| High arsenic (As) groundwater has become one of the most serious environmental problems nowadays, threatening the lives of more than140million people. At present, high As groundwater has been found in more than17Countries including Bangladesh, India, Vietnam, Argentina, Chile, Japan, USA and China. In China, high As groundwater are mainly distributed in Xinjiang, Shanxi, Inner Mongolia, Taiwan and Guizhou, with more than3million people under threat. Long term intake of high As groundwater can cause many chronic diseases and severe cancers such as vascular disease, skin cancer, respiration and heart cancer. Nowadays, many studies have been conducted concerning geogenic As groundwater and focused on hydrogeochemistry, mineralogy and deposit conditions. Previous studies indicated that As release and mobilization in aquifers are integrated results of series of microbially mediated reactions and geochemical processes. Microorganisms as one kind of important geological mediators, their resistance, and oxidizing or reducing capacity can allow them to function and affect mobilization of absorbed As. At present, three mechanisms of As enrichment in groundwater systems have been proposed in microbes can play important roles:oxidation of As-rich sulfide mineral, reductive dissolution of Fe(III) oxyhydroxides, and dissimilatory reduction of As. Therefore, to better understand the microbially mediated mechanism of As mobilization and release, it is worthy of studying the in situ microbial community structures and diversities and the functional microbial populations in high As aquifers.In this study, microbially mediated As release and mobilization in strongly reducing aquifers of Hetao Plain were studied using both geochemical and microbial molecular methods, to analyze the geochemistry of groundwater and aquifer sediments, to investigate microbial community structure and diversity in sediments and groundwater with different geochemical characteristics, and to explore functional microbial communities in strongly reducing As groundwater. The main results are as follows:1. Geochemical characteristics of high As groundwaters and sediments in Hetao Plain.Twenty-one high As groundwater samples and thirty-two sediment samples from three boreholes which included two high As borehole and one control borehole were collected for geochemical analysis including As speciation, Fe, S and TOC. Most groundwater samples had negative Eh values under near neutral or weakly alkaline conditions. Concentrations of total As and As(Ⅲ) varied in the range of65.6-1088.7μg/L and14.5-560.3μg/L, respectively. As(Ⅲ) concentrations in more than half of the samples were higher than As(V) and concentrations of As(Ⅲ) and total As showed positive linear relationship. Total iron concentrations changed between107μg/L and2257μg/L. Methane concentrations increased with total As concentrations, with the highest value of568.20μg/L. Sulfate concentrations were relatively low in all samples while distinctly elevated only when total As concentrations were low. TOC concentrations ranged from1.9mg/L to11.9mg/L. TOC concentrations and Fe(Ⅱ)/Fe(Ⅲ) values showed positive correlation with total As concentrations, while sulfate concentrations presented negative exponential correlation with total As. Most of high As groundwaters were characterized with high concentrations of As(Ⅲ), TOC, Fe(Ⅱ)/Fe(Ⅲ) and low concentrations of sulfate and nitrate.Arsenic contents in high As sediments varied between33.6mg/kg and77.6mg/kg while in low As sediments ranged from1.5mg/kg to5.8mg/kg. MMAV and DMAV were under the detection limits in all sediment samples. In high As boreholes, As(Ⅲ) contents increased with depth, but As(Ⅴ) dominated in all samples. The concentrations of Fe(Ⅱ) and Fe(Ⅲ) in the high As sediments lay in the range0.05-7.01g/kg and0.38-5.29g/kg, the ratios of Fe(Ⅱ) to total Fe increased with depth. Positive correlation was found between total Fe and total As, which was consistent with the results of previous studies. TOC contents in high As boreholes ranged from0.002to1.854wt.%, and positively correlated with total As. Most of the high As sediments were clay or silty clay samples, characterized with high concentrations of total sulfur, iron and high TOC contents.2. Spatial distribution of microbial communities in high As aquifers of Hetao Plain, Inner Mongolia.Based on the geochemistry results, several groundwater and sediment samples were selected for the analysis of structure and diversity of microbial communities using PCR-DGGE,16S rRNA gene clone library and Illumina MiSeq sequencing approaches.DGGE analysis results indicated that community structures in high As sediments were complex and distinctly different from low As sediment samples. In high As sediment samples, DGGE bands related with Pseudomonas and Acinebacter faded out as the total As concentrations decrease and Thiobacillus appeared as concentrations of sulfate increase. The phylogenetic relationship and closest relatives in GenBank divided these bands sequences from high As boreholes into four groups:Alpha-, Beta-, Gamma-proteobacteria and Firmicutes. Some bands were identified as genus