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Item Subsurface hydrocarbon degradation strategies in low- and high-sulfate coal seam communities identified with activity-based metagenomics(Springer Science and Business Media LLC, 2022-02) Schweitzer, Hannah D.; Smith, Heidi J.; Barnhart, Elliott P.; McKay, Luke J.; Gerlach, Robin; Cunningham, Alfred B.; Malmstrom, Rex R.; Goudeau, Danielle; Fields, Matthew W.Environmentally relevant metagenomes and BONCAT-FACS derived translationally active metagenomes from Powder River Basin coal seams were investigated to elucidate potential genes and functional groups involved in hydrocarbon degradation to methane in coal seams with high- and low-sulfate levels. An advanced subsurface environmental sampler allowed the establishment of coal-associated microbial communities under in situ conditions for metagenomic analyses from environmental and translationally active populations. Metagenomic sequencing demonstrated that biosurfactants, aerobic dioxygenases, and anaerobic phenol degradation pathways were present in active populations across the sampled coal seams. In particular, results suggested the importance of anaerobic degradation pathways under high-sulfate conditions with an emphasis on fumarate addition. Under low-sulfate conditions, a mixture of both aerobic and anaerobic pathways was observed but with a predominance of aerobic dioxygenases. The putative low-molecular-weight biosurfactant, lichysein, appeared to play a more important role compared to rhamnolipids. The methods used in this study—subsurface environmental samplers in combination with metagenomic sequencing of both total and translationally active metagenomes—offer a deeper and environmentally relevant perspective on community genetic potential from coal seams poised at different redox conditions broadening the understanding of degradation strategies for subsurface carbon.Item Addressing wellbore integrity and thief zone permeability using microbially-induced calcium carbonate precipitation (MICP): A field demonstration(Elsevier BV, 2020-02) Kirkland, Catherine M.; Thane, Abby; Hiebert, Randy; Hyatt, Robert; Kirksey, Jim; Cunningham, Alfred B.; Gerlach, Robin; Spangler, Lee; Philips, Adrienne J.Microbially-induced calcium carbonate precipitation (MICP) is an emerging biotechnology for wellbore integrity applications including sealing defects in wellbore cement and modifying the permeability of rock formations. The goal of this field demonstration was to characterize a failed waterflood injection well and provide proof of principle that MICP can reduce permeability in the presence of oil using conventional oilfield fluid delivery methods. We compared well logs performed at the time the well was drilled with ultrasonic logs, sonic cement evaluation, and temperature logs conducted after the well failed. Analysis of these logs suggested that, rather than entering the target waterflood formation, injectate was traveling through defects in the well cement to a higher permeability sandstone layer above the target formation. Sporosarcina pasteurii cultures and urea-calcium media were delivered 2290 ft (698 m) below ground surface using a 3.75 gal (14.2 L) slickline dump bailer to promote mineralization in the undesired flow paths. By Day 6 and after 25 inoculum and 49 calcium media injections, the injectivity [gpm/psi] had decreased by approximately 70%. This demonstration shows that 1) common well logs can be used to identify scenarios where MICP can be employed to reduce system permeability, remediate leakage pathways, and improve waterflood efficiency, and 2) MICP can occur in the presence of hydrocarbons.Item Kinetics of Calcite Precipitation by Ureolytic Bacteria under Aerobic and Anaerobic Conditions(2019-05) Mitchell, Andrew C.; Espinosa-Ortiz, Erika J.; Parks, Stacy L.; Phillips, Adrienne J.; Cunningham, Alfred B.; Gerlach, RobinThe kinetics of urea hydrolysis (ureolysis) and induced calcium carbonate (CaCO3) precipitation for engineering use in the subsurface was investigated under aerobic conditions using Sporosarcina pasteurii (ATCC strain 11859) as well as Bacillus sphaericus strains 21776 and 21787. All bacterial strains showed ureolytic activity inducing CaCO3 precipitation aerobically. Rate constants not normalized to biomass demonstrated