Environmental Biogeochemistry Of Arsenic In Tengchong Geothermal Area,China

Arsenic (As) is a ubiquitous toxicant and carcinogen in the environment and can be released through various natural processes such as rock weathering, hydrothermal and volcanic emissions, or by anthropogenic activities such as use of arsenical pesticides and herbicides, release of industrial wastes, incineration and combustion of fossil fuels and mining. In geothermal systems, arsenic concentrations typically range from 1 mg/L to 10 mg/L, sometimes up to 50 mg/L. As(III) is predominant in reducing geothermal reservoir, and is rapidly oxidized to As(V) by microbial populations once a rising geothermal fluid is mixed with shallow oxygenated groundwater or exposed to the land surface. As a consequence, groundwater and river systems receiving geothermal fluids contain elevated As concentrations, leading to some potential environmental risks. In the last decade, arsenic migration and transformation from geothermal fluids has drawn great attention and been widely investigated around the world including USA, Canada, Japan, New Zealand, Philippines, Iceland, France, Spain, Russia, Turkey, and some Latin American countries such as Mexico and Chile. However, microbially-mediated As environmental geochemistry in various types of hot springs has yet to be fully understood.In this study, environmental biogeochemistry of As in Tengchong geothermal area of Yunnan, China, was studied with an integrated approaches including hydrogeochemical measurement,16S rRNA gene Illumina MiSeq sequencing, functional gene clone libraries, Q-PCR, enrichment and pure cultures. These results listed below not only improve our understanding of microbially mediated As mobilization in China geothermal areas, but also provide basis for the remediation of As contamination from geothermal fluid sources and exploitation of thermophilic microorganism resources.1. Arsenic geochemical characteristics in Tengchong geothermal areaGeothermal water in Tengchong geothermal area mainly belonged to three water types including SO4 and Na-Cl-HCO3 types in Rehai and Na-HCO3 type in Ruidian. Arsenic concentrations of geothermal water widely ranged from 22.1 μg/L to 1150.3 μg/L. Acid SO4 type geothermal water samples, formed by separating of a vapor phase rich in H2S and subsequent condensing and oxidizing in shallow oxygen-rich groundwater or surface water, was characterized with low As concentrations (22.1-153.2 μg/L, on averaged:77.1 μg/L). Alkaline Na-Cl-HCO3 type geothermal water in Rehai geothermal field and closely neutral Na-HCO3 type geothermal water in Ruidian geothermal field, as the mixing products of geothermal reservoir water and shallow groundwater or surface water, had high As concentrations (156.7-1150.3 μg/L, on averaged:495.1 μg/L) as well as high concentrations of K, Na, F, Cl, Li and B. Rock leaching through water-rock interaction in geothermal reservoir was the main source of As enrichment in geothermal water. The As(Ⅲ)/AsTot widely ranged from 0.01 to 0.97. The oxidation of As(Ⅲ) mainly occurred in geothermal water mixed with shallow groundwater or surface water. Arsenic had a significantly positive correlation with HCO3 concentrations in geothermal water (R=0.81, P=0.01), which implied that shallow groundwater or surface water in Tengchong geothermal area was probably contaminated by As due to the mixing of geothermal water.2. Arsenic biogeochemistry at the outflow of an acid vapor-formed ZhenzhuquanThe Zhenzhuquan pool contained low concentrations of sulfide (0.03-0.05 mg/L) and As (62.26-66.83 μg/L), but had high As(Ⅴ)/AsTot (0.73-0.86). Different from other acid SO4-Cl hot springs where sulfide inhibits microbially-mediated As(Ⅲ) oxidation, the absence of sulfide in the Zhenzhuquan pool allowed microbial As(Ⅲ) oxidation. Along the outflow channel, coupled with iron oxidation (Fe(Ⅲ)/FeTot increased from 0.09 to 0.34) and sulfur oxidation (SO42- concentrations increased from 120.05 mg/L to 158.21 mg/L), arsenic and Fe co-deposited in downstream sediments. There were significantly elevated concentrations of AsTot (10.53-16.44 g/kg) and FeTot (0.05-4.55 g/kg) in the sediments. The extremely high As/Fe mole ratios (2.70-6.72) of the sediments suggested that As was adsorbed by Fe oxides and clay minerals. Temperature, TOC and DO significantly shaped the microbial community structure of upstream and downstream samples. Results of 16S rRNA gene Illumina MiSeq sequencing and aioA gene clone library displayed that above