Scholarly Work - Chemical & Biological Engineering
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Item Mineralogy of microbially induced calcium carbonate precipitates formed using single cell drop-based microfluidics(2020-10) Zambare, Neerja M.; Naser, Nada Y.; Gerlach, Robin; Chang, Connie B.Microbe-mineral interactions are ubiquitous and can facilitate major biogeochemical reactions that drive dynamic Earth processes such as rock formation. One example is microbially induced calcium carbonate precipitation (MICP) in which microbial activity leads to the formation of calcium carbonate precipitates. A majority of MICP studies have been conducted at the mesoscale but fundamental questions persist regarding the mechanisms of cell encapsulation and mineral polymorphism. Here, we are the first to investigate and characterize precipitates on the microscale formed by MICP starting from single ureolytic E. coli MJK2 cells in 25 µm diameter drops. Mineral precipitation was observed over time and cells surrounded by calcium carbonate precipitates were observed under hydrated conditions. Using Raman microspectroscopy, amorphous calcium carbonate (ACC) was observed first in the drops, followed by vaterite formation. ACC and vaterite remained stable for up to 4 days, possibly due to the presence of organics. The vaterite precipitates exhibited a dense interior structure with a grainy exterior when examined using electron microscopy. Autofluorescence of these precipitates was observed possibly indicating the development of a calcite phase. The developed approach provides an avenue for future investigations surrounding fundamental processes such as precipitate nucleation on bacteria, microbe-mineral interactions, and polymorph transitions.Item Importance of specific substrate utilization by microbes in microbially enhanced coal-bed methane production: A modelling study(2020-07) Emmert, Simon; Class, Holger; Davis, Katherine J.; Gerlach, RobinThis study addresses a major gap in the understanding and control of microbially enhanced coal-bed methane (MECBM) production. A mathematical and conceptual model comprises a food-web that includes two types of bacteria and three types of archaea representing substrate-specific members of the community; the microbial community members are potentially interacting by competing for or being inhibited by substrates or products of other microbial community members. The model was calibrated using data sets from two different experimental setups. The calibrated model effectively predicted the methane concentrations within a 7% range of deviation from the experimental results. The results of additional batch experiments using varied conditions are also reproduced in an attempt to validate the model and to test the hypothesis of amendment-induced stimulation of microbial community members capable of converting coal into substrates available to methane producing microbes. This study significantly enhances the understanding of the complex interactions between microbial activity, substrate-specificity and bio-availability of coal for methane production, and provides the basis for including hydraulic flow and transport processes into future mathematical models important for the design and implementation of more sustainable methods of harvesting methane from un-mineable coalbeds.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 Darcy-scale modeling of microbially induced carbonate mineral precipitation in sand columns(2012-07) Ebigbo, Anozie; Phillips, Adrienne J.; Gerlach, Robin; Helmig, Rainer; Cunningham, Alfred B.; Class, Holger; Spangler, Lee H.This investigation focuses on the use of microbially induced calcium carbonate precipitation (MICP) to set up subsurface hydraulic barriers to potentially increase storage security near wellbores of CO2 storage sites. A numerical model is developed, capable of accounting for carbonate precipitation due to ureolytic bacterial activity as well as the flow of two fluid phases in the subsurface. The model is compared to experiments involving saturated flow through sand-packed columns to understand and optimize the processes involved as well as to validate the numerical model. It is then used to predict the effect of dense-phase CO2 and CO2-saturated water on carbonate precipitates in a porous medium.Item Taxis toward hydrogen gas by Methanococcus maripaludis(2013-11) Brileya, Kristen A.; Connolly, James M.; Downey, Carey; Gerlach, Robin; Fields, Matthew W.Knowledge of taxis (directed swimming) in the Archaea is currently expanding through identification of novel receptors, effectors, and proteins involved in signal transduction to the flagellar motor. Although the ability for biological cells to sense and swim toward hydrogen gas has been hypothesized for many years, this capacity has yet to be observed and demonstrated. Here we show that the average swimming velocity increases in the direction of