Ecosystems biology of the hidden sulfur cycle in rice paddy soil - phylogenetic, functional, and proteogenomic analysis of key players with focus on sulfate reducers

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Rice paddies are man-made wetlands that are essential for staple food production. The prevailing anoxic conditions in these water-logged soils make them one of the most important sources of the greenhouse gas methane. Understanding their biogeochemistry and microbial ecology is therefore of general importance and indispensable to foresee their response to global change. The proposed project targets to elucidate the identity and physiological potential of sulfate reducing microorganisms (SRM) in rice paddy soil. These microorganisms drive a highly active but hidden sulfur cycle that is not apparent from the low standing pools of sulfate and thus has been severely understudied so far. As a consequence little is known about SRM themselves. This is especially true since a large diversity of novel deep-branching dsrAB genes, which serve as functional marker for SRM, can be found in rice paddy soil. Since sulfate reduction effectively competes with the methanogenic degradation of organic matter, SRM have a control function on methane production in rice paddies. To identify active SRM against the background of microorganism involved in parallel biogeochemical pathways, we will apply a multiphasic approach under conditions that stimulate the hidden sulfur cycle in the rhizosphere and bulk soil around rice plants grown in a greenhouse until the early reproductive phase. This will be achieved by gypsum (CaSO4) amendment to rice paddy soil in amounts relevant for rice agriculture and subsequent comparison to unamended controls. SRM active in the rhizosphere will be targeted by stable isotope probing (SIP) using 13C-labeled root exudates that were synthesized by the rice plant itself from provided 13CO2. Parallel high-troughput amplicon sequencing will identify additional active SRM in the bulk soil, where insufficient amounts of 13C-labeled substrates for SIP can be expected, and SRM in the rhizosphere that use no or too little 13C-labeled substrates for anabolism. In both approaches, besides the 16S rRNA gene also the functional marker gene dsrB and/or their transcripts will be targeted to differentiate between SRM and co-targeted sulfur oxidizers. To get initial insights into the genomic content and most actively expressed proteins of SRM and sulfur oxidizers, we will analyze the metagenomes and metaproteomes of all samples. The metaproteome analysis will be conducted in combination with Protein-SIP to extend results obtained by standard SIP. This ecosystems approach will be flanked by targeted cultivation of SRM throughout the project to get stable enrichment or pure cultures of putatively novel SRM represented by deep-branching dsrAB lineages. We hypothesize that both, microorganism harboring novel dsrAB as well as SRM previously identified by molecular methods or cultivation but whose ecological function has yet to be demonstrated, are driving sulfate reduction and thus fuel the hidden sulfur cycle in rice paddy soil.