slightly higher-rate coefficients for both ureolysis (kurea) and CaCO3 precipitation (kprecip) for B. sphaericus 21776 (kurea=0.10±0.03 h−1, kprecip=0.60±0.34 h−1) compared to S. pasteurii (kurea=0.07±0.02 h−1, kprecip=0.25±0.02 h−1), though these differences were not statistically significantly different. B. sphaericus 21787 showed little ureolytic activity but was still capable of inducing some CaCO3 precipitation. Cell growth appeared to be inhibited during the period of CaCO3 precipitation. Transmission electron microscopy (TEM) images suggest this is due to the encasement of cells and was reflected in lower kurea values observed in the presence of dissolved Ca. However, biomass regrowth could be observed after CaCO3 precipitation ceased, which suggests that ureolysis-induced CaCO3 precipitation is not necessarily lethal for the entire population. The kinetics of ureolysis and CaCO3 precipitation with S. pasteurii was further analyzed under anaerobic conditions. Rate coefficients obtained in anaerobic environments were comparable to those under aerobic conditions; however, no cell growth was observed under anaerobic conditions with NO−3, SO2−4 or Fe3+ as potential terminal electron acceptors. These data suggest that the initial rates of ureolysis and ureolysis-induced CaCO3 precipitation are not significantly affected by the absence of oxygen but that long-term ureolytic activity might require the addition of suitable electron acceptors. Variations in the ureolytic capabilities and associated rates of CaCO3 precipitation between strains must be fully considered in subsurface engineering strategies that utilize microbial amendments.Item Finding a Balance between Accuracy and Effort For Modeling Biomineralization(2016-11) Hommel, Johannes; Ebigbo, Anozie; Gerlach, Robin; Cunningham, Alfred B.; Helmig, Rainer; Class, HolgerMicrobially induced calcite precipitation (MICP) is a technology aiming at the mitigation of potential leakage from underground gas storage sites. A numerical model for MICP was previously developed and validated. The model complexity leads to high computation times, prohibiting at the moment the use of the model for designing field-scale MICP applications. This study investigates savings of the computational time by well-chosen model simplifications. Additionally, this approach is motivated by the high uncertainty of relevant input-parameters. Excessively detailed equations are unnecessary burdens to the MICP model, whose reliability is influenced by the input-parameter uncertainty.Item Investigating the influence of the initial biomass distribution and injection strategies on biofilm-mediated calcite precipitation in porous media(2016-09) Hommel, Johannes; Lauchnor, Ellen G.; Gerlach, Robin; Cunningham, Alfred B.; Ebigbo, Anozie; Helmig, Rainer; Class, HolgerAttachment of bacteria in porous media is a complex mixture of processes resulting in the transfer and immobilization of suspended cells onto a solid surface within the porous medium. Quantifying the rate of attachment is difficult due to the many simultaneous processes possibly involved in attachment, including straining, sorption, and sedimentation, and the difficulties in measuring metabolically active cells attached to porous media. Preliminary experiments confirmed the difficulty associated with measuring active Sporosarcina pasteurii cells attached to porous media. However, attachment is a key process in applications of biofilm-mediated reactions in the subsurface such as microbially induced calcite precipitation. Independent of the exact processes involved, attachment determines both the distribution and the initial amount of attached biomass and as such the initial reaction rate. As direct experimental investigations are difficult, this study is limited to a numerical investigation of the effect of various initial biomass distributions and initial amounts of attached biomass. This is performed for various injection strategies, changing the injection rate as well as alternating between continuous and pulsed injections. The results of this study indicate that, for the selected scenarios, both the initial amount and the distribution of attached biomass have minor influence on the Ca2+2+ precipitation efficiency as well as the distribution of the precipitates compared