Accretionary prisms are thick layers of sedimentary material piled up at convergent plate boundaries. Large amounts of anaerobic groundwater and methane (CH4) are contained in the deep aquifers associated with accretionary prisms. In order to identify microbial activity and CH4 production processes in the deep aquifers associated with the Cretaceous accretionary prism in Okinawa Island, Japan, we performed geochemical and microbiological studies using anaerobic groundwater and natural gas (mainly CH4) samples collected through four deep wells. Chemical and stable hydrogen and oxygen isotope analyses of groundwater samples indicated that the groundwater samples obtained from each site originated from ancient seawater and a mixture of rainwater and seawater, respectively. Additionally, the chemical and stable carbon isotopic signatures of groundwater and natural gas samples suggested that CH4 in the natural gas samples was of a biogenic origin or a mixture of biogenic and thermogenic origins. Microscopic observations and a 16S rRNA gene analysis targeting microbial communities in groundwater samples revealed the predominance of dihydrogen (H2)-producing fermentative bacteria and H2-utilizing methanogenic archaea. Moreover, anaerobic cultures using groundwater samples suggested a high potential for CH4 production by a syntrophic consortium of H2-producing fermentative bacteria and H2-utilizing methanogenic archaea through the biodegradation of organic substrates. Collectively, our geochemical and microbiological data support the conclusion that the ongoing biodegradation of organic matter widely contributes to CH4 production in the deep aquifers associated with the Cretaceous accretionary prism.

Accretionary prisms are thick masses of sedimentary material scraped from the oceanic crust and piled up at convergent plate boundaries found across large regions of the world. Large amounts of anoxic groundwater and natural gas, mainly methane (CH 4), are contained in deep aquifers associated with these accretionary prisms. To identify the subsurface environments and potential for CH 4 production by the microbial communities in deep aquifers, we performed chemical and microbiological assays on groundwater and natural gas derived from deep aquifers associated with an accretionary prism and its overlying sedimentary layers. Physicochemical analyses of groundwater and natural gas suggested wide variations in the features of the six deep aquifers tested. On the other hand, a stable carbon isotope analysis of dissolved inorganic carbon in the groundwater and CH 4 in the natural gas showed that the deep aquifers contained CH 4 of biogenic or mixed biogenic and thermogenic origins. Live/dead staining of microbial cells contained in the groundwater revealed that the cell density of live microbial cells was in the order of 10 4 to 10 6 cells mL-1 , and cell viability ranged between 7.5 and 38.9%. A DNA analysis and anoxic culture of microorganisms in the groundwater suggested a high potential for CH 4 production by a syntrophic consortium of hydrogen (H 2)-producing fermentative bacteria and H 2-utilizing methanogenic archaea. These results suggest that the biodegradation of organic matter in ancient sediments contributes to CH 4 production in the deep aquifers associated with this accretionary prism as well as its overlying sedimentary layers.