Engineering thermostable Parageobacillus thermoglucosidasius for CO₂ biomineralization

In 2023, global CO₂ emissions reached an estimated 37.4 billion tons, with over 70% attributed to industrial activities such as power generation, combustion, and construction. One promising carbon capture strategy is bio-catalyzed conversion of CO₂ to CaCO₃ through biomineralization, producing a stable and environmentally benign final product. This process involves a three-step chemical reaction: (1) CO₂ + H₂O ⇔ HCO₃^- + H⁺; (2) HCO₃- ⇔ CO₃²- + H⁺; (3) Ca²⁺ + CO₃²-⇒ CaCO₃. The rate-limiting first step, CO₂ hydration, can be catalyzed by carbonic anhydrases (CAs), which significantly enhances the reaction rate. Despite numerous studies on robust, high-performing CAs, their implementation at pilot or industrial scales has faced challenges, including high temperatures of industrial effluent gases and specific CaCO₃ crystallization requirements, such as nucleation sites and alkaline pH.

This Ph.D. thesis develops a novel bio-based technology to curb CO₂ emissions from industrial processes through genetic engineering of the thermophilic bacterium Parageobacillus thermoglucosidasius. This bacterium, which exhibits rapid growth and heat tolerance up to 70°C, was chosen as an ideal host for the secretion and surface display of SazCA, the most efficient CA to date, isolated from Sulfurihydrogenibium azorense. The system was further optimized by inactivating host proteases and implementing surface-displayed calcium-binding proteins to create a calcium-rich environment conducive to nucleation. Additionally, adaptive laboratory evolution under alkaline stress was conducted to enhance the strain's alkaline tolerance. This process identified mutations that could reveal mechanisms of alkaline tolerance, pinpointing potential targets for future reverse genetic engineering experiments.

The engineered strain achieved a CO₂ hydration activity of 170 U/mL/OD₆₀₀, surpassing the current benchmark of 26.6 U/mL/OD600, and retained over 50% activity at 70°C for five days, compared to the previous 12 hours. It also maintained significant activity within the relevant pH range of 8-9.5. Importantly, characterization of the CaCO₃ precipitated by P. thermoglucosidasius revealed a product suitable for commercialization. Our system produced a mixture of calcite and vaterite, suggesting that both construction and biomedical applications markets could be accessible for making carbon capture economically sustainable.