Controlling microbial arsenite oxidation and mobilization in arsenite-adsorbed iron minerals: The Influence of pH conditions and mineralogical composition

Role of Bacteria in Arsenic Removal from an Aqueous Environment

Microbial arsenite oxidation is an essential biogeochemical process whereby more toxic arsenite is oxidized to the less toxic arsenate. Thiomonas strains represent an important arsenite oxidizer found ubiquitous in acid mine drainage. In the present study, the arsenite oxidase gene (aioBA) was cloned from Thiomonas delicata DSM 16361, expressed heterologously in E. coli and purified to homogeneity. The purified recombinant Aio consisted of two subunits with the respective molecular weights of 91 and 21 kDa according to SDS-PAGE. Aio catalysis was optimum at pH 5.5 and 50–55 °C. Aio exhibited stability under acidic conditions (pH 2.5–6). The Vmax and Km values of the enzyme were found to be 4 µmol min−1 mg−1 and 14.2 µM, respectively. SDS and Triton X-100 were found to inhibit the enzyme activity. The homology model of Aio showed correlation with the acidophilic adaptation of the enzyme. This is the first characterization studies of Aio from a species belonging to the Thiomonas genus. The arsenite oxidase was found to be among the acid-tolerant Aio reported to date and has the potential to be used for biosensor and bioremediation applications in acidic environments.

The identification and characterization of genes involved in the microbial oxidation of arsenite will contribute to our understanding of factors controlling As cycling in natural systems. Towards this goal, we recently characterized the widespread occurrence of aerobic arsenite oxidase genes (aroA-like) from pure-culture bacterial isolates, soils, sediments and geothermal mats, but were unable to detect these genes in all geothermal systems where we have observed microbial arsenite oxidation. Consequently, the objectives of the current study were to measure arsenite-oxidation rates in geochemically diverse thermal habitats in Yellowstone National Park (YNP) ranging in pH from 2.6 to 8, and to identify corresponding 16S rRNA and aroA genotypes associated with these arsenite-oxidizing environments. Geochemical analyses, including measurement of arsenite-oxidation rates within geothermal outflow channels, were combined with 16S rRNA gene and aroA functional gene analysis using newly designed primers to capture previously undescribed aroA-like arsenite oxidase gene diversity. The majority of bacterial 16S rRNA gene sequences found in acidic (pH 2.6-3.6) Fe-oxyhydroxide microbial mats were closely related to Hydrogenobaculum spp. (members of the bacterial order Aquificales), while the predominant sequences from near-neutral (pH 6.2-8) springs were affiliated with other Aquificales including Sulfurihydrogenibium spp., Thermocrinis spp. and Hydrogenobacter spp., as well as members of the Deinococci, Thermodesulfobacteria and beta-Proteobacteria. Modified primers designed around previously characterized and newly identified aroA-like genes successfully amplified new lineages of aroA-like genes associated with members of the Aquificales across all geothermal systems examined. The expression of Aquificales aroA-like genes was also confirmed in situ, and the resultant cDNA sequences were consistent with aroA genotypes identified in the same environments. The aroA sequences identified in the current study expand the phylogenetic distribution of known Mo-pterin arsenite oxidase genes, and suggest the importance of three prominent genera of the order Aquificales in arsenite oxidation across geochemically distinct geothermal habitats ranging in pH from 2.6 to 8.

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.

