An in situ culturing device was incubated within a flowing borehole in a mafic sill at 1.474 km depth in Evander Au mine, South Africa. The device was designed to enrich methanogenic, Fe3+-reducing and SO42- -reducing microorganisms using acetate, formate, methanol, Fe3+-citrate and SO42- - enriched agar and sand cartridges. At the end of the 33 day incubation geochemical analyses detected elevated H2, acetate, CH4 and Fe concentrations and depleted SO42- - concentrations. 16S rDNA sequences and PLFA analyses revealed that the microbial community composition of the substrate-bearing cartridges were distinct from that of the original borehole water and the non-substrate-bearing control cartridge. 16S rDNA and dissimilatory sulfite reductase, dsrAB, gene sequences indicated the device successfully targeted SO42- - reducing bacteria (SRB), which were not detected in the original borehole water. 16S rDNA sequences also revealed a shift in the microbial community from one relying on H2 based methanogenesis to one suggestive of H2 based acetogenesis supporting aceticlastic methanogenesis and SO42- - reduction compatible with the subsurface lithoautotrophic hypothesis. © Taylor & Francis Group, LLC.