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Subsurface microorganisms may respond to increased CO 2  levels in ways that significantly affect pore fluid chemistry. Changes in CO 2  concentration or speciation may result from the injection of supercritical CO 2  (scCO 2 ) into deep aquifers. Therefore, understanding subsurface microbial responses to scCO 2 , or unnaturally high levels of dissolved CO 2 , will help to evaluate the use of geosequestration to reduce atmospheric CO 2  emissions. This study characterized microbial community changes at the 16S rRNA gene level during a scCO 2  geosequestration experiment in the 1.4 km-deep Paaratte Formation of the Otway Basin, Australia. One hundred and fifty tons of mixed scCO 2  and groundwater was pumped into the sandstone Paaratte aquifer over 4 days. A novel U-tube sampling system was used to obtain groundwater samples under  in situ  pressure conditions for geochemical analyses and DNA extraction. Decreases in pH and temperature of 2.6 log units and 5.8°C, respectively, were observed. Polyethylene glycols (PEGs) were detected in the groundwater prior to scCO 2  injection and were interpreted as residual from drilling fluid used during the emplacement of the CO 2  injection well. Changes in microbial community structure prior to scCO 2  injection revealed a general shift from  Firmicutes  to  Proteobacteria  concurrent with the disappearance of PEGs. However, the scCO 2  injection event, including changes in response to the associated variables (e.g., pH, temperature and salinity), resulted in increases in the relative abundances of  Comamonadaceae  and  Sphingomonadaceae  suggesting the potential for enhanced scCO 2  tolerance of these groups. This study demonstrates a successful new  in situ  sampling approach for detecting microbial community changes associated with an scCO 2  geosequestration event.

Changes in the deep subsurface microbial biosphere resulting from a field-scale CO2 geosequestration experiment