motile rod. The results presented here demonstrate that this strain could represent a good candidate for arsenic remediation in heavily polluted sites.     

Remodulation of central carbon metabolic pathway in response to arsenite exposure in Rhodococcus sp. strain NAU‐1

Arsenite‐tolerant bacteria were isolated from an organic farm of Navsari Agricultural University (NAU), Gujarat, India (Latitude: 20°55′39.04″N; Longitude: 72°54′6.34″E). One of the isolates, NAU‐1 (aerobic, Gram‐positive, non‐motile, coccobacilli), was hyper‐tolerant to arsenite (As^III, 23 mM) and arsenate (As^V, 180 mM). 16S rRNA gene of NAU‐1 was 99% similar to the 16S rRNA genes of Rhodococcus (Accession No. HQ659188). Assays confirmed the presence of membrane bound arsenite oxidase and cytoplasmic arsenate reductase in NAU‐1. Genes for arsenite transporters (arsB and ACR3(1)) and arsenite oxidase gene (aoxB) were confirmed by PCR. Arsenite oxidation and arsenite efflux genes help the bacteria to tolerate arsenite. Specific activities of antioxidant enzymes (catalase, ascorbate peroxidase, superoxide dismutase and glutathione S‐transferase) increased in dose‐dependent manner with arsenite, whereas glutathione reductase activity decreased with increase in As^III concentration. Metabolic studies revealed that Rhodococcus NAU‐1 produces excess of gluconic and succinic acids, and also activities of glucose dehydrogenase, phosphoenol pyruvate carboxylase and isocitrate lyase were increased, to cope with the inhibited activities of glucose‐6‐phosphate dehydrogenase, pyruvate dehydrogenase and α‐ketoglutarate dehydrogenase enzymes respectively, in the presence of As^III. Enzyme assays revealed the increase in direct oxidative and glyoxylate pathway in Rhodococcus NAU‐1 in the presence of As^III.     

Most-Probable-Number Rapid Viability PCR method to detect viable spores of Bacillus anthracis in swab samples

A comparison of Most-Probable-Number Rapid Viability (MPN RV) PCR and traditional culture methods for the quantification of Bacillus anthracis Sterne spores in macrofoam swabs from a multi-center validation study was performed. The purpose of the study was to compare environmental swab processing methods for recovery, detection, and quantification of viable B. anthracis spores from surfaces. Results show that spore numbers provided by the MPN RV-PCR method were typically within 1-log of the values from a plate count method for all three levels of spores tested (3.1 × 10⁴, 400, and 40 spores sampled from surfaces with swabs) even in the presence of debris. The MPN method tended to overestimate the expected result, especially at lower spore levels. Blind negative samples were correctly identified using both methods showing a lack of cross contamination. In addition to detecting low levels of spores in environmental conditions, the MPN RV-PCR method is specific, and compatible with automated high-throughput sample processing and analysis protocols, enhancing its utility for characterization and clearance following a biothreat agent release.