Pseudomonas, Brevundimonas, and Sporosarcina which were highly similar with microorganisms capable of As resistance. Some other bands showed high similarity to Pseudomonas and Thiobacillus. As reported by previous studies, these two species were closely related with bacteria with sulfur-oxidizing or denitrification in freshwater and soil system. The dominant populations in the control borehole were mostly similar with bacteria found in cold arctic marine sediment or permafrost wetland. Populations within sediment samples from control borehole presented higher diversity than in high As sediments, which was consistent with the results reported in Bangladesh aquifers. The result of16S rRNA gene clone libraries in high As sediment samples showed that most of bacteria in Hetao Plain were affiliated with the Betaproteobacteria, which was similar with that of previous studies on high As streambed sediments. Among all the bacterial populations, more than70%clones were closed related (100%similarity) with a thiosulfate-oxidizing bacterium Thiobacillus thioparus. Besides, Pseudomonas, Brevundimonas and Hydrogenophaga were also the dominant populations in high As sediments. In our previous study, a large amount of As containing pyrite (FeS2) was found in the sampling site. The release of As, sulfate and Fe might be related to the oxidation of As-containing pyrite by the microbial populations such as Thiobacillus, and this would result in the accumulation of As(Ⅴ) onto surfaces of the oxidized minerals. The absorbed arsenate was reduced into arsenite which was more mobile because of its poor affinity for mineral surfaces. This might be the reason for the dominance of As(Ⅴ) in high sediment samples as well as for that of As(Ⅲ) in high As groundwater.The results of16S rRNA gene clone library of three representative groundwater samples indicated that bacteria in high As groundwater could be grouped into twelve bacterial classes: Alpha-, Beta-Delta-, Epsilon-, and Gammaproteobacteria, Chlorobi, Bacteroidetes, Firmicutes, Chloroflexi, Actinobacteria, and Deinococcus-Thermus. The sample with the highest As(Ⅲ)/total As ratio showed the highest bacterial diversity and was mainly dominated by Alphaproteobacteria and Betaproteobacteria, which was different from other two samples that dominated by Gammaproteobacteria. But at the genus level, all of these three samples were dominated by Acinetobacter and Pseudomonas with different relative abundances. Generally speaking, dominant populations in high As groundwater included Acinetobacter, Pseudomonas, Brevundimonas, Geobacter and Aquabacterium. Similarly, the archaeal16S rRNA gene clone library result showed that archaeal populations in the sample with the lowest As concentration was more diverse than the other two samples, suggesting that As toxicity can decrease archaeal diversity. Archaea populations in high As groundwater fell into Thaumarchaeota, Crenarchaeota and Euryarchaeota. Among them, Thaumarchaeota was a newly established phylum containing all known ammonia-oxidizing archaea. DGGE result showed that the number of Thaumarchaeota-related sequences decreased gradually as nitrate concentrations decreased and As concentrations increased. Dominant archaeal populations in high As groundwater included Methanosaeta (belong to Euryarchaeota), Nitrosophaera (belong to Thaumarchaeota) and Thermoprotei (belong to Crenarchaeota). These results suggested that microbe-mediated ammonia oxidation and sulfur reduction might be involved in arsenic mobilization in groundwater systems.Illumina MiSeq sequencing results revealed that groundwater samples with different geochemical characteristics presented significantly different microbial community structures. Most groundwater samples were dominated by populations including Acinetobacter, Rheinheimera, Pseudomonas, Methylotenera, Aquabacterium, Hydrogenophaga, Crenothrix and LCP-6, which was mostly consistent with the result of16S rDNA clone libraries. However, Rheinheimera, Methylotenera, Crenothrix and LCP-6were not found in the clone library analysis, which might imply the possible bias between these two methods. The top ten OTUs for dissimilarity between high As groundwaters and low As groundwaters included Acinetobacter, Pseudomonas, Psedudomonadaceae, Alishewanella, Psychrobacter which were more abundant in high As groundwater, and Rheinheimera, Aquabacterium, Comamonadaceae and Gallionella which dominated in low As groundwater.Statistical analysis showed that samples with different As concentrations presented distinctly different values of other geochemical parameters. Besides, microbial communities were different in high As groundwater as compared to low As samples, and some other environmental variables had also effects with microbial communities.Evaluating the results of microbial communities in both high As sediment and groundwater in the same location, it can be concluded that the microbial community structure in high As sediments was totally different from that of high As groundwater. The distribution difference of As(III) and As(V) as well as the microbial populations between high As sediments and groundwater implied