Abstract. Sequestration of CO₂ via biological sinks is a matter of great scientific importance due to their potential to lower atmospheric CO₂ levels. In this study a custom built incubation chamber was used to cultivate a soil microbial community, under ideal conditions, to investigate soil chemoautotrophy. The internal atmospheric CO₂ concentrations were monitored and used to estimate the mass of CO₂ uptake. It was found after careful background corrections that 256.4 μg CO₂ kg⁻¹ dry soil was removed from the chamber atmosphere over 16 h. Comparisons were made to photosynthetic controls (i.e. grass and soil algae) whereupon it was observed that the chemoautotrophic fraction sequestered 2.6 and 5.4 % of that taken up by grass and soil algae respectively. Using isotopically labelled ¹³CO₂ and GCMS-IRMS it was also possible to extract and identify labelled fatty acids after a short incubation time, hence confirming the CO₂ uptake potential of the soil slurry. Provided with favourable conditions, chemoautotrophic soil bacteria have the potential to make a significant impact on inorganic carbon sequestration within the environment. The results of this in vivo study have provided ground work for future studies intending to mimic the in situ environment by providing a reliable method for investigating CO₂ uptake by soil microorganisms.