to the influence of the injection strategy. The influence of the initial biomass distribution on the resulting final distribution of the precipitated calcite is limited, except for the continuous injection at intermediate injection rate. But even for this injection strategy, the Ca2+2+ precipitation efficiency shows no significant dependence on the initial biomass distribution.Item Microbially enhanced carbon capture and storage by mineral-trapping and solubility-trapping(2010-07) Mitchell, Andrew C.; Dideriksen, K.; Spangler, Lee H.; Cunningham, Alfred B.; Gerlach, RobinThe potential of microorganisms for enhancing carbon capture and storage (CCS) via mineral-trapping (where dissolved CO2 is precipitated in carbonate minerals) and solubility trapping (as dissolved carbonate species in solution) was investigated. The bacterial hydrolysis of urea (ureolysis) was investigated in microcosms including synthetic brine (SB) mimicking a prospective deep subsurface CCS site with variable headspace pressures [p(CO2)] of 13C-CO2. Dissolved Ca2+ in the SB was completely precipitated as calcite during microbially induced hydrolysis of 5-20 g L-1 urea. The incorporation of carbonate ions from 13C-CO2 (13C-CO32-) into calcite increased with increasing p(13CO2) and increasing urea concentrations: from 8.3% of total carbon in CaCO3 at 1 g L-1 to 31% at 5 g L-1, and 37% at 20 g L-1. This demonstrated that ureolysis was effective at precipitating initially gaseous [CO2(g)] originating from the headspace over the brine. Modeling the change in brine chemistry and carbonate precipitation after equilibration with the initial p(CO2) demonstrated that no net precipitation of CO2(g) via mineral-trapping occurred, since urea hydrolysis results in the production of dissolved inorganic carbon. However, the pH increase induced by bacterial ureolysis generated a net flux of CO2(g) into the brine. This reduced the headspace concentration of CO2 by up to 32 mM per 100 mM urea hydrolyzed because the capacity of the brine for carbonate ions was increased, thus enhancing the solubility-trapping capacity of the brine. Together with the previously demonstrated permeability reduction of rock cores at high pressure by microbial biofilms and resilience of biofilms to supercritical CO2, this suggests that engineered biomineralizing biofilms may enhance CCS via solubility-trapping, mineral formation, and CO2(g) leakage reduction.Item Reducing the risk of well bore leakage using engineered biomineralization barriers(2011-04) Cunningham, Alfred B.; Gerlach, Robin; Spangler, Lee H.; Mitchell, Andrew C.; Park, Saehan; Phillips, Adrienne J.If CO2 is injected in deep geological formations it is important that the receiving formation hassufficient porosity and permeability for storage and transmission and be overlain by a suitable low-permeability cap rock formation. When the resulting CO2 plume encounters a well bore, leakage may occur through various pathways in the “disturbed zone†surrounding the well casing. Gasda et al.[9], propose a method for determining effective well bore permeability from a field pressure test. If permeability results from such tests prove unacceptably large, strategies for in situ mitigation of potential leakage pathways become important. To be effective, leakage mitigation methods must block leakage pathways on timescales longer than the plume will be mobile, be able to be delivered without causing well screen plugging, and be resistant to supercritical CO2 (ScCO2) challenges. Traditional mitigation uses cement, a viscous fluid that requires a large enough aperture for delivery and that also must bond to the surrounding surfaces in order to be effective. Technologies that can be delivered via low viscosity fluids and that can effectively plug small aperture pathways, or even the porous rock surrounding the well could have significant advantages for some leakage scenarios.We propose a microbially mediated method for plugging preferential leakage pathways and/or porous media, thereby lowering the risk of unwanted upward migration of CO2, similar to thatdiscussed by Mitchell et al.