that the microbially mediated mechanism of As mobilization was potential different in sediments and groundwater.3. Functional microbial communities in strongly reducing As groundwaterAnalysis of the diversity and abundance of functional populations in groundwater were essential for understanding the microbial ecology and biogeochemical processes in aquifer systems. Hydrogeochemical parameters indicated that the high As groundwater in Hetao Plain was under strongly reducing conditions, along with high concentrations of As, TOC and CH4and low concentrations of sulfate. Therefore, community structure and abundance of sulfate-reducing bacteria, methanogenic archaea, As-oxidizing and reducing bacteria in high As groundwater of Hetao Plain were investigated using corresponding functional genes. Analysis of aioA gene clone library indicated that As-oxidizing bacteria in high As groundwater of Hetao Plain were all belonged to Proteobacteria including Betaproteobacteria, Gammaproteobacteria and Alphaproteobacteria. All clones in Betaproteobacteria belonged to Burkholderiales which contained Rhodoferax ferrireducens, Leptothrix sp., and Acidovorax sp.. Previously studies showed that Rhodoferax ferrireducens was one kind of anaerobic bacterium that could grow from dissimilatory Fe(III) reduction, and Leptothnix sp. and Acidovorax sp. were isolated from As-contaminated sediment and As-contaminated mining site. Clones in Gammaproteobacteria showed high similarity with species of Pseudomonas and Acinetobacter, which was consistent with the abundant existence of Pseudomonas and Acinetobacter when investigating the distribution of microbial communities in high As groundwater. Clones affiliated with Alphaproteobacteria were closely related to the order Rhizobiales, such as Xanthobacter autotrophicus, Bradyrhizobium sp., Bosea sp., Aminobacter sp. and Nitrobacter hamburgensis, which were previously found in As-contaminated soil or As-mining sites. As-oxidizing bacteria in high As groundwater in Hetao Plain were dominated by Rhodoferax ferrireducens, Leptothrix sp., Acidovorax sp., Pseudomonas sp. and Acinetobacter sp.. As-reducing populations in high As groundwater mainly occurred in Deltaproteobacteria, Betaproteobacteria, Chrysiogenetes, Firmicutes and an uncultured group, among which the uncultured group accounted for about one-third. Dominant populations included Geobacter sp., Desulfosporosinus sp., Chrysiogenes and Desulfurispirillum. Most of these populations were found capable of dissimilatory arsenate reducing or sulfur oxidizing in previously studies. The uncultured group was highly similar with uncultured dissimilatory arsenate reducing bacteria in high As sediment and groundwater of West Bengal and north Utah. The concurrence of As-oxidizing bacteria and As-reducing bacteria in high As groundwater implied the complexity of microbially mediated As mobilization in high As aquifers.The analysis of methanogenic archaea in18high As groundwaters showed that the abundance of mcrA gene ranged from3.01×103copies/L to3.80×106copies/L and presented higher abundance in samples with high CH4concentrations. The relative abundance of mcrA gene to total archaea was0.0-30.2%, and presented positive correlations with total As and Fe(II). This could imply that methanogens were more abundant in samples with high As concentrations and were related to dissolution of As-containing minerals. Besides, the relative abundance of mcrA gene to total archaea was negative exponentially related to the sulfate concentrations, which showed consistency with previous results that the consumption of sulfate in high As groundwater could accelerate CH4accumulation. Clone library result of mcrA gene indicated methanogens in high As groundwater were mainly composed of Methanomicrobia, Methanobacteria and the uncultured group, among which83%clones belonged to Methanomicrobia including Methanomicrobiales and Methanosarcinales. Previous studies showed that most of these methanogenic archaea were from peat bog, permafrost and metal-contaminated sediments. Besides, some clones in this study showed extremely low similarity with previously identified mcrA gene sequences, implying the possible presence of special methanogenic populations in high As groundwater.The abundance of bacterial16S gene and dsrB gene in high As groundwater were8.74×106-4.12×109copies/L and0-4.9×106copies/L respectively. The highest percentage of dsrB gene copies to bacterial16S rRNA gene copies was2.1%. The abundance of dsrB gene was positive exponentially correlated with As concentrations, which could imply sulfate reduction occurs simultaneously with As and Fe reduction, and might result in increased As release and mobilization when As is not incorporated into iron sulfides. DGGE result showed that sulfate reducing bacteria in high As groundwater belonged to either Firmicutes or Deltaproteobacteria, and were mainly dominated by Desulfotomaculum, Desulfobulbus, Desulfosarcina and Desulfobacca. Desulfotomaculum was previously found in As contaminated environment capable of metabolizing using As as electron acceptor. |
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