Impact of Arsenite on the Bacterial Community Structure and Diversity in Soil.

Arsenic is a toxic metalloid that ranks 20th in abundance in the earth’s crust and is distributed ubiquitously in various natural systems (24). Arsenic in drinking water poses the greatest threat to human health in many parts of the world, including Bangladesh and West Bengal (27, 28). Arsenic contamination has also been reported in some paddy fields located downstream of mining areas (48). The transfer of arsenic from paddy soil to rice may amplify the risk of arsenic poisoning among the inhabitants of these areas. Since arsenic contamination in ground water and soil is a serious environmental and health hazard, the effects of arsenic have received increased attention (9). The predominant forms of arsenic in soil and water are inorganic arsenite (As[III]) and arsenate (As[V]). The toxicity, mobility, and bioavailability of arsenic depend on its oxidation state (5); therefore, it is important to examine arsenic species under various environmental conditions. Under oxic conditions, As(V) is predominant and strongly binds to various soil minerals including iron, manganese, and aluminum (hydr)oxides. In contrast, under reducing conditions, arsenic mostly occurs as As(III), which is more mobile and toxic than As(V) (3, 42). As(III) specifically adsorbs iron and amorphous aluminum (hydr)oxides (6, 13). Manganese oxide minerals are also important minerals because they readily oxidize As(III) to As(V) abiotically (31). Microbial redox transformations of arsenic, i.e., As(V) reduction and As(III) oxidation, are key reactions in the biogeochemical cycling of arsenic (30, 47). Numerous studies have revealed that microbial activity is indispensable for the reduction of As(V) to As(III) under reducing conditions (29, 45). Microbial As(III) oxidation has also been observed in various environments. Yamamura et al. (46) incubated five soil slurry types with 1,000 μM As(III) and found that As(III) was oxidized to As(V) microbiologically under aerobic conditions. Another study also showed the abiotic oxidation of As(III) by a poorly crystalline manganese oxide mineral occurring in soils (16). We recently reported that microbial As(III) oxidation accounted for more than 30% of total As(III) oxidation in natural paddy soils to which no exogenous arsenic was added (8). These findings suggest that the capacity for As(III) oxidation is widespread in natural soil microbial communities. To date, a large number of As(III)-oxidizing bacteria have been isolated from diverse environments (47). Some of these bacteria are heterotrophic As(III) oxidizers and require organic compounds for growth. In contrast, certain autotrophic bacteria have the ability to derive metabolic energy for growth from As(III) oxidation (36, 39). As(III)-oxidizing bacteria are phylogenetically diverse, but all perform As(III) oxidation by means of the enzyme As(III) oxidase, which belongs to the dimethyl sulfoxide (DMSO) reductase family (10, 17). As(III) oxidase consists of a large subunit, AioA, and a small subunit, AioB. The former contains a molybdenum site and [3Fe-4S] cluster, while the latter contains a Rieske-type [2Fe-2S] site. Genes encoding As(III) oxidase have been characterized in As(III)-oxidizing bacteria from various prokaryotic groups, including Alpha-, Beta-, and Gammaproteobacteria (30, 34). These genes have been used to monitor As(III) oxidizers in natural environments (14, 30, 34). It currently remains unclear whether As(III) has an impact on natural microbial communities, particularly As(III)-oxidizing bacterial communities. Quemeneur et al. (35) determined the diversity and structure of As(III)-oxidizing bacteria in arsenic-polluted waters collected from disused mines in France. Arsenic levels affected the structure of the aioA-carrying bacterial population, which consisted of members of Alpha-, Beta-, and Gammaproteobacteria, and the highest copy number of the aioA gene was found in the most polluted locations. Lami et al. (20) conducted soil column experiments in order to investigate the effects of the addition of As(III) (200 μM) on soil microbial communities. The 16S rRNA gene analysis revealed that diversity was the lowest in As(III)-spiked soil, while the highest diversity was observed in the initial soil. Although the diversity of aioA genes did not differ significantly between spiked and initial soils, the copy number of the aioA gene increased in As(III)-spiked soil. These findings suggest the potential impact of As(III) on the soil bacterial community and As(III)-oxidizing bacterial community. However, it remains unclear how As(III) affects the natural soil bacterial community when the same soil is exposed to different levels of As(III). The aim of the present study was to determine the impact of As(III) on the structure and diversity of the soil bacterial community. Soil slurries were incubated with 50, 500, and 5,000 μM As(III) under oxic conditions, and As(III) oxidizing rates were determined under these conditions. PCR-denaturing gradient gel electrophoresis (DGGE) targeting the 16S rRNA genes was performed to monitor the soil bacterial community. In addition, in order to understand the structure and diversity of the As(III)-oxidizing bacterial community, aioA was amplified using two sets of PCR primers. The possible mechanisms responsible for bacterial community shifts induced by As(III) and the bacterial contribution to As(III) oxidation in soil were also discussed.