Microbes play essential roles in arsenic transformation in the environment. Microbial arsenite oxidation is catalyzed by either of two distantly related arsenite oxidases, referred to as AIO and ARX. The arx genes encoding ARX and its regulatory proteins were originally defined in the genomes of gammaproteobacteria isolated from an alkaline soda lake. The arx gene cluster has been identified in a limited number of bacteria, predominantly in gammaproteobacteria isolated from lakes characterized by high pH and high salinity. In the present study, a novel arsenite-oxidizing betaproteobacterium, strain M52, was isolated from a hot spring microbial mat. The strain oxidized arsenite under both microaerophilic and nitrate-reducing conditions at nearly neutral pH. Genome analysis revealed that the strain possesses the arx gene cluster in its genome and lacks genes encoding AIO. Inspection of the bacterial genomes available in the GenBank database revealed that the presence of this gene cluster is restricted to genomes of Proteobacteria, mainly in the classes Gammaproteobacteria and Betaproteobacteria. In these genomes, the structure of the gene cluster was generally well-conserved, but genes for regulatory proteins were lacking in genomes of strains belonging to a specific lineage. Phylogenetic analysis suggested that ARX encoded in the genomes can be divided into three groups, and strain M52 belongs to a group specific for organisms living in low-salt environments. The ArxA protein encoded in the genome of strain M52 was characterized by the presence of a long insertion, which was specifically observed in the same group of ARX. In clone library analyses with a newly designed primer pair, a diverse ArxA sequence with a long insertion was detected in samples of lake water and hot spring microbial mat, characterized by low salinity and a nearly neutral pH. Among the isolated bacterial strains whose arsenite oxidation has been demonstrated, strain M52 is the first betaproteobacterium that possesses the arx genes, the first strain encoding ARX of the group specific for low-salt environments, and the first organism possessing the gene encoding ArxA with a long insertion.

چکیده آلودگی های نفتی، از جمله رایج ترین و خطرناک ترین آلاینده هایی هستند که باعث آلودگی محیط زیست می شوند. تکنولوژی نو ظهور گیاه-پالایی به منظور پالایش برخی آلودگی ها، از جمله آلودگی های نفتی، به دلایل هزینه ی کم و پایین بودن تکنولوژی مورد نیاز، در دنیا بسیار مورد توجه قرار گرفته است. در این پژوهش با هدف پاک‌سازی مناطق آلوده به هیدروکربن های نفتی، سه گیاه جو، ماش و لوبیا از دو خانواده ی گرس و لگومینه انتخاب و در سه سطح تراکم І، Π و Ш که به‌ترتیب بیشتر می شد ( گیاه جو و ماش به‌ترتیب10-15-20 و لوبیا 5-10-15 عدد بذر در هر گلدان) در قالب طرح بلوک کامل تصادفی، آزمایش فاکتوریل، در سه تکرار در خاک آلوده و غیر‌آلوده کشت گردیدند. آلودگی اولیه و آلودگی خاک در سه مرحله ی جوانه زنی، رویشی و زایشی رشد گیاه اندازه‌گیری گردید و نتایج با استفاده از نرم افزار SAS تجزیه و تحلیل گردید. نتایج نشان داد که حداکثر نرخ پالایش مربوط به گیاه جو در سطح تراکم Ш بود که میزان آلودگی را تا 78/45 درصد کاهش داد. به‌طور‌کلی به‌ علت مقاومت بالای گیاه جو به آلودگی در طول رشد و نرخ پالایش بالای آن، در بین تیمارهای مورد آزمون مناسب ترین تیمار جهت گیاه پالایی، تشخیص داده شد. واژه‌های کلیدی: آلودگی، جو، خاک، لوبیا، ماش، نفت

 The oxidation of arsenite (AsIII) to arsenate (AsV) as an effective pre-treatment step was usually considered in order to ultimatly remove arsenic (As) from reducing aquatic environment such as groundwater. The present work selected quartz sand and pozzolan as two support materials filled into the parallel up-flow fixed-bed reactors. Concentrated AsIII was completly oxidized with two support materials after inoculation of acclimatized arsenite-oxidzing bacteria (AsOB) with various HRT of 6 h, 3 h and 1 h. Moreover, pozzolan was more efficient than quartz sand for AsIII oxidation even at a HRT of 40 min. Batch tests demonstrated that AsIII oxidation rate was negatively correlated with the axial length of reactors, which was closely related with the AsOB distribution in the fixed-bed reactor. Finally, high efficient removal of As realized through the microbial AsIII oxidation followed with the fixed-bed reactors filled with zero valent iron (ZVI) or negative ion-exhange resin.

