Assessing the contribution of the rhizosphere microbiome in arsenic biogeochemical cycle in rice fields

1. Introduction In order to develop this PhD project, two main parameters affecting arsenic (As) biogeochemistry in rice fields are analyzed: water management and sulfate (SO42-) amendment. The purposes of the activities are to: A) Investigate the role of water management on the composition of rice rhizospheric microbial populations involved in As cycle B) Identify the microbial populations responsible of the decrease of As bioavailability as a consequence of SO42- amendment in rice cultivated under continuous flooding 2. Arsenic contamination of rice grains Arsenic contamination of rice is an issue of global concern (EFSA 2009). Recent studies revealed that Italy, together with Asian countries like Bangladesh, is one of the countries mostly affected by As contamination of rice grain in Europe (Meharg et al. 2009). Furthermore, from January 2016 specific limits for As concentration in rice have been established (Commission regulation (EU) 2015/1006). Since Italy is the European leader of rice production, it is important to find effective solutions to produce rice with As contents below these limits. Arsenic contamination affects rice more than other crops as a consequence of agronomic practices used in rice cultivation (Spanu et al. 2012). All over the world, rice is mostly cultivated under continuous flooding. In this condition, oxygen is rapidly depleted in the soil, with the reduction of the redox potential in the system. At highly reduced redox potentials, As is rapidly released into the porewater as a consequence of two main processes: the dissolution of FeIII-minerals, where As is firmly bound, and the reduction of arsenate (AsV) to arsenite (AsIII), a highly soluble and toxic oxidation state of As that can be rapidly taken up by the plants and transferred to the grain (Yamaguchi et al. 2014). Changes in the water management of rice paddies have been proposed as possible options to reduce As uptake by rice plants (Spanu et al. 2012). Another important aspect is that, in the presence of sulfide, AsIII co-precipitate with the formation of AsnSn minerals (Fisher et al. 2008). Previous studies indicated that the addition of a sulfur source in soil promotes the decrease of As content in rice grains (Hu et al. 2007), suggesting sulfur amendment as another hypothetical mechanism to reduce As bioavailability in rice paddies. 3. Microbial metabolisms able to influence As biogeochemistry Since the early stages of evolution, microbes have evolved different strategies to survive in the presence of different As forms (Slyemi and Bonnefoy 2012). The highly toxic AsIII can be oxidized to the less toxic AsV by AsIII-oxidizing bacteria (AOB), with AsV-oxidases encoded by the aio operon (Cavalca et al. 2013). In other microrganisms AsV is reduced to AsIII by enzymes encoded by the ars operon. AsIII is then extruded from the cells by specific AsIII-efflux membrane pumps encoded either by the ars or the ACR operons (Andres et al. 2016). Arsenic can also be used by some microorganisms in their energy metabolism. AsV can be used as electron acceptor by anaerobic AsV-respiring bacteria carrying arr genes (dissimilatory AsV-reducing bacteria, DARB). On the other hand AsIII can be used as electron donor by chemolitotrophic aerobic or anaerobic bacteria (Zhu et al. 2014). Arsenic is also influenced by the presence of Fe and sulfur minerals. Therefore, microorganisms that use Fe or sulfur for their metabolic activities can indirectly influence As biogeochemistry in the environment. FeOB and SO42--reducing bacteria (SRB) promote the co-precipitation of As with FeIII and sulfide minerals, whereas FeIII-reducing bacteria (FeRB) and sulfide-oxidizing bacteria (SOB) contribute to the release of As in anoxic environments. Although some work has been performed in order to elucidate the role of microorganisms in As contamination of Asian rice (Somenahally et al 2001, Ma et al 2014), this aspects have still to be clarified for Italian rice paddies. 