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 A Microfluidic-Based Investigation of Bacterial Attachment in Ureteral Stents(2020-04) De Grazia, Antonio; LuTheryn, Gareth; Meghdadi, Alireza; Mosayyebi, Ali; Espinosa-Ortiz, Erika J.; Gerlach, Robin; Carugo, DarioObstructions of the ureter lumen can originate from intrinsic or extrinsic factors, such as kidney stones, tumours, or strictures. These can affect the physiological flow of urine from the kidneys to the bladder, potentially causing infection, pain, and kidney failure. To overcome these complications, ureteral stents are often deployed clinically in order to temporarily re-establish urinary flow. Despite their clinical benefits, stents are prone to encrustation and biofilm formation that lead to reduced quality of life for patients; however, the mechanisms underlying the formation of crystalline biofilms in stents are not yet fully understood. In this study, we developed microfluidic-based devices replicating the urodynamic field within different configurations of an occluded and stented ureter. We employed computational fluid dynamic simulations to characterise the flow dynamic field within these models and investigated bacterial attachment (Pseudomonas fluorescens) by means of crystal violet staining and fluorescence microscopy. We identified the presence of hydrodynamic cavities in the vicinity of a ureteric occlusion, which were characterised by low levels of wall shear stress (WSS < 40 mPa), and observed that initiation of bacterial attachment occurred in these specific regions of the stented ureter. Notably, the bacterial coverage area was directly proportional to the number of cavities present in the model. Fluorescence microscopy confirmed that the number density of bacteria was greater within cavities (3 bacteria·mm-2) when compared to side-holes of the stent (1 bacterium·mm-2) or its luminal surface (0.12·bacteria mm-2). These findings informed the design of a novel technological solution against bacterial attachment, which reduces the extent of cavity flow and increases wall shear stress over the stent's surface.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 Reducing the risk of well bore leakage using engineered biomineralization barriers(2011-04) Cunningham, Alfred B.; Gerlach, Robin; Spangler, Lee H.; Mitchell, Andrew C.; Parks, Stacy L.; Phillips, Adrienne J.If CO2 is injected in deep geological formations it is important that the receiving formation has sufficient 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. , 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 that discussed by Mitchell et al. .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 Potential role of multiple carbon fixation pathways during lipid accumulation in Phaeodactylum tricornutum(2012-06) Valenzuela, Jacob J.; Mazurie, Aurélien J.; Carlson, Ross P.; Gerlach, Robin; Cooksey, Keith E.; Bothner, Brian; Peyton, Brent M.; Fields, Matthew W.Background Phaeodactylum tricornutum is a unicellular diatom in the class Bacillariophyceae. The full genome has been sequenced (<30 Mb), and approximately 20 to 30% triacylglyceride (TAG) accumulation on a dry cell basis has been reported under different growth conditions. To elucidate P. tricornutum gene expression profiles during nutrient-deprivation and lipid-accumulation, cell cultures were grown with a nitrate to phosphate ratio of 20:1 (N:P) and whole-genome transcripts were monitored over time via RNA-sequence determination. Results The specific Nile Red (NR) fluorescence (NR fluorescence per cell) increased over time; however, the increase in NR fluorescence was initiated before external nitrate was completely exhausted. Exogenous phosphate was depleted before nitrate, and these results indicated that the depletion of exogenous phosphate might be an early trigger for lipid accumulation that is magnified upon nitrate depletion. As expected, many of the genes associated with nitrate and phosphate utilization were up-expressed. The diatom-specific cyclins cyc 7 and cyc 10 were down-expressed during the nutrient-deplete state, and cyclin B1 was up-expressed during lipid-accumulation after growth cessation. While many of the genes associated with the C3 pathway for photosynthetic carbon reduction were not significantly altered, genes involved in a putative C4 pathway for photosynthetic carbon assimilation were up-expressed as the cells depleted nitrate, phosphate, and exogenous dissolved inorganic carbon (DIC) levels. P. tricornutum has multiple, putative carbonic anhydrases, but only two were significantly up-expressed (2-fold and 4-fold) at the last time point when exogenous DIC levels had increased after the cessation of growth. Alternative pathways that could utilize HCO3- were also suggested by the gene expression profiles (e.g., putative propionyl-CoA and methylmalonyl-CoA decarboxylases). Conclusions The results indicate that P. tricornutum continued carbon dioxide reduction when population growth was arrested and different carbon-concentrating mechanisms were used dependent upon exogenous DIC levels. Based upon overall low gene expression levels for fatty acid synthesis, the results also suggest that the build-up of precursors to the acetyl-CoA carboxylases may play a more significant role in TAG synthesis rather than the actual enzyme levels of acetyl-CoA carboxylases per se. The presented insights into the types and timing of cellular responses to inorganic carbon will help maximize photoautotrophic carbon flow to lipid accumulation.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.