Groundwater is the most important source for drinking, cooking and other household purposes. Agriculture is also largely dependent on groundwater. In this study, arsenic was detoxified by microbial oxidation using arsenic-resistant bacteria (ARB) in synthetic water. Two bacterial strains of Bacillus cereus (SP21) and Bacillus toyonensis (SP23), which are resistant to arsenic (As3+), were tested well up to 100 ppm. Optimization of arsenic for oxidation using different carbon sources (glucose, cellulose and starch), of which 1.5% cellulose was the best concentration, considering different temperatures and pH values. Oxidation of arsenite (As3+ to As5+) through a lab-scale column filled with immobilized bacterial cells and a synthetic medium through a lab-scale bioreactor. Samples were taken at regular intervals and processed for the oxidation of arsenite to arsenate (846 nm). The reduction of arsenate to arsenite (869 nm) was determined according to the corresponding protocol using the molybdenum blue method with a UV spectrophotometer. The selected arsenic-tolerant bacterial strains Bacillus cereus SP21 and Bacillus toyonensis SP23 achieved maximum removal of arsenic by the immobilized bacterial cell inclusion method.

Saccharomyces boulardii is a probiotic with proven health benefits. However its survival is challenged by gastrointestinal transit, and a ratio between 1 and 3% of living yeast is recovered in the feces after oral administration. The aim of the study was to determine to what extent the yeast was sensitive to gastrointestinal pH conditions. Therefore we explored the survival of different concentrations of S. boulardii in conditions mimicking the stomach pH (pH 1.1 0.1 N HCl) and the intestinal pH (pH 6.8 phosphate buffer) in vitro. The probiotic being commercialized as a freeze-dried powder obtained from an aqueous suspension, both forms were evaluated. In phosphate buffer pH 6.8, the viability remained stable for both forms of S. boulardii for 6 h. In HCl pH 1.1, viability of both forms (200 mg L(-1)) significantly decreased from 5 min. Observation under scanning/transmission electron microscopy showed morphological damages and rupture of the yeast wall. Threshold value from which S. boulardii viability was unaltered was pH 4. At the highest concentration of 200 g L(-1), the initial pH value of 1.1 rose to 3.2, exerting a protective effect. In conclusion, although the yeast in aqueous suspension was less sensitive than the freeze-dried yeast to acidic conditions, a gastric protection for improvement of oral bioavailability of viable S. boulardii appears necessary.

Influence of pH conditions on metabolic regulations in serine alkaline protease production by Bacillus licheniformis

The flexography ink has disadvantage in a deinking process because it tends to form too fine particles in alkali condition to be removed in flotation deinking. The influence of pH conditions on the particle size of phthalocyanine cyan ink used for flexo-printing was investigated to see the effect of pH conditions on flexography ink dispersion. Flexography ink particles prepared by grinding dried ink films were used in this experiment. Greater reduction of the ink particle size was noticed under alkaline pH condition, which was attributed to dissolution of resin component of the ink. Adsorption behavior of flexography ink onto pigment particles was examined using clay and talc as substrate pigments. Pretreatment of inorganic pigments with a cationic poly-DADMAC increased the surface adsorption of flexography ink particles, which improved the removal of the inks by centrifugal sedimentation of inorganic pigments. Most efficient removal of the ink particles was achieved when an optimal addition level of the cationic polymer was used for pretreatment of inorganic pigments, and this optimal addition level corresponds to the surface saturation point of the polyelectrolyte. Adsorption of flexography ink particles onto inorganic pigments improved the ink removal in flotation deinking since the pigment particles has the optimal particle size for flotation deinking.