[12].We examine the concept of using engineered microbial biofilms which are capable of precipitating crystalline calcium carbonate using the process of ureolysis. The resulting combination of biofilm plus mineral deposits, if targeted near points of CO2 injection, may result in the long-term sealing of preferential leakage pathways. Successful development of these biologically-based concepts could result in a CO2 leakage mitigation technology which can be applied either before CO2 injection or as a remedial measure. Results from laboratory column studies are presented which illustrate how biomineralization deposits can be developed along packed sand columns at length scales of 2.54 cm and 61 cm. Strategies for controlling mineral deposition of uniform thickness along the axis of flow are also discussed.Item Bulk electric conductivity response to soil and rock CO2 concentration during controlled CO2 release experiments: Observations & analytic modeling(2015-09) Jewell, Scott; Zhou, Xiaobing; Apple, Martha E.; Dobeck, Laura M.; Spangler, Lee H.; Cunningham, Alfred B.To develop monitoring technologies for geologic CO2 storage, controlled CO2 release experiments at the Zero Emissions Research and Technology (ZERT) site in Bozeman, Montana, USA, were carried out in 2009-2011. To understand the impact on the electric properties of soil and sediment rock due to possible CO2 leakage, we have developed an analytical model to explain and predict the electric conductivity (EC) for CO2 impacted soil and sedimentary rock. Results from the model were compared with the measurements at the ZERT site during 2009–2011 and the CO2-Vadose Project site in France in 2011-2012 after model calibration at each site. The model was calibrated using the saturation (n) and cementation (m) exponents contained in Archie's equation, and a chemistry coefficient (pKc) as tuning parameters that minimized the misfit between observed and modeled soil/rock bulk conductivity data. The calibration resulted in n=3.15, m=2.95, and pKc=4.7 for the ZERT site, which was within the range of values in the literature. All the ZERT data sets had rms errors of 0.0115-0.0724. For the CO2-Vadose site, calibration resulted in n=3.6-9.85 and m=2.5-4.2, pKc=4.80-5.65, and the rms error of 0.0002-0.0003; the cementation exponents were consistent with the literature. These results found that the model predicted the bulk EC reasonably well in soil and rock once the unmeasurable model parameters (n, m, and pKc) were calibrated.Item Whole cell kinetics of ureolysis by Sporosarcina pasteurii(2015-06) Lauchnor, Ellen G.; Topp, D. M.; Cunningham, Alfred B.; Gerlach, RobinAims Ureolysis drives microbially induced calcium carbonate precipitation (MICP). MICP models typically employ simplified urea hydrolysis kinetics that do not account for cell density, pH effect or product inhibition. Here, ureolysis rate studies with whole cells of Sporosarcina pasteurii aimed to determine the relationship between ureolysis rate and concentrations of (i) urea, (ii) cells, (iii) and (iv) pH (H+ activity). Methods and Results Batch ureolysis rate experiments were performed with suspended cells of S. pasteurii and one parameter was varied in each set of experiments. A Michaelis–Menten model for urea dependence was fitted to the rate data (R2 = 0·95) using a nonlinear mixed effects statistical model. The resulting half-saturation coefficient, Km, was 305 mmol l−1 and maximum rate constant, Vmax, was 200 mmol l−1 h−1. However, a first-order model with k1 = 0·35 h−1 fit the data better (R2 = 0·99) for urea concentrations up to 330 mmol l−1. Cell concentrations in the range tested (1 × 107–2 × 108 CFU ml−1) were linearly correlated with ureolysis rate (cell dependent = 6·4 × 10−9 mmol CFU−1 h−1). Conclusions Neither pH (6–9) nor ammonium concentrations up to 0·19 mol l−1 had significant effects on the ureolysis rate and are not necessary in kinetic modelling of ureolysis. Thus, we conclude that first-order kinetics with respect to urea and cell concentrations are likely sufficient to describe urea hydrolysis rates at most relevant concentrations. Significance and Impact of the Study These results can be used in simulations of ureolysis driven processes such as microbially induced mineral precipitation and they verify that under the stated conditions, a simplified first-order rate for ureolysis can be employed. The study shows that the kinetic models developed for enzyme kinetics of urease do not apply to whole cells of S. pasteurii.