Towards standardized microbial hydrogen consumption testing in the subsurface: harmonized field sampling and enrichment approaches.

Hydrogen (H₂) will play a crucial role in Europe's green energy transition, necessitating efficient storage solutions such as underground storage in salt caverns or porous media. However, the potential microbial H₂ consumption in these subsurface environments poses risks to storage stability and safety, and its magnitude remains relatively unexplored. Within the HyLife-CETP project, we developed a brine sampling protocol for the field operators and tested a standardized laboratory procedure for estimating microbial hydrogen consumption rates in these original brine samples, combining precise gas, chemical, and genetic analyses. Four labs tested and compared the developed enrichment protocol in a round-robin-like test using artificial brine and the hydrogen-consuming, sulfate-reducer Oleidesulfovibrio alaskensis as a reference strain. This test revealed consistent trends in microbial hydrogen consumption and corresponding pH increase across labs, indicating that the developed protocol effectively captures the overall microbial activity. However, inter-laboratory variability in the reported H₂ consumption quantity, ranging from 19.8 to 61%, highlights how metabolic activity, analytical approaches, and sample handling variations can affect results. Most importantly, abiotic hydrogen loss, particularly leakage from experimental bottles, was identified as a significant factor affecting accurate hydrogen consumption estimation, and we tested different measures to reduce abiotic losses. Our developed protocol effectively detected microbial hydrogen consumption and identified associated metabolic processes, supporting its robustness for studying microbial activity in underground storage systems. In addition, the findings underscore the natural biological variability inherent to growth tests, particularly in hydrogen-utilizing systems.