a source of hydrogen gas for the methanogen Methanococcus maripaludis using a capillary assay with anoxic gas-phase control and time-lapse microscopy. The results indicate that a methanogen couples motility to hydrogen concentration sensing, and is the first direct observation of hydrogenotaxis in any domain of life. Hydrogenotaxis represents a strategy that would impart a competitive advantage to motile microorganisms that compete for hydrogen gas and would impact the C, S and N cycles.Item Archaeal and bacterial communities in three alkaline hot springs in Heart Lake Geyser Basin, Yellowstone National Park(2013-11) Bowen De León, Kara; Gerlach, Robin; Peyton, Brent M.; Fields, Matthew W.The Heart Lake Geyser Basin (HLGB) is remotely located at the base of Mount Sheridan in southern Yellowstone National Park (YNP), Wyoming, USA and is situated along Witch Creek and the northwestern shore of Heart Lake. Likely because of its location, little is known about the microbial community structure of springs in the HLGB. Bacterial and archaeal populations were monitored via small subunit (SSU) rRNA gene pyrosequencing over 3 years in 3 alkaline (pH 8.5) hot springs with varying temperatures (44°C, 63°C, 75°C). The bacterial populations were generally stable over time, but varied by temperature. The dominant bacterial community changed from moderately thermophilic and photosynthetic members (Cyanobacteria and Chloroflexi) at 44°C to a mixed photosynthetic and thermophilic community (Deinococcus-Thermus) at 63°C and a non-photosynthetic thermophilic community at 75°C. The archaeal community was more variable across time and was predominantly a methanogenic community in the 44 and 63°C springs and a thermophilic community in the 75°C spring. The 75°C spring demonstrated large shifts in the archaeal populations and was predominantly Candidatus nitrosocaldus, an ammonia-oxidizing crenarchaeote, in the 2007 sample, and almost exclusively Thermofilum or Candidatus caldiarchaeum in the 2009 sample, depending on SSU rRNA gene region examined. The majority of sequences were dissimilar (≥10% different) to any known organisms suggesting that HLGB possesses numerous new phylogenetic groups that warrant cultivation efforts.Item Phevalin (aureusimine B) Production by Staphylococcus aureus Biofilm and Impacts on Human Keratinocyte Gene Expression(2012-07) Secor, Patrick R.; Jennings, Laura K.; James, Garth A.; Kirker, Kelly R.; deLancey Pulcini, Elinor; McInnerney, Kathleen; Gerlach, Robin; Livinghouse, Tom; Hilmer, Jonathan K.; Bothner, Brian; Fleckman, Philip; Olerud, John E.; Stewart, Philip S.Staphylococcus aureus biofilms are associated with chronic skin infections and are orders of magnitude more resistant to antimicrobials and host responses. S. aureus contains conserved nonribosomal peptide synthetases that produce the cyclic dipeptides tyrvalin and phevalin (aureusimine A and B, respectively). The biological function of these compounds has been speculated to be involved in virulence factor gene expression in S. aureus, protease inhibition in eukaryotic cells, and interspecies bacterial communication. However, the exact biological role of these compounds is unknown. Here, we report that S. aureus biofilms produce greater amounts of phevalin than their planktonic counterparts. Phevalin had no obvious impact on the extracellular metabolome of S. aureus as measured by high-performance liquid chromatography-mass spectrometry and nuclear magnetic resonance. When administered to human keratinocytes, phevalin had a modest effect on gene expression. However, conditioned medium from S. aureus spiked with phevalin amplified differences in keratinocyte gene expression compared to conditioned medium alone. Phevalin may be exploited as potential biomarker and/or therapeutic target for chronic, S. aureus biofilm-based infections.Item Estimation of a biofilm-specific reaction rate: kinetics of bacterial urea hydrolysis in a biofilm(2015-09) Connolly, James M.; Jackson, Benjamin; Rothman, Adam P.; Klapper, Isaac; Gerlach, RobinBackground/Objectives: Biofilms and specifically urea-hydrolysing biofilms are of interest to the medical community (for example, urinary tract infections), scientists and engineers (for example, microbially induced carbonate precipitation). To appropriately model these systems, biofilm-specific reaction rates are required. A simple method for determining biofilm-specific reaction rates is described and applied to a urea-hydrolysing biofilm. Methods: Biofilms were grown in small silicon tubes and influent and effluent urea concentrations were determined. Immediately after sampling, the tubes were thin sectioned to estimate the biofilm thickness profile along the length of the tube. Urea concentration and biofilm thickness data were used to construct an inverse model for the estimation of the urea hydrolysis rate. Results/Conclusions: It was found that urea hydrolysis