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Substrate-induced respiration (SIR) was used to estimate microbial respiration and microbial biomass in soils from Stavešinci natural CO2 spring (mofette) exposed to different geological CO2 concentrations. SIR measurements clearly demonstrated higher microbial respiration and microbial biomass in control sites compared to high soil CO2 sites. Sampling in two different locations and in three different years also confirmed long-term stability of this pattern, which was found for both locations and in different sampling periods.