(743d) Increasing the Temperature and pH Range of Urease to Enable Biomineralization Sealing of Leaky Wells

Urease Induced Calcium Carbonate Precipitation (UICP) has been shown to seal sub-surface fractures around leaky wells (Cunningham et al., 2011; Kirkland et al., 2021, in review; Phillips et al., 2016). UICP could be useful in carbon capture and storage (CCS) to seal leaking wells. CCS sites tend to have a low pH due to the mixing of supercritical CO₂ and water and sometimes elevated reservoir temperatures. These conditions may be inhibitory to the urease enzyme that is involved in the UICP process, which could limit the application of sealing leakage pathways in CCS wells. We investigated enzyme immobilization as a strategy to increase the stability of urease at low pH and increased temperatures.

Jack Bean Meal (JBM) suspensions as the source of urease were prepared from JBM powder in deionized water (DI) and mixed for 16 hours at 22°C. To test the potential increase in enzyme stability, immobilized JBM was prepared by sorption to a porous ceramic proppant. After 24 hours, the liquid was decanted, retaining the ceramic proppant in the flask. Phosphate buffered saline solution (PBS) was added to rinse the ceramic proppant before being decanted again. This rinse procedure was repeated twice to remove residual suspended enzyme.

Four different pH buffers were prepared, three with acetic acid and one with phosphate. After preparation of these buffers, pH of JBM solutions ranged from 3.7 to 7.1. First, 5 mL of each buffer was transferred to triplicate glass vials. Second, 5 mL of 10 g/L suspended JBM solution was added. In the case of immobilized enzyme samples, solid proppant particles were added as the enzyme sources and 5 mL of sterile water were added to keep buffer concentrations identical with suspended enzyme experiments. The suspended or immobilized enzyme was exposed to the pH conditions at 22°C or 60°C. After one hour the pH was adjusted to 7, urea was added, and the glass vials were placed in a water bath at 30°C and agitated at 120 RPM. Samples were taken for urea analysis after 0, 0.5, 1, 3, 8, 24, and 48 hours.

A first order rate model was used to describe the ureolytic activity.

-dA/dt=kA

Using the integrated rate form, a plot of ln(A) versus time was made.

ln(A)=ln(A₀) - kt

Using linear regression, the first order rate constant, k, was found. The first order rate coefficient, k, was taken as a measure for the inactivation that occurred during the 1-hour pH and/or temperature exposure. Residual activities were normalized to the 1-hour exposure to pH 7. Suspended and immobilized JBM were exposed to pH conditions 3.7, 4.1, 4.7, and 7.1 at 22°C and 60°C.

Urease appeared to be protected from inactivation by immobilization (Figure 1). The immobilized JBM exposed to pH values of 3.7, 4.1, 4.7, and 7.1 at 22°C for one hour remained active. This is an improvement in comparison to the suspended JBM exposure, where suspended enzyme became fully inactivated at pH 3.7 (Figure 1 and 2). The immobilized JBM urease showed activity after exposures to 60°C for 1-hour and pH conditions 4.1, 4.7, and 7.1, however higher enzyme inactivation was observed in combination with high temperature. There was no remaining activity observed for the immobilized enzyme exposed to pH condition 3.7 at 60 °C for 1-hour. The immobilization allowed the enzyme to retain activity when exposed to pH 4.1 at 60°C, however less activity than after exposure to higher pH values.

Attached as Abstract.PNG

Figure 1: Urea concentrations over time during incubation at 30°C at pH 7, after 1-hour exposure of suspended and immobilized urease to various pH conditions and temperatures.

Attached as Abstract.PNG

Figure 2: First order reaction rate coefficients determined from urea hydrolysis experiments at 30°C at pH 7, after 1-hour exposure of urease to pH values and temperatures indicated on the x-axis and the legend, respectively.

After a 1-hour exposure of urease to pH 3.7, 4.1, and 4.7 at 22°C the activity of the suspended enzyme decreased by 100% (complete inactivation), 57% and -7% (negative number indicates increase), while the activity of immobilized enzyme decreased by only 13.5%, -8%, and -30%, respectively. When the pH exposures were performed at 60°C, to mimic potential conditions in the deep subsurface, the extent of irreversible enzyme inactivation increased. After a 1-hour exposure of suspended urease to pH 3.7, 4.1, and 4.7 at 60°C, the activity decreased by 100%, 100% and 72%, while the immobilized enzyme activity decreased by 100%, 98%, and 42% respectively.

The pH range, for which the enzyme retained significant activity after exposure, was narrower during 60°C exposure than during 22°C exposure. The combination of heat and low pH completely inactivated the suspended enzyme exposed to pH 3.7 and 4.1 at 60°C and decreased the activity when exposed to pH 4.1 at 22°C. Meanwhile, immobilized urease remained active after exposure to pH values as low as 3.7 at 22°C and as low as 4.1 at 60°C.

Inactivation appeared to be irreversible after the 1-hour exposure to the low pH buffers since the subsequent activity assessments were performed at pH values and temperatures ideal for urease activity (pH 7 and 30°C). The extent of inactivation was increased significantly for the suspended enzyme between pH 3.7-4.7 at room temperature and elevated temperature exposure; however immobilized enzyme was able to remain active even at pH 3.7 during exposure at 22°C.

Immobilization decreased enzyme inactivation due to, both, high temperature and low pH exposure. Immobilized urease retained ureolytic activity even after exposure to pH 3.7 at 22°C. The ability of urease to remain active after exposure to low pH conditions and elevated temperatures indicates its potential usefulness in sealing leakage pathways in geologic CO₂ sequestration scenarios.

Ureolysis can occur under low pH conditions. This study suggests that using enzyme- catalyzed UICP in CCS reservoirs may be possible even if the enzyme is exposed to high temperatures, acidity. This study demonstrates the possibility for ureolysis to occur even after exposure to harsh subsurface conditions. The exposure to pH 3.7 did not fully inactivate the urease at room temperature when immobilized; at 60°C urease was completely inactivated after 1-hour long exposure at pH 3.7 even if immobilized. The combination of temperature and low pH appears to cause faster (and thus more complete) inactivation.

References:

Cunningham, A. B., Gerlach, R., Spangler, L., Mitchell, A. C., Parks, S., & Phillips, A. (2011). Reducing the risk of well bore leakage of CO2 using engineered biomineralization barriers. Energy Procedia, 4, 5178-5185. doi:10.1016/j.egypro.2011.02.495

Kirkland, C., Akyel, A., Hiebert, R., McCloskey, J., Kirksey, J., Cunningham, A. B., Phillips, A. (2021, in review). Ureolysis-induced calcium carbonate precipitation (UICP) in the presence of CO2-affected brine: a field demonstration. International Journal of Greenhouse Gas Control.

Phillips, A. J., Cunningham, A. B., Gerlach, R., Hiebert, R., Hwang, C. C., Lomans, B. P., Spangler, L. (2016). Fracture Sealing with Microbially-Induced Calcium Carbonate Precipitation: A Field Study. Environmental Science & Technology, 50(7), 4111-4117. doi:10.1021/acs.est.5b05559