Scholarly Work - Center for Biofilm Engineering
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Item 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.Item Struvite Stone Formation by Ureolytic Biofilms(2019) Espinosa-Ortiz, Erika J.; Gerlach, RobinThis chapter describes the role ureolytic biofilms (communities of microbes attached to surfaces) play in struvite stone formation in the urinary tract. The formation of struvite stones (MgNH4PO4·6H2O), commonly known as infection stones, is associated with urinary tract infections, particularly, with ureolytic microorganisms. Establishment of ureolytic biofilms in the urinary tract can result in increased microbial resistance to medical treatment and development of the necessary urine conditions to promote struvite (or other mineral) precipitation possibly leading to stone formation. Ureolytic microorganisms produce urease, an enzyme that breaks down urea (CO(NH2)2) generating ammonium (NH4+) and alkalizing urine, which changes urine chemistry to potentially promote struvite and other mineral precipitation. This chapter describes the series of steps involved in biofilm development and struvite precipitation leading to stone formation. Furthermore, this chapter presents an overview of controlled laboratory experiments and computer simulations currently used in different disciplines to study microbe-fluid-mineral interactions. We conclude that an interdisciplinary approach including the disciplines of engineering, mathematics, chemistry, microbiology and medicine will provide a more comprehensive understanding of the process of stone formation in the urinary tract and will ultimately allow for the development of improved management and prevention strategies for infection stones.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 Current insights into the mechanisms and management of infection stones(2018-11) Espinosa-Ortiz, Erika J.; Eisner, Brian H.; Lange, Dirk; Gerlach, RobinInfection stones are complex aggregates of crystals amalgamated in an organic matrix that are strictly associated with urinary tract infections. The management of patients who form infection stones is challenging owing to the complexity of the calculi and high recurrence rates. The formation of infection stones is a multifactorial process that can be driven by urine chemistry, the urine microenvironment, the presence of modulator substances in urine, associations with bacteria, and the development of biofilms. Despite decades of investigation, the mechanisms of infection stone formation are still poorly understood. A mechanistic understanding of the formation and growth of infection stones — including the role of organics in the stone matrix, microorganisms, and biofilms in stone formation and their effect on stone characteristics — and the medical implications of these insights might be crucial for the development of improved treatments. Tools and approaches used in various disciplines (for example, engineering, chemistry, mineralogy, and microbiology) can be applied to further understand the microorganism–mineral interactions that lead to infection stone formation. Thus, the use of integrated multidisciplinary approaches is imperative to improve the diagnosis, prevention, and treatment of infection stones.Item Field-scale modeling of microbially induced calcite precipitation(2018-11) Cunningham, Alfred B.; Class, Holger; Ebigbo, Anozie; Gerlach, Robin; Phillips, Adrienne J.; Hommel, JohannesThe biogeochemical process known as microbially induced calcite precipitation (MICP) is being investigated for engineering and material science applications. To model MICP process behavior in porous media, computational simulators must couple flow, transport, and relevant biogeochemical reactions. Changes in media porosity and permeability due to biomass growth and calcite precipitation, as well as their effects on one another must be considered. A comprehensive Darcy-scale model has been developed by Ebigbo et al. (Water Resour. Res. 48(7), W07519, 2012) and Hommel et al. (Water Resour. Res. 51, 3695–3715, 2015) and validated at different scales of observation using laboratory experimental systems at the Center for Biofilm Engineering (CBE), Montana State University (MSU). This investigation clearly demonstrates that a close synergy between laboratory experimentation at different scales and corresponding simulation model development is necessary to advance MICP application to the field scale. Ultimately, model predictions of MICP sealing of a fractured sandstone formation, located 340.8 m below ground surface, were made and compared with corresponding field observations. Modeling MICP at the field scale poses special challenges, including choosing a reasonable model-domain size, initial and