MICROBIAL ARSENIC CYCLING IN ITALIAN RICE PADDIES: AN ECOLOGICAL PERSPECTIVE

Abstract

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…