The role of carbonic anhydrase in rock weathering and pH regulation by the soil bacterium Burkholderia thailandensis E264
Enhanced rock weathering (ERW) is increasingly recognized as a way to sequester atmospheric carbon dioxide (CO2) to slow global warming, but its effectiveness needs to be optimized. Carbonic anhydrase (CA), an enzyme capable of accelerating rock weathering both in vitro and in soil, offers a valuable target due to its ability to convert CO2 into carbonic acid. This conversion promotes rock dissolution, enabling immediate CO2 absorption through cation release and charge-balance mechanisms. Studies have shown that bacteria grown in axenic, rock-amended media increase CA gene expression, but the influence of bacterial CA on rock dissolution rates remains unclear. To investigate this, we used a reverse genetics approach with the phosphate-solubilizing bacterium Burkholderia thailandensis E264. We examined three CA-inactivated mutants alongside the wildtype, growing them in minimal media with basalt rock dust (0-10% w/v) at an initial pH of 6.0. After 7 days, we measured weathering potential through elemental concentrations, pH, and dissolved inorganic carbon. In 1% basalt medium, inactivation of the CA1 gene (BTH_I1052) significantly reduced base cation weathering by 41% compared to the wildtype, whereas inactivation of CA2 (BTH_I0345) and CA3 (BTH_I1199) had no significant effect. In the highly buffered, 10% basalt medium, CA1 had a minor role in weathering, and both CA2 and CA3 had no effect. These findings suggest that CA genes in B. thailandensis operate differently and that CA1s effect is pH-dependent. Surprisingly, CA1 was localized intracellularly, raising questions about how intracellular CAs might influence mineral dissolution, potentially through acidity export or abiontic enzyme activity after cell lysis. ImportanceWhile purified carbonic anhydrase (CA) protein has been shown to increase mineral dissolution rates in mineral-amended media in vitro, it remains unclear if the bacterial CA gene directly drives this process. This study used CA-inactivated mutants of the soil bacterium Burkholderia thailandensis in basalt-amended liquid media and found that only one of the three CA genes influenced mineral dissolution rates. This finding supports prior evidence that bacterial CAs may contribute to mineral dissolution in soils. Importantly, it also showed that not all CA genes in a bacterium may activate under the same conditions, which could impact how soil bacterial CAs are leveraged to enhance weathering. Furthermore, cellular localisation predictions indicated that all three CA genes in B. thailandensis are cytosolic, challenging the common focus on extracellular CAs and suggesting that CA proteins may influence the external environment without needing to be actively exported from the cell.