boundary conditions, and determining the initial distribution of porosity and permeability. In the presented study, model predictions of deposited calcite volume agree favorably with corresponding field observations of increased injection pressure during the MICP fracture sealing test in the field. Results indicate that the current status of our MICP model now allows its use for further subsurface engineering applications, including well-bore cement sealing and certain fracture-related applications in unconventional oil and gas production.Item Selenate removal in biofilm systems: effect of nitrate and sulfate on selenium removal efficiency, biofilm structure and microbial community(2018-08) Tan, Lea Chua; Espinosa-Ortiz, Erika J.; Nancharaiah, Yarlagadda V.; van Hullebusch, Eric D.; Gerlach, Robin; Lens, Piet N. L.BACKGROUND Selenium (Se) discharged into natural waterbodies can accumulate over time and have negative impacts on the environment. Se‐laden wastewater streams can be treated using biological processes. However, the presence of other electron acceptors in wastewater, such as nitrate (NO3‐) and sulfate (SO42‐), can influence selenate (SeO42‐) reduction and impact the efficiency of biological treatment systems. RESULTS SeO42‐ removal by biofilms formed from an anaerobic sludge inoculum was investigated in the presence of NO3‐ and SO42‐ using drip flow reactors operated continuously for 10 days at pH 7.0 and 30 °C. The highest total Se (∼60%) and SeO42‐ (∼80%) removal efficiencies were observed when the artificial wastewater contained SO42‐. A maximum amount of 68 μmol Se cm‐2 was recovered from the biofilm matrix in SO42‐ + SeO42‐ exposed biofilms and biofilm mass was 2.7‐fold increased for biofilms grown in the presence of SO42‐. When SeO42‐ was the only electron acceptor, biofilms were thin and compact. In the simultaneous presence of NO3‐ or SO42‐, biofilms were thicker (> 0.6 mm), less compact and exhibited gas pockets. CONCLUSION The presence of SO42‐ had a beneficial effect on biofilm growth and the SeO42‐ removal efficiency, while the presence of NO3‐ did not have a significant effect on SeO42‐ removal by the biofilms.Item Carbon-dependent chromate toxicity mechanism in an environmental Arthrobacter isolate(2018-08) Field, Erin K.; Blaskovich, John P.; Peyton, Brent M.; Gerlach, RobinArthrobacter spp. are widespread in soil systems and well-known for their Cr(VI) reduction capabilities making them attractive candidates for in situ bioremediation efforts. Cellulose drives carbon flow in soil systems; yet, most laboratory studies evaluate Arthrobacter-Cr(VI) interactions solely with nutrient-rich media or glucose. This study aims to determine how various cellulose degradation products and biostimulation substrates influence Cr(VI) toxicity, reduction, and microbial growth of an environmental Arthrobacter sp. isolate. Laboratory culture-based studies suggest there is a carbon-dependent Cr(VI) toxicity mechanism that affects subsequent Cr(VI) reduction by strain LLW01. Strain LLW01 could only grow in the presence of, and reduce, 50 μM Cr(VI) when glucose or lactate were provided. Compared to lactate, Cr(VI) was at least 30-fold and 10-fold more toxic when ethanol or butyrate was the sole carbon source, respectively. The addition of sulfate mitigated toxicity somewhat, but had no effect on the extent of Cr(VI) reduction. Cell viability studies indicated that a small fraction of cells were viable after 8 days suggesting cell growth and subsequent Cr(VI) reduction may resume. These results suggest when designing bioremediation strategies with Arthrobacter spp. such as strain LLW01, carbon sources such as glucose and lactate should be considered over ethanol and butyrate.Item Biological treatment of selenium-laden wastewater containing nitrate and sulfate in an upflow anaerobic sludge bed reactor at pH 5.0(2018-11) Tan, Lea Chua; Nancharaiah, Yarlagadda V.; Lu, Shipeng; van Hullebusch, Eric D.; Gerlach, Robin; Lens, Piet N. L.This study investigated the removal of selenate (SeO42−), sulfate (SO42−) and nitrate (NO3−) at different influent pH values ranging from 7.0 to 5.0 and 20 °C in an upflow anaerobic sludge blanket (UASB) reactor using lactate as an electron donor. At pH 5.0, the UASB reactor showed a 20–30% decrease in reactor performance compared to operation at pH 5.5 to 7.0, reaching removal efficiencies of 79%, 15%, 43% and 61% for NO3−, SO42−, Setotal and Sediss, respectively. However, the reactor stability was an issue upon lowering the pH to 5.0 and further experiments are recommended. The sludge formed during low pH operation had a fluffy, floc-like appearance with filamentous structure, possibly due to the low polysaccharide (PS) to protein (PN) ratio (0.01 PS/PN) in the soluble