in Escherichia coli MJK2 biofilms is well approximated by first-order kinetics between urea concentrations of 0.003 and 0.221 mol/l (0.186 and 13.3 g/l). The first-order rate coefficient (k1) was estimated to be 23.2±6.2 h−1. It was also determined that advection dominated the experimental system rather than diffusion, and that urea hydrolysis within the biofilms was not limited by diffusive transport. Beyond the specific urea-hydrolysing biofilm discussed in this work, the method has the potential for wide application in cases where biofilm-specific rates must be determined.Item A revised model for microbially induced calcite precipitation: Improvements and new insights based on recent experiments(2015-05) Hommel, Johannes; Lauchnor, Ellen G.; Phillips, Adrienne J.; Gerlach, Robin; Cunningham, Alfred B.; Helmig, Rainer; Ebigbo, Anozie; Class, HolgerThe model for microbially induced calcite precipitation (MICP) published by Ebigbo et al. (2012) has been improved based on new insights obtained from experiments and model calibration. The challenge in constructing a predictive model for permeability reduction in the underground with MICP is the quantification of the complex interaction between flow, transport, biofilm growth, and reaction kinetics. New data from Lauchnor et al. (2015) on whole-cell ureolysis kinetics from batch experiments were incorporated into the model, which has allowed for a more precise quantification of the relevant parameters as well as a simplification of the reaction kinetics in the equations of the model. Further, the model has been calibrated objectively by inverse modeling using quasi-1D column experiments and a radial flow experiment. From the postprocessing of the inverse modeling, a comprehensive sensitivity analysis has been performed with focus on the model input parameters that were fitted in the course of the model calibration. It reveals that calcite precipitation and concentrations of inline image and inline image are particularly sensitive to parameters associated with the ureolysis rate and the attachment behavior of biomass. Based on the determined sensitivities and the ranges of values for the estimated parameters in the inversion, it is possible to identify focal areas where further research can have a high impact toward improving the understanding and engineering of MICP.Item Optimized Inorganic Carbon Regime for Enhanced Growth and Lipid Accumulation in Chlorella Vulgaris(2015-06) Lohman, Egan J.; Gardner, Robert D.; Pedersen, Todd C.; Peyton, Brent M.; Cooksey, Keith E.; Gerlach, RobinBackground Large-scale algal biofuel production has been limited, among other factors, by the availability of inorganic carbon in the culture medium at concentrations higher than achievable with atmospheric CO 2 . Life cycle analyses have concluded that costs associated with supplying CO 2 to algal cultures are significant contributors to the overall energy consumption. Results A two-phase optimal growth and lipid accumulation scenario is presented, which (1) enhances the growth rate and (2) the triacylglyceride (TAG) accumulation rate in the oleaginous Chlorophyte Chlorella vulgaris strain UTEX 395, by growing the organism in the presence of low concentrations of NaHCO 3 (5 mM) and controlling the pH of the system with a periodic gas sparge of 5 % CO 2 (v/v). Once cultures reached the desired cell densities, which can be “fine-tuned” based on initial nutrient concentrations, cultures were switched to a lipid accumulation metabolism through the addition of 50 mM NaHCO 3 . This two-phase approach increased the specific growth rate of C. vulgaris by 69 % compared to cultures sparged continuously with 5 % CO 2 (v/v); further, biomass productivity (g L −1 day −1 ) was increased by 27 %. Total biodiesel potential [assessed as total fatty acid methyl ester (FAME) produced] was increased from 53.3 to 61 % (FAME biomass −1 ) under the optimized conditions; biodiesel productivity (g FAME L −1 day −1 ) was increased by 7.7 %. A bicarbonate salt screen revealed that American Chemical Society (ACS) and industrial grade NaHCO 3 induced the highest TAG accumulation (% w/w), whereas Na 2 CO 3 did not induce significant TAG accumulation. NH 4 HCO 3 had a negative effect on cell health presumably due to ammonia toxicity. The raw, unrefined form of trona, NaHCO 3 ∙Na 2 CO 3 (sodium sesquicarbonate) induced TAG accumulation, albeit to a slightly lower extent than the more refined forms of sodium bicarbonate. Conclusions The strategic addition of sodium bicarbonate was found to enhance growth and lipid accumulation rates in cultures of C. vulgaris, when compared to traditional culturing strategies, which rely on continuously sparging algal cultures with elevated concentrations of CO 2(g) . This work presents a two-phased, improved photoautotrophic growth and lipid accumulation approach, which may result in an overall increase in algal biofuel productivity.