The specific aim of this Large-Scale EMSL proposal is to image the metabolic interactions of deep subsurface anaerobic CH4-cycling microbial consortia: 1) at high pressure and temperature; 2) at high temperature and salinity, and 3) during in situ colonization of solid substrates.
We propose doing this by combining our new fluorescent technique, FISH-TAMB, with NanoSIMS analyses on samples we have recently obtained from deep subseafloor sediments (40 MPa and 70-80 degrees C) and will be obtaining from the continental deep subsurface hypersaline fracture water (250 g/L and 55 degrees C) that contain micro-biofilms where in situ syntrophic microbial cycling of CH4 can be mapped. We will perform 13C-enriched laboratory incubations on subsurface sediments, biofilms collected during borehole incubations, and planktonic communities concentrated from fracture water. We propose using EMSL facilities to analyze these samples to address the following hypotheses:
1. At high temperatures (70-8 degree C) and hypersalinity (240 g/L), CH4-oxidizing microorganisms and consortia that utilize electron acceptors with high free energy yields (e.g. nitrate as opposed to sulfate) will be selected, because of the greater maintenance energy demand rates at these conditions1.
2. Syntrophic anaerobic CH4 and HS- oxidizing consortia form the core of micro-biofilms that are the foundation of deep subsurface multi-trophic ecosystems and exhibit spatial arrangements that maximize interspecies electron flux.
The data will be used by various other scientific communities as follows:
Combined FISH-TAMB and NanoSIMS analyses of deep subsurface syntrophic consortia will provide ecologists with data they can use to model the assembly and evolution of subsurface communities.
Discovering how novel anaerobic CH4-oxidizing microorganisms or consortia utilize new combinations of electron donor and acceptor pathways or direct electron transfer at high temperatures and salinity will expand our understanding of the different environmental conditions under which CH4 cycling occurs, which directly relates to EMSL's and BER's mission of understanding global carbon and biogeochemical cycles. BER-supported research has modeled how subsurface sessile and planktonic microbial communities interact and evolve by neutral drift, dispersal and habitat selection in order to construct simulate biogeochemical processes, but the role of microbial syntrophy in these models remains to be explored. NanoSIMS has provided enormous insight into how microbial aggregates cycle C and N substrates using isotopically labeled substrate uptake. FISH-TAMB can improve the applicability of NanoSIMS by 1) identifying which cells are metabolically the most active even if they represent a tiny minority, 2) identifying which metabolic pathway is active thereby resolving the confounding signals from label transfer through metabolite exchange, and 3) determining when a specific metabolic pathway is active to when sufficient biomass turnover has occurred to be isotopically detectable. For these reasons the proposed research will develop protocols that directly relate to EMSL's mission to lead molecular level discoveries of environmental challenges.