oxidations might be correlated with the appearance of some putative functional microbial populations, such as Aquificae and Pseudomonas (As oxidation), Sulfolobus (S and Fe oxidation), Metallosphaera and Acidicaldus (Fe oxidation). In addition, accumulated As in downstream sediments appeared to significantly constrain their microbial community diversity.3. Arsenic biogeochemistry at the outflow of an alkaline sulfide-rich ZimeiquanThe Zimeiquan pools contained high concentrations of sulfide (5.00-5.87 mg/L), As (408.32-624.10 μg/L), and high As(V)/AsTot (0.73-0.86). As(III) oxidation should have taken place in the pools, which indicated the distinct role of sulfide on As(III) oxidation in acidic and alkaline hot springs. Along the outflow channel, Assum (the combined concentrations of As(III) and As(V)) increased from 5.45 μmol/L to 13.86 μmol/L, and Ssum (the combined concentrations of sulfide and sulfate) increased from 292.02 μmol/L to 364.28 μmol/L. Based on the detection of monothioarsenate (H3AsSO3), dithioarsenate (H3AsS2O2) and tetrathioarsenate (H3AsS4) in the pools and As(III) and As(V) concentrations elevations upstream and downstream respectively, increase in As concentration along the outflow channel were attributed to thioarsenic transformation. Temperature, sulfide, As and DO significantly shaped the microbial communities between not only the pools and downstream samples, but also water and sediment samples. Results of 16S rRNA gene Illumina MiSeq sequencing, aioA gene clone library, enrichments culture experiments showed that the dominant Thermocrinis upstream responded to the transformation of thioarsenic to As(III) and the downstream Thermits contributed to the oxidation of As(III) to As(V).4. Diversity and abundance of the As(III) oxidase gene aioAIn Tengchong geothermal area, aioA abundance ranged from 1.63×101 to 7.08x103 per ng of DNA, with an average of 1.52×10 copies per ng DNA. The aioA-relative abundances in the Ruidian geothermal area were distinctly higher than those of Rehai. aioA abundance positively correlated with As(V)/Asrot and sulfide (R= 0.85, P< 0.05) concentrations. And if the acidic spring samples were removed, the R would rise to 0.99, suggesting that sulfide could enhance the As(III) oxidation in alkaline hot springs. Based on qPCR estimates of 16S rRNA gene abundance, aioA harboring organisms comprised as much as-15%of the total community. Phylogenetically, the major aioA sequences in the acidic hot springs (pH 3.3-4.4) were affiliated with Aquificales and Rhizobiales, while those in neutral or alkaline springs (pH 6.6-9.1) were inferred to be primarily bacteria related to Thermales and Burkholderiales. Interestingly, aioA gene abundance at Zhenzhuquan greatly exceeded bacterial 16S rRNA gene abundance, suggesting the aioA genes were archaeal, even though phylogenetically these aioA sequences were most similar to the Aquificales.5. Isolation, dentification and characterization of As(III) oxidizing bacteriaTwo As(III)-oxidizing bacteria TCZ10 and TCC9-4 were isolated from two hot springs in Tengchong geothermal area of Yunnan, China. The strain TCC9-4 was a facultative chemolithoautotrophic bacterium, and could grow with As(III) as an energy source, HCO3- as a carbon source and oxygen as the electron acceptor in a minimal salts medium. Under chemolithoautotrophic condition,1.33 mM As(III) could be oxidized by the strain in 36 hours. Temperature was an important environmental factor that strongly influenced As(III) oxidation rate and As(III) oxidase (Aio) activity. The highest As(III) oxidation rate (37.11 μM/hour) and Aio activity (0.037 U/mg) were found at the temperature of 40℃. Addition of yeast extract enhanced the growth significantly, but delayed the As(III) oxidation. The strain TCZ10 was a heterotrophic bacterium with optimal temperature 68℃ and pH 8.2. It can completely oxidize 2.5 mM As(III) in 36 h and 5 mM As(Ⅲ) in 72 h at 68℃, with an averaged As(III) oxidation rate of 76.35 μM/hour (5.72 mg/L/hour). On the basis of 16S rRNA gene sequence analysis, strains TCC9-4 and TCZ10 were identified as the genera Anoxybacillus and Geobacillus in the phylum Firmicutes, respectively. We failed to obtain the aioA genes in bacteria TCC9-4 and TCZ10 by PCR amplifying with the present seven referred primers, which implies that these genes might be quite novel relative to currently known gene sequences. To our best knowledge, this was the first report of As(III) oxidation ocurring in the genera Geobacillus and Anoxybacillus.