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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 Ureolysis-induced calcium carbonate precipitation (UICP) in the presence of CO2-affected brine: A field demonstration(Elsevier BV, 2021-07) Kirkland, Catherine M.; Akyel, Arda; Hiebert, Randy; McCloskey, JayBiomineralization is an emerging biotechnology for subsurface engineering applications like remediating leaky wellbores. The process relies on ureolysis to induce precipitation of calcium carbonate in undesired flow paths. In geologic storage of CO2, there is a potential for leakage and low pH conditions, thus, ureolysis-induced calcium carbonate precipitation (UICP) was tested at field scale to seal a channel in the wellbore cement annulus in the presence of CO2-affected brine. Conventional oil field methods were used to deliver UICP-promoting fluids downhole to the treatment zone approximately 1000 feet (305 m) below ground surface (bgs). Over 4 days, 242 L (64 gal) of heat-treated Sporosarcina pasteurii cultures (22 bailers) and 329 L (87 gal) of urea – calcium chloride solution (30 bailers) were injected. The UICP treatment resulted in a 94% reduction of injectivity and ultrasonic well logging showed a noticeable increase in the percentage of solids in the channel outside the casing, including more than 30 m (100 ft) above the injection point. Subsequent well logging 11 months after the field demonstration showed that a significant portion of the new solids remained but the seal was compromised following sustained pumping. The results of this experiment suggest that UICP can be promoted in the presence of CO2-affected brine to seal leakage pathways. Additional research is required to optimize long term seal integrity to ensure storage of CO2 in geologic carbon sequestration scenarios.Item A Numerical Model for Enzymatically Induced Calcium Carbonate Precipitation(MDPI, 2020-06) Hommel, Johannes; Akyel, Arda; Frieling, Zachary; Phillips, Adrienne J.; Gerlach, Robin; Cunningham, Alfred B.; Class, HolgerEnzymatically induced calcium carbonate precipitation (EICP) is an emerging engineered mineralization method similar to others such as microbially induced calcium carbonate precipitation (MICP). EICP is advantageous compared to MICP as the enzyme is still active at conditions where microbes, e.g., Sporosarcina pasteurii, commonly used for MICP, cannot grow. Especially, EICP expands the applicability of ureolysis-induced calcium carbonate mineral precipitation to higher temperatures, enabling its use in leakage mitigation deeper in the subsurface than previously thought to be possible with MICP. A new conceptual and numerical model for EICP is presented. The model was calibrated and validated using quasi-1D column experiments designed to provide the necessary data for model calibration and can now be used to assess the potential of EICP applications for leakage mitigation and other subsurface modifications.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.