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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 Influence of pH on 2,4,6-trinitrotoluene degradation by Yarrowia lipolytica(2010-04) Ziganshin, Ayrat M.; Naumova, R. P.; Pannier, Andy J.; Gerlach, RobinThe microbial reduction of the aromatic ring of 2,4,6-trinitrotoluene (TNT) can lead to its complete destruction. The acid-tolerant yeast Yarrowia lipolytica AN-L15 transformed TNT through hydride ion-mediated reduction of the aromatic ring (as the main pathway), resulting in the accumulation of nitrite and nitrate ions, as well as through nitro group reduction (as minor pathway), resulting in hydroxylamino- and aminoaromatics. TNT transformation depended on the yeasts' ability to acidify the culture medium through the production of organic acids. Aeration and a low medium buffer capacity favored yeast growth and resulted in rapid acidification of the medium, which influenced the rate and extent of TNT transformation. This is the first time that nitrate has been detected as a major product of microbial TNT degradation, and this work demonstrates the importance of pH on TNT biotransformation. The ability of Y. lipolytica AN-L15 to reduce the TNT aromatic ring to form TNT-hydride complexes, followed by their denitration, makes this strain a potential candidate for bioremediation of sites contaminated with explosives. (c) 2010 Elsevier Ltd. All rights reserved.Item Application of molecular techniques to elucidate the influence of cellulosic waste on the bacterial community structure at a simulated low level waste site(2010-03) Field, E. K.; D'Imperio, Seth; Miller, A. R.; VanEngelen, Michael R.; Gerlach, Robin; Lee, Brady D.; Apel, William A.; Peyton, Brent M.Low-level radioactive waste sites, including those at various U.S. Department of Energy (DOE) sites, frequently contain cellulosic waste in the form of paper towels, cardboard boxes, or wood contaminated with heavy metals and radionuclides such as chromium and uranium. To understand how the soil microbial community is influenced by the presence of cellulosic waste products, multiple soil samples were obtained from a non-radioactive model low-level waste test pit at the Idaho National Laboratory. Samples were analyzed using 16S rRNA gene clone libraries and 16S rRNA gene microarray (PhyloChip) analyses. Both methods revealed changes in the bacterial community structure with depth. In all samples, the PhyloChip detected significantly more Operational Taxonomic Units (OTUs), and therefore relative diversity, than the clone libraries. Diversity indices suggest that diversity is lowest in the Fill (F) and Fill Waste (FW) layers and greater in the Wood Waste (WW) and Waste Clay (WC) layers. Principal coordinates analysis and lineage specific analysis determined that Bacteroidetes and Actinobacteria phyla account for most of the significant differences observed between the layers. The decreased diversity in the FW layer and increased members of families containing known cellulose degrading microorganisms suggests the FW layer is an enrichment environment for these organisms. These results suggest that the presence of the cellulosic material significantly influences the bacterial community structure in a stratified soil system.Item Identification and characterization of a novel member of the radical AdoMet enzyme superfamily and implications for the biosynthesis of the Hmd hydrogenase active site cofactor(2009-11) McGlynn, Shawn E.; Boyd, Eric S.; Shepard, Eric M.; Lange, Rachel K.; Gerlach, Robin; Broderick, Joan B.; Peters, John W.The genetic context, phylogeny, and biochemistry of a gene flanking the H2-forming methylene-H4-methanopterin dehydrogenase gene (hmdA), here designated hmdB, indicate that it is a new member of the radical S-adenosylmethionine enzyme superfamily. In contrast to the characteristic CX3CX2C or CX2CX4C motif defining this family, HmdB contains a unique CX5CX2C motif.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 Modeling biofilm growth in the presence of carbon dioxide and water flow in the subsurface(2010-07) Ebigbo, Anozie; Helmig, Rainer; Cunningham, Alfred B.; Class, Holger; Gerlach, RobinThe concentration of greenhouse gases—particularly carbon dioxide (CO2)—in the atmosphere has been on the rise in the past decades. One of the methods which have been proposed to help reduce anthropogenic CO2 emissions is the capture of CO2 from large, stationary point sources and storage in deep geological formations. The caprock is an impermeable geological layer which prevents the leakage of stored CO2, and its integrity is of utmost importance for storage security. Due to the high pressure build-up during injection, the caprock in the vicinity of the well is particularly at risk of fracturing. Biofilms could be used as biobarriers which help prevent the leakage of CO2 through the caprock in injection well vicinity by blocking leakage pathways. The biofilm could also protect well cement from corrosion by CO2-rich brine.The goal of this paper is to develop and test a numerical model which is capable of simulating the development of a biofilm in a CO2 storage reservoir. This involves the description of the growth of the biofilm, flow and transport in the geological formation, and the interaction between the biofilm and the flow processes. Important processes which are accounted for in the model include the effect of biofilm growth on the permeability of the formation, the hazardous effect of supercritical CO2 on suspended and attached bacteria, attachment and detachment of biomass, and two-phase fluid flow processes. The model is tested by comparing simulation results to experimental data.Item Imaging biologically induced mineralization in fully hydrated flow systems(2011) Schultz, Logan N.; Pitts, Betsey; Mitchell, Andrew C.; Cunningham, Alfred B.; Gerlach, RobinA number of proposed technologies involve the controlled implementation of biologically induced carbonate mineral precipitation in the geologic subsurface. Examples include the enhancement of soil stability [1], immobilization of groundwater contaminants such as strontium and uranium [2], and the enhancement of oil recovery and geologic carbon sequestration via controlled permeability reduction [3]. The most significant challenge in these technologies remains to identify and better understand an industrially, environmentally, and economically viable carbonate precipitation route.One of the most promising routes is ureolytic biomineralization, because of the ample availability of urea and the controllable reaction rate. In this process, ureolytic bacteria hydrolyze urea, leading to an increase in pH. In the presence of calcium, this process favors the formation of solid calcium carbonate, as illustrated in the following equations:CO(NH2)2 + H2O → NH2COOH + NH3→ 2 NH3 + CO2 (Urea hydrolysis) (1)2 NH3 + 2 H2O ↔ 2NH4+ + 2OH– (pH increase) (2)CO2 + 2 OH– ↔ CO32– + H2O(Carbonate ion formation) (3)CO32– + Ca2+ ↔ CaCO3 (solid)(Precipitation is favored at high pH) (4)This process relies on molecular-level chemical and biological processes that must be better understood for large-scale implementation.Researchers at the Center for Biofilm Engineering at Montana State University (USA) and Aberystwyth University (UK) have conducted several biomineralization experiments in simulated porous media reactors. Microscopy has proven to be one of the most useful analytical tools in these studies, providing the ability to non-invasively visualize, differentiate, and quantify the various components, including the cells, cell matrix, and mineral precipitates. Because of the possibility of real-time observation and the lack of dehydration artifacts, microscopy has been tremendously useful for elucidating the temporal and spatial relationships of these components.Item Reduction of environmental and energy footprint of microalgal biodiesel production through material and energy integration(2012-03) Chowdhury, R.; Viamajala, Sridhar; Gerlach, RobinThe life cycle impacts were assessed for an integrated microalgal biodiesel production system that facilitates energy- and nutrient- recovery through anaerobic digestion, and utilizes glycerol generated within the facility for additional heterotrophic biodiesel production. Results show that when external fossil energy inputs are lowered through process integration, the energy demand, global warming potential (GWP), and process water demand decrease significantly and become less sensitive to algal lipid content. When substitution allocation is used to assign additional credit for avoidance of fossil energy use (through utilization of recycled nutrients and biogas), GWP and water demand can, in fact, increase with increase in lipid content. Relative to stand-alone algal biofuel facilities, energy demand can be lowered by 3–14 GJ per ton of biodiesel through process integration. GWP of biodiesel from the integrated system can be lowered by up to 71% compared to petroleum fuel. Evaporative water loss was the primary water demand driver.Item Comparison of CO2 and bicarbonate as inorganic carbon sources for triacylglycerol and starch accumulation in Chlamydomonas reinhardtii(2013-01) Gardner, Robert D.; Lohman, Egan J.; Gerlach, Robin; Cooksey, Keith E.; Peyton, Brent M.Microalgae are capable of accumulating high levels of lipids and starch as carbon storage compounds. Investigation into the metabolic activities involved in the synthesis of these compounds has escalated because these compounds can be used as precursors for food and fuel. Here, we detail the results of a comprehensive analysis of Chlamydomonas reinhardtii using high or low inorganic carbon concentrations and speciation between carbon dioxide and bicarbonate, and the effects these have on inducing lipid and starch accumulation during nitrogen depletion. High concentrations of CO2 (5%;v/v) produced the highest amount of biofuel precursors, transesterified to fatty acid methyl esters, but exhibited rapid accumulation and degradation characteristics. Low CO2 (0.04%;v/v) caused carbon limitation and minimized triacylglycerol (TAG) and starch accumulation. High bicarbonate caused a cessation of cell cycling and accumulation of both TAG and starch that was more stable than the other experimental conditions. Starch accumulated prior to TAG and then degraded as maximum TAG was reached. This suggests carbon reallocation from starch-based to TAG-based carbon storage.Item Potential CO2 leakage reduction through biofilm-induced calcium carbonate precipitation(2013-01) Phillips, Adrienne J.; Lauchnor, Ellen G.; Eldring, Joseph; Esposito, R.; Mitchell, Andrew C.; Gerlach, Robin; Cunningham, Alfred B.; Spangler, Lee H.Mitigation strategies for sealing high permeability regions in cap rocks, such as fractures or improperly abandoned wells, are important considerations in the long term security of geologically stored carbon dioxide (CO2). Sealing technologies using low-viscosity fluids are advantageous in this context since they potentially reduce the necessary injection pressures and increase the radius of influence around injection wells. Using aqueous solutions and suspensions that can effectively promote microbially induced mineral precipitation is one such technology. Here we describe a strategy to homogenously distribute biofilm-induced calcium carbonate (CaCO3) precipitates in a 61 cm long sandfilled column and to seal a hydraulically fractured, 74 cm diameter Boyles Sandstone core. Sporosarcina pasteurii biofilms were established and an injection strategy developed to optimize CaCO3 precipitation induced via microbial urea hydrolysis. Over the duration of the experiments, permeability decreased between 2 and 4 orders of magnitude in sand column and fractured core experiments, respectively. Additionally, after fracture sealing, the sandstone core withstood three times higher well bore pressure than during the initial fracturing event, which occurred prior to biofilm-induced CaCO3 mineralization. These studies suggest biofilm-induced CaCO3 precipitation technologies may potentially seal and strengthen fractures to mitigate CO2 leakage potential.