H2 produced from renewable energies will play a central role in both greenhouse gas reduction and decarbonization by 2050. Nonetheless, to improve H2 diffusion and utilization as a fuel, large storage capacity systems are needed. Underground storage of natural gas in depleted reservoirs, aquifers and salt caverns is a well-established technology. However, new challenges arise when it comes to storing hydrogen due to the occurrence and activity of indigenous microbial populations in deep geological formations. In a previous study, four Italian natural gas reservoirs were characterized both from a hydro-chemical and microbiological point of view, and predictive functional analyses were carried out with the perspective of underground hydrogen storage (UHS). In the present work, formation waters from the same reservoirs were used as inoculant during batch cultivation tests to characterize microbial activity and its effects on different gas mixtures. Results evidence a predominant acidogenic/acetogenic activity, whilst methanogenic and sulfate reducing activity were only marginal for all tested inoculants. Furthermore, the microbial activation of tested samples is strongly influenced by nutrient availability. Obtained results were fitted and screened in a computational model which would allow deep insights in the study of microbial activity in the context of UHS.