MICROBIAL ARSENIC CYCLING IN ITALIAN RICE PADDIES: AN ECOLOGICAL PERSPECTIVE

Arsenic (As) contamination of rice is an issue of global concern. Italy, although representing the European leader of rice production, is one of the countries mostly affected by As contamination of rice grain. Rice is mainly cultivated under continuous flooding, with the rapid depletion of oxygen in the soil. At the consequent highly reduced redox potentials, As is released into the porewater by the dissolution of iron-arsenic (Fe-As) minerals, and by the reduction of arsenate [As(V)] to arsenite [As(III)], a soluble compound that is rapidly taken up by the plants. In the presence of sulfide, As(III) co-precipitate with the formation of AsnSn minerals. Microorganisms are known to actively oxidize and reduce As, as well as to convert inorganic to organic As via methylation. Furthermore, microorganisms that use Fe or sulfur for their metabolic activities indirectly influence As biogeochemistry in the environment. In this study, the role of two different practices, suggested to reduce As contamination in rice fields, in shaping rice rhizospheric microbial communities were investigated. Specifically, changes in the water management and use of sulfate (SO42-) as fertilizer were tested. To analyze the influence of the water regime in rice rhizosphere microbiota, a semi-field experiment was set up. Plants were grown in rice field soil from Pavia (containing 18 mg kg-1 of As) in box plots managed with three water regimes: continuous flooding, continuous flooding with 2 weeks of drainage before flowering, and watering after complete soil drying (“aerobic rice”). In rhizosphere soil and in rhizoplane, aioA, arsC, arsM and arrA genes, encoding for different types of As transformation, as well as 16S rRNA genes belonging to dissimilatory Fe-reducing bacteria (DFeRB) and Fe-oxidizing bacteria (FeOB), were amplified and quantified with Real Time quantitative PCR (RT-qPCR). To analyze the whole active bacterial community, RNA was reverse-transcribed and 16S rRNA was amplified and sequenced by 454-pyrosequencing. The presence of DFeRB and FeOB was also highlighted in rhizoplane samples from plants at flowering stage with Fluorescence In Situ Hybridization (FISH). Furthermore, enrichment cultures of FeOB from roots cultivated under continuous flooding and from aerobic rice were set up on Fe(II) gradient tubes and exposed to either As(V) or As(III). Bacterial growth and related Fe(III) oxides were analyzed with Scanning Electron Microscopy (SEM) combined with Energy Dispersive X-ray Spectrometry (EDS), and used for 16S rRNA gene clone library preparation. To test the effect of SO42- amendment on As dissolution into the porewater, a greenhouse experiment was set up with rice plants grown in single pots on rice field soil from Carpiano (MI) (containing 30 mg kg-1 of As). Different pots with and without plants and with and without 0.13 % (w/w) calcium sulfate (CaSO4) amendment were installed. Microbial As genes were quantified with RT-qPCR in i bulk and rhizosphere soil. In a similar experiment performed using rice field soil from Vercelli, the genome belonging to a new putative SO42--reducing species of the Nitrospirae phylum was isolated from a metagenomic library by differential genome binning. The phylogenetic affiliation of this species as well as its metabolic features were characterized by the analysis of specific marker genes and expressed proteins. In continuous flooding, active DFeRB, As(V)-reducing and sulfur-oxidizing bacteria were stimulated, potentially contributing to the release of As into the porewater. The RT-qPCR quantification confirmed that DFeRB belonging to the genus Geobacter significantly increased when rice was cultivated under continuous flooding, in concomitance with a significant increase of As in the porewater over time. This supported the hypothesis that Geobacter, by dissolving Fe(III) minerals, promoted As solubilization. In aerobic rice, genera able to oxidize Fe(II) and/or As(III) were selected. Quantification with RT-PCR confirmed that aioA genes, encoding for As(III)-oxidase, were among the most abundant As genes, increasing when drainage was applied before flowering and in aerobic rice. In Fe(II) gradient tubes, As(V) promoted the enrichment of the nitrate-reducing FeOB genus Azospira from roots developed under continuous flooding, whereas As(III) addition inhibited the growth of FeOB. The SEM-EDS analysis revealed the presence of microorganisms covered by putative Fe encrustation as well as As-Fe oxides crystals. FISH analysis on rice rhizoplane confirmed the presence of FeRB belonging to the family Geobacteraceae and of both microaerophilic and nitrate-reducing FeOB, respectively belonging to the family Gallonellaceae and to the genus Thiobacillus. The addition of SO42- to rice field soil led, on the one hand, to a lower As release into the porewater, on the other hand, to a lower translocation of the metalloid in the plants. The bulk and rhizosphere soil bacterial community was enhanced by the addition of SO42-, but the abundance of genes involved in As transformation did not change significantly. The analysis of a genome retrieved in a metagenomic library prepared on rice bulk and rhizosphere soil from a similar SO42--addition experiment, revealed the presence of a novel species belonging to the Nitrospirae phylum in Vercelli rice field soils. These microorganisms carry the whole genetic background for dissimilatory reduction of SO42- and nitrate. Through the Wood-Ljungdahl pathway, they likely use acetate as electron donor. Amendment of SO42- in the soil promoted the expression of SO42- respiration, whereas in the control treatments genes for nitrate respiration were expressed. These outcomes confirm and elucidate the role of the microbial community living in the rhizosphere of rice plants in decreasing As solubility when changes on the water regime are applied. Future research should be focused the possible role of endophytic bacteria on the decrease of As translocation when is rice plants are fertilized with SO42-.