4. Experimental Procedure The effect of two different parameters on rice rhizosphere microbiome were evaluated: water management and SO42- amendment. To analyze the influence of the water regime in rice rhizosphere microbiome, a semi-field experiment was set up. Plants were grown in box plots managed with three water regimes: continuous flooding for the whole life cycle, continuous flooding with a 2 weeks drainage before flowering (treatment referred to as “mid-raising-drainage”), and watering after complete soil drying (“aerobic rice”). The soil used for the experiment (containing 18 mg kg-1 of As), rhizosphere soil and rhizoplane samples were collected for the analysis of the microbial communities. The chemical analysis of the porewater and of rice grains were performed by Maria Martin (Università degli studi di Torino), Gian Maria Beone (Istituto di Chimica Agraria e Ambientale, Scienze Agrarie, Alimentari e Ambientali, Università Cattolica del Sacro Cuore, Piacenza) and Marco Romani (Rice Research Centre, Ente Nazionale Risi, Castello d’Agogna, Pavia). The microbial communities living in the rhizosphere of the plants were analysez using molecular and microscopy techniques. To test the effect of SO42- amendment on As dissolution into the porewater, a greenhouse experiment is currently set up where rice plants are grown in single pots. Rice field soil containing 30 mg kg-1 of As sampled in Carpiano (MI) has been used. Different pots with and without plants and with and without gypsum (CaSO4) have been installed. Plants germinated on soaked paper were transferred onto the pots and flooded after two weeks of acclimatization. Arsenic in the poreweater is being monitored over time, whereas the microbial communities in the soil used for the experiment and in bulk and rhizosphere soil during late vegetative phase will be characterized. 5. Materials and Methods RNA and DNA were isolated using PowerSoil RNA isolation kit and DNA elution kit (MOBIO). Genes encoding aioA, arsC, arsM and arrA were amplified and quantified with Real Time quantitative Polymerase Chain Reaction (RT-qPCR). With the same method, 16S rRNA genes belonging to FeIII-reducing bacteria belonging to the genera Geobacter and Shewanella and Gallionella-like FeII-oxidizing bacteria were amplified and quantified. To analyze tho whole bacterial community, RNA was reverse-transcribed and the V1-V3 region of 16S rRNA was amplified and sequenced using, amplified and sequenced using the 454-pyrosequencing technique. The sequences were analyzed with the software QIIME (http://qiime.org/). Rhizosphere samples from plants at flowering stage were fixed in paraformaldehyde 3% and processed for DAPI and FISH analysis according to Bertraux et al. 2007. Specific probes have been used to highlight the presence of different population of FeIII-reducing and FeII-oxidizing bacteria. Enrichment cultures of FeOB from roots cultivated under continuous flooding and from aerobic rice were set up on FeII gradient tubes as described by Emerson & Moyer 1997. For each treatment, three lines of enrichment cultures were prepared: one without As amendment, one with 30 mM AsV and one with 3 mM AsIII. After three transfers, the portion of culture characterized by the presence of orange precipitates (FeIII oxides) was sampled from each tubes. One set of samples were analyzed with Scanning Electron Microscopy (SEM) combined with Energy Dispersive X-ray Spectrometry (EDS). Another set of sample was used for DNA isolation, 16S rRNA gene amplification, cloning using the TOPO® TA Cloning® Kit (Invitrogen) and sequencing. In the gypsum experiment, AsV and AsIII dissolved in the porewater are separated using WATERS Sep-Pak Plus Acell Plus QMA cartridge (Waters Corporation), according to Corsini et al. 2010. Total As, AsV and AsIII were then quantified using Inductively Coupled Plasma Mass Spectrometry (ICP-MS). Aerobic and anaerobic heterotrophic colony forming units (CFU) are counted by plating serial dilutions of soil samples on R2A agar plates. Plates for anaerobic growth are incubated in Gas-Pak jars. Three lines of counting plates are set up, one without As addition, one with 20 mM As and one with 3 mM AsIII. 