Rishi Ram Adhikari, Jens Kallmeyer

• The subsurface harbors a large fraction of Earth's living biomass, forming complex microbial ecosystems. Without a profound knowledge of the ongoing biologically mediated processes and their reaction to anthropogenic changes it is difficult to assess the long-term stability and feasibility of any type of geotechnical utilization, as these influence subsurface ecosystems. Despite recent advances in many areas of subsurface microbiology, the direct quantification of turnover processes is still in its infancy, mainly due to the extremely low cell abundances. We provide an overview of the currently available techniques for the quantification of microbial turnover processes and discuss their specific strengths and limitations. Most techniques employed so far have focused on specific processes, e.g. sulfate reduction or methanogenesis. Recent studies show that processes that were previously thought to exclude each other can occur simultaneously, albeit at very low rates. Without the identification of the respective processes it isThe subsurface harbors a large fraction of Earth's living biomass, forming complex microbial ecosystems. Without a profound knowledge of the ongoing biologically mediated processes and their reaction to anthropogenic changes it is difficult to assess the long-term stability and feasibility of any type of geotechnical utilization, as these influence subsurface ecosystems. Despite recent advances in many areas of subsurface microbiology, the direct quantification of turnover processes is still in its infancy, mainly due to the extremely low cell abundances. We provide an overview of the currently available techniques for the quantification of microbial turnover processes and discuss their specific strengths and limitations. Most techniques employed so far have focused on specific processes, e.g. sulfate reduction or methanogenesis. Recent studies show that processes that were previously thought to exclude each other can occur simultaneously, albeit at very low rates. Without the identification of the respective processes it is impossible to quantify total microbial activity. Even in cases where all simultaneously occurring processes can be identified, the typically very low rates prevent quantification. In many cases a simple measure of total microbial activity would be a better and more robust measure than assays for several specific processes. Enzyme or molecular assays provide a more general approach as they target key metabolic compounds. Depending on the compound targeted a broader spectrum of microbial processes can be quantified. The two most promising compounds are ATP and hydrogenase, as both are ubiquitous in microbes. Technical constraints limit the applicability of currently available ATP-assays for subsurface samples. A recently developed hydrogenase radiotracer assay has the potential to become a key tool for the quantification of subsurface microbial activity.…