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Item Improving pH and temperature stability of urease for ureolysis-induced calcium carbonate precipitation(Montana State University - Bozeman, College of Engineering, 2022) Akyel, Arda; Chairperson, Graduate Committee: Robin Gerlach and Adrienne Phillips (co-chair); This is a manuscript style paper that includes co-authored chapters.Ureolysis-induced calcium carbonate (CaCO 3) precipitation (UICP) is a promising technology that takes advantage of urea hydrolysis. During UICP, the enzyme urease hydrolyzes urea, and calcium carbonate can precipitate in the presence of calcium (Ca 2+). This process is also known as biomineralization, and urease is found in several bacterial and plant cells. Urease must be active to enable biomineralization engineering applications such as sealing leakage pathways around wells for CO 2 sequestration. However, biotechnological reactions are limited by physicochemical conditions (temperature, pH, toxic compounds, etc.), and conditions in practice can be suboptimal. Sporosarcina pasteurii and jack bean meal (JBM) ureolytic activities were investigated while simulating potential environmental stresses such as high temperature and pH conditions. Urease was extracted from bacterial cells to evaluate bacterial urease as an alternative to plantbased ureases. Ureolytic activities and thermal inactivation for both bacterial- and plant-based ureases were similar. Urease became thermally inactivated at elevated temperatures (> 50 °C), and urease activity also decreased when pH values moved away from circumneutral pH conditions, i.e., at pH values < 5 and > 9. Urease stability was improved through immobilization for temperatures up to 60 °C and pH values between 3.7 and 4.7. While suspended urease did not demonstrate any residual activity after a one-hour exposure to pH 4.1 at 60 °C, immobilized urease remained active after the exposure. The studies presented here suggest that UICP technology may be used in a broad range of applications, and urease stability can be improved. The use of bacterially derived urease could be cost-competitive. UICP technology not only has the potential to solve various engineering challenges, but it also has the potential to replace traditional cement technologies and contribute to a more sustainable future.Item Temperature‐dependent inactivation and catalysis rates of plant‐based ureases for engineered biomineralization(Wiley, 2020-11) Feder, Marnie J.; Akyel, Arda; Morasko, Vincent J.; Gerlach, Robin; Phillips, Adrienne J.Engineered (bio)mineralization uses the enzyme urease to catalyze the hydrolysis of urea to promote carbonate mineral precipitation. The current study investigates the influence of temperature on ureolysis rate and degree of inactivation of plant-sourced ureases over a range of environmentally relevant temperatures. Batch experiments at 30◦C demonstrated that jack bean meal (JBM) has a 1.7 to 56 times higher activity (844 μmol urea hydrolyzed g−1 JBM min−1) than the other tested plant-sourced ureases (soybean, pigeon pea and cottonseed). Hence, ureolysis and enzyme inactivation rates were evaluated for JBM at temperatures between 20◦C and 80◦C. A combined first-order urea hydrolysis and first-order enzyme inactivation model described the inactivation of urease over the investigated range of temperatures. The temperature-dependent rate coefficients (kurea) increased with temperature and ranged from 0.0018 at 20◦C to 0.0249 L g−1 JBM min−1 at 80◦C; JBM urease became ≥50% inactivated in as little as 5.2 minutes at 80◦C and in as long as 2238 minutes at 50◦C. The combined urea hydrolysis kinetics and enzyme inactivation model provides a mathematical relationship useful for the design of biomineralization technologies and can be incorporated into reactive transport modelsItem Facultative and anaerobic consortia of haloalkaliphilic ureolytic micro-organisms capable of precipitating calcium carbonate(Wiley, 2019-08) Skorupa, Dana J.; Akyel, Arda; Fields, Matthew W.; Gerlach, RobinAims Development of biomineralization technologies has largely focused on microbially induced carbonate precipitation (MICP) via Sporosarcina pasteurii ureolysis; however, as an obligate aerobe, the general utility of this organism is limited. Here, facultative and anaerobic haloalkaliphiles capable of ureolysis were enriched, identified and then compared to S. pasteurii regarding biomineralization activities. Methods and Results Anaerobic and facultative enrichments for haloalkaliphilic and ureolytic micro‐organisms were established from sediment slurries collected at Soap Lake (WA). Optimal pH, temperature and salinity were determined for highly ureolytic enrichments, with dominant populations identified via a combination of high‐throughput SSU rRNA gene sequencing, clone libraries and Sanger sequencing of isolates. The enrichment cultures consisted primarily of Sporosarcina‐ and Clostridium‐like organisms. Ureolysis rates and direct cell counts in the enrichment cultures were comparable to the S. pasteurii (strain ATCC 11859) type strain. Conclusions Ureolysis rates from both facultatively and anaerobically enriched haloalkaliphiles were either not statistically significantly different to, or statistically significantly higher than, the S. pasteurii (strain ATCC 11859) rates. Work here concludes that extreme environments can harbour highly ureolytic active bacteria with potential advantages for large scale applications, such as environments devoid of oxygen. Significance and Impact of the Study The bacterial consortia and isolates obtained add to the possible suite of organisms available for MICP implementation, therefore potentially improving the economics and efficiency of commercial biomineralization.