5. Results and Discussion 5.1 Quantification of target genes with qPCR in different water managements and FISH analysis The quantification of bacteria related to Fe cycle, revealed that FeRB belonging to the genus Geobacter significantly increased to the order of 109 copies per g of dwt when rice was cultivated under continuous flooding. With this water regime, an significant increase of As to 190 g L-1 was measured in the porewater over time, supporting the hypothesis that Geobacter, by dissolving FeIII minerals, promoted this release. Among arsenic genes retrieved in rice rhizosphere, aioA genes, encoding for AsIII-oxidase, were the most abundant, reaching the order of 108 genes per g dwt in the rhizoplane of plants cultivated under mid-raising drainage and aerobic rice. This observation, coupled with a lower release of As into the porewater and a lower accumulation of As in rice grains, leads to the hypothesis that the oxidation of AsIII to AsV derived by the expression of aioA genes could have favored the immobilization of AsV in the soil with FeIII minerals, chemically formed in MRD and AR plants by the presence of oxygen. Fluorescence in situ hybridization (FISH) of rice rhizosphere soil samples with specific probes confirmed the presence of FeIII-reducing bacteria belonging to the families Geobacteraceae and Shewanellaceae. Both microaerophilic and nitrate-reducing FeII-oxidizing bacteria were present, respectively belonging to the family Gallonellaceae and to the genus Thiobacillus. 5.2 Microbial ecology in rice rhizosphere in relation to water management The characterization of the microbial communities inhabiting rice rhizosphere by 16S rRNA pyrosequencing revealed that continuous flooding promoted bacterial species potentially involved with the release of As into the porewater. These genera were potentially able to reduce AsV and/or FeII and/or to oxidize sulfur. In aerobic rice, genera potentially able to oxidize FeII and/or AsIII were selected. These outcomes confirm the active role of specific populations in the release of As into rice field porewater when anoxic conditions are established during continuous flooding. 5.3 FeOB enrichment cultures From rice roots of plants cultivated under continuous flooding and in aerobic conditions different species of FeOB where enriched. In gradient tubes containing AsV, from roots developed under continuous flooding, nitrate-reducing iron-oxidizing bacteria belonging to the genus Azospira have been enriched. Different strains of Pseudomonas were enriched from both treatments. When AsIII was added to the enrichment cultures, no genera belonging to known FeOB were retrieved. In all treatments a carry-over of sulfur-oxidizing bacteria was observed. In continuously-flooded roots these organisms were represented by Thiobacillus thioparus, whereas from aerobic rice roots Sulfuricella denitrificans was enriched. Scanning Electron Microscopy performed on culture samples revealed the presence of microorganisms covered by putative Fe encrustation. Moreover, EDS analysis showed the presence of As on Fe oxides crystals. This analysis confirmed the identity of FeOB actively playing an important role in the reduction of As solubilization in aerobic rice rhizosphere, confirming the co-precipitation of As with Fe oxides. 5.4 Greenhouse experiment with gypsum amendment The first outcomes of the analysis of the soil used for the experiment confirmed the presence of microorganisms resistant to the tested concentration of AsV and AsIII. After the first weeks of growth, a decrease in As concentration in the porewater was already detectable in pots with plants where gypsum was amended. 6. Conclusions and Future Perspectives The outcomes of this PhD confirm and elucidate the role of the microbial community living in the rhizosphere of rice plants in the determination of As fate under different environmental conditions. The typically employed continuous flooding of rice fields promote the presence of specific populations of DARB, FeRB and SOB such as Geobacter spp. and Thiobacillus spp., which promote the dissolution of As from Fe plaques. On the other hand, in aerobic rice rhizosphere several genera of AOB and FeOB plays an important role in the maintenance of As stably co-precipitated with Fe minerals. Furthermore, already during the first phase of plant growth, As solubility was decreased by gypsum amendment. In the forthcoming weeks, the microbial populations responsible for this dynamics will be identified. The use of this strategies should be tested on a large scale to evaluate their suitability in terms of costs and production of an acceptable amount of high-quality rice.