BackgroundArsenic is known as a toxic metalloid, which primarily exists in inorganic form [As(III) and As(V)] and can be transformed by microbial redox processes in the natural environment. As(III) is much more toxic and mobile than As(V), hence microbial arsenic redox transformation has a major impact on arsenic toxicity and mobility which can greatly influence the human health. Our main purpose was to investigate the distribution and diversity of microbial arsenite-resistant species in three different arsenic-contaminated soils, and further study the As(III) resistance levels and related functional genes of these species.ResultsA total of 58 arsenite-resistant bacteria were identified from soils with three different arsenic-contaminated levels. Highly arsenite-resistant bacteria (MIC > 20 mM) were only isolated from the highly arsenic-contaminated site and belonged to Acinetobacter, Agrobacterium, Arthrobacter, Comamonas, Rhodococcus, Stenotrophomonas and Pseudomonas. Five arsenite-oxidizing bacteria that belonged to Achromobacter, Agrobacterium and Pseudomonas were identified and displayed a higher average arsenite resistance level than the non-arsenite oxidizers. 5 aoxB genes encoding arsenite oxidase and 51 arsenite transporter genes [18 arsB, 12 ACR3(1) and 21 ACR3(2)] were successfully amplified from these strains using PCR with degenerate primers. The aoxB genes were specific for the arsenite-oxidizing bacteria. Strains containing both an arsenite oxidase gene (aoxB) and an arsenite transporter gene (ACR3 or arsB) displayed a higher average arsenite resistance level than those possessing an arsenite transporter gene only. Horizontal transfer of ACR3(2) and arsB appeared to have occurred in strains that were primarily isolated from the highly arsenic-contaminated soil.ConclusionSoils with long-term arsenic contamination may result in the evolution of highly diverse arsenite-resistant bacteria and such diversity was probably caused in part by horizontal gene transfer events. Bacteria capable of both arsenite oxidation and arsenite efflux mechanisms had an elevated arsenite resistance level.

This study was conducted to evaluated seawater bacteria and their seasonal characteristics in the arsenic contaminated coastal seawater of Yeosu Bay, the Republic of Korea. Arsenite-oxidizing bacteria play an important role in the seawater of the arsenic contaminated bay, with a variety of arsenic resistance system (ars) genotypes being present during summer. Specifically, Bacillus sp. strain SeaH-As22w (FJ607342), isolated from the bay, were found to contain the arsB, arrA and aoxR type operons, which are involved in arsenic resistance. The isolated bacteria showed relatively high tolerance to sodium arsenite (III; ) at concentrations as high as 50 mM. Additionally, batch seawater experiments showed that Bacillus sp. strain SeaH-As22w completely oxidized 1 mM of As (III) to As (V) within 10 days. Ecologically, the arsenic-oxidizing potential plays an important role in arsenic toxicity and mobility in As-contaminated coastal seawater of Yeosu Bay during all seasons because it facilitates the activity of Bacillus sp. groups.

Arsenic (As) mobility in the environment is greatly affected by its oxidation state and the degree to which it is sorbed on metal oxide surfaces. Manganese oxides (Mn oxides) have the ability to decrease overall As mobility both by oxidizing toxic arsenite (As(III)) to less toxic arsenate (As(V)), and by sorbing As. However, the effect of competing ions on the mobility of As sorbed on Mn-oxide surfaces is not well understood. In this study, desorption of As(V) and As(III) from a poorly crystalline phyllomanganate (δ-MnO(2)) by two environmentally significant ions is investigated using a stirred-flow technique and X-ray absorption spectroscopy (XAS). As(III) is not observed in solution after desorption under any conditions used in this study, agreeing with previous studies showing As sorbed on Mn-oxides exists only as As(V). However, some As(V) is desorbed from the δ-MnO(2) surface under all conditions studied, while neither desorptive used in this study completely removes As(V) from the δ-MnO(2) surface.

Chapter 18 - Role of microbial consortia in remediation of soil, water and environmental pollution caused by indiscriminate use of chemicals in agriculture: Opportunities and challenges