extracellular polymeric substances (EPS) matrix of the biomass. Scanning electron microscopy with energy dispersive X-ray spectroscopy (SEM-EDX) analysis of the sludge confirmed Se oxyanion reduction and deposition of Se0 particles inside the biomass. Microbial community analysis using Illumina MiSeq sequencing revealed that the families of Campylobacteraceae and Desulfomicrobiaceae were the dominant phylotypes throughout the reactor operation at approximately 23% and 10% relative abundance, respectively. Furthermore, approximately 10% relative abundance of both Geobacteraceae and Spirochaetaceae was observed in the granular sludge during the pH 5.0 operation. Overall, this study demonstrated the feasibility of UASB operation at pH values ranging from 7.0 to 5.0 for removing Se and other oxyanions from wastewaters.Item Enhancing wellbore cement integrity with microbially induced calcite precipitation (MICP): A field scale demonstration(2018-12) Phillips, Adrienne J.; Troyer, E.; Hiebert, R.; Kirkland, Catherine M.; Gerlach, Robin; Cunningham, Alfred B.; Spangler, Lee H.; Kirksey, J.; Rowe, W.; Esposito, R.The presence of delaminations, apertures, fractures, voids and other unrestricted flow channels in the wellbore environment substantially reduces wellbore integrity. Compromised cement may cause a loss of zonal isolation leading to deleterious flow of fluids between zones or to the surface with multiple potential negative impacts including: loss of resource production, reduction of sweep efficiency in EOR operations, and regulatory non-compliance. One potential solution to enhance wellbore integrity is microbially induced calcite precipitation (MICP) to plug preferential flow pathways. MICP is promoted with micrometer-sized organisms and low viscosity (aqueous) solutions thereby facilitating fluid transport into small aperture, potentially tortuous leakage flow paths within the cement column. In this study, MICP treatment of compromised wellbore cement was demonstrated at a depth interval of 310.0–310.57 m (1017–1019 feet) below ground surface (bgs) using conventional oil field subsurface fluid delivery technologies (packer, tubing string, and a slickline deployed bailer). After 25 urea/calcium solution and 10 microbial (Sporosarcina pasteurii) suspension injections, injectivity was reduced from the initial 0.29 cubic meters per hour (m3/h) (1.28 gallons per minute (gpm)) to less than 0.011 m3/h (0.05 gpm). The flow rate was decreased while maintaining surface pumping pressure below a maximum pressure of 81.6 bar (1200 psi) to minimize the potential for fracturing a shale formation dominant in this interval. The pressure decay immediately after each injection ceased decreased after MICP treatment. Comparison of pre- and post-test cement evaluation logs revealed substantial deposition of precipitated solids along the original flow channel. This study suggests MICP is a promising tool for enhancing wellbore cement integrity.Item Biogenic coal-to-methane conversion efficiency decreases after repeated organic amendment stimulation(2018-01) Davis, Katherine J.; Barnhart, Elliott P.; Fields, Matthew W.; Gerlach, RobinAddition of organic amendments to coal-containing systems can increase the rate and extent of biogenic methane production for 60−80 days before production slows or stops. Understanding the effect of repeated amendment additions on the rate and extent of enhanced coal-dependent methane production is important if biological coal-to-methane conversion is to be enhanced on a commercial scale. Microalgal biomass was added at a concentration of 0.1 g/L to microcosms with and without coal on days 0, 76, and 117. Rates of methane production were enhanced after the initial amendment but coal-containing treatments produced successively decreasing amounts of methane with each amendment. During the first amendment period, 113% of carbon added as amendment was recovered as methane, whereas in the second and third amendment periods, 39% and 32% of carbon added as amendment was recovered as methane, respectively. Additionally, algae-amended coal treatments produced ∼38% more methane than unamended coal treatments and ∼180% more methane than amended coal-free treatments after one amendment. However, a second amendment addition resulted in only an ∼25% increase in methane production for coal versus noncoal treatments and a third amendment addition resulted in similar methane production in both coal and noncoal treatments. Successive amendment additions appeared to result in a shift from coal-to-methane conversion to amendment-to-methane conversion. The reported results indicate that a better understanding is needed of the potential impacts and efficiencies of repeated stimulation for enhanced coal-to-methane conversion.