Biogeochemical processes in a clay formation in situ experiment: Part D – Microbial analyses – Synthesis of results

► Geochemical parameters were measured in Opalinus Clay porewater. ► Unexpected microbial activity occurred in porewater. ► Microorganisms were sustained by organic contamination. ► Microbial activity affected geochemical conditions. ► Fe(III)- reduction, sulphate-reduction and methanogenesis occurred. The purpose of the Porewater Chemistry (PC) experiment at the Mont Terri (MT) Underground Rock Laboratory (URL) was to measure geochemical parameters, such as pH, Eh and pCO 2, in the porewater of the Opalinus Clay formation. Although the PC experiment was designed and implemented carefully from a geochemical perspective, conditions were not sterile and some microbial and nutrient contamination likely occurred. Microbial activity in the added synthetic porewater in the borehole was apparent shortly after initiation of the experiment and affected the geochemical parameters observed in the porewater. This paper summarizes the results from microbial analyses of post-termination PC water and overcore clay samples, conducted to attempt to elucidate the role of microbial activity in the evolution of the geochemical conditions in the PC experiment. Microbial analyses of the PC borehole water, and of clay overcore samples from around the borehole, were carried out at three laboratories and included both molecular biology and culturing methods. Results indicated the presence of heterotrophic aerobic and anaerobic organisms that resulted likely from the initial, non-sterile conditions, sustained by suspected contamination with organic matter (glycerol, acetone). The results also indicated the presence of NO 3-reducers, Fe-reducers, SO 4-reducers and methanogens (i.e., Bacteria as well as Archaea), suggesting a reducing environment with Fe(III)- and SO 4 reduction, and methanogenesis occurring in the PC water and adjacent clay. A black precipitate containing pyrite (identified by XRD and SEM) and a strong H 2S smell in the porewater confirmed the occurrence of SO 4 reduction. Microorganisms identified in the porewater included Pseudomonas stutzeri, Bacillus licheniformis, Desulfosporosinus spp. and Hyphomonas spp. Species identified in enrichment cultures from the overcore samples included Pseudomonas stutzeri, three species of Trichococcus spp., Caldanaerocella colombiensis, Geosporobacter subterrenus and Desulfosporosinus lacus. Overall the results indicated a thriving microbial community in the PC water and adjacent clay in contrast to “undisturbed” Opalinus Clay fo