Scholarly Work - Center for Biofilm Engineering
Permanent URI for this collectionhttps://scholarworks.montana.edu/handle/1/9335
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Item Activity-based, genome-resolved metagenomics uncovers key populations and pathways involved in subsurface conversions of coal to methane(Springer Science and Business Media LLC, 2021-10) McKay, Luke J.; Smith, Heidi J.; Barnhart, Elliott P.; Schweitzer, Hannah D.; Malmstrom, Rex R.; Goudeau, Danielle; Fields, Matthew W.Microbial metabolisms and interactions that facilitate subsurface conversions of recalcitrant carbon to methane are poorly understood. We deployed an in situ enrichment device in a subsurface coal seam in the Powder River Basin (PRB), USA, and used BONCAT-FACS-Metagenomics to identify translationally active populations involved in methane generation from a variety of coal-derived aromatic hydrocarbons. From the active fraction, high-quality metagenome-assembled genomes (MAGs) were recovered for the acetoclastic methanogen, Methanothrix paradoxum, and a novel member of the Chlorobi with the potential to generate acetate via the Pta-Ack pathway. Members of the Bacteroides and Geobacter also encoded Pta-Ack and together, all four populations had the putative ability to degrade ethylbenzene, phenylphosphate, phenylethanol, toluene, xylene, and phenol. Metabolic reconstructions, gene analyses, and environmental parameters also indicated that redox fluctuations likely promote facultative energy metabolisms in the coal seam. The active "Chlorobi PRB" MAG encoded enzymes for fermentation, nitrate reduction, and multiple oxygenases with varying binding affinities for oxygen. "M. paradoxum PRB" encoded an extradiol dioxygenase for aerobic phenylacetate degradation, which was also present in previously published Methanothrix genomes. These observations outline underlying processes for bio-methane from subbituminous coal by translationally active populations and demonstrate activity-based metagenomics as a powerful strategy in next generation physiology to understand ecologically relevant microbial populations.Item Evaluation of the Antimicrobial Efficacy of N-Acetyl-l-Cysteine, Rhamnolipids, and Usnic Acid—Novel Approaches to Fight Food-Borne Pathogens(MDPI, 2021) Chlumsky, Ondrej; Smith, Heidi J.; Parker, Albert E.; Brileya, Kristen; Wilking, James N.; Purkrtova, Sabina; Michova, Hana; Ulbrich, Pavel; Viktorova, Jitka; Demnerova, KaterinaIn the food industry, the increasing antimicrobial resistance of food-borne pathogens to conventional sanitizers poses the risk of food contamination and a decrease in product quality and safety. Therefore, we explored alternative antimicrobials N-Acetyl-L-cysteine (NAC), rhamnolipids (RLs), and usnic acid (UA) as a novel approach to prevent biofilm formation and reduce existing biofilms formed by important food-borne pathogens (three strains of Salmonella enterica and two strains of Escherichia coli, Listeria monocytogenes, Staphylococcus aureus). Their effectiveness was evaluated by determining minimum inhibitory concentrations needed for inhibition of bacterial growth, biofilm formation, metabolic activity, and biofilm reduction. Transmission electron microscopy and confocal scanning laser microscopy followed by image analysis were used to visualize and quantify the impact of tested substances on both planktonic and biofilm-associated cells. The in vitro cytotoxicity of the substances was determined as a half-maximal inhibitory concentration in five different cell lines. The results indicate relatively low cytotoxic effects of NAC in comparison to RLs and UA. In addition, NAC inhibited bacterial growth for all strains, while RLs showed overall lower inhibition and UA inhibited only the growth of Gram-positive bacteria. Even though tested substances did not remove the biofilms, NAC represents a promising tool in biofilm prevention.Item Sulfide product inhibition of desulfovibrio desulfuricans in batch and continuous cultures(1995-02) Okabe, Satoshi; Nielsen, P. H.; Jones, Warren L.; Characklis, William G.Sulfide product inhibition kinetics for growth and activity of Desulfovibrio desulfuricans was investigated in batch and continuous cultures at pH = 7.0. A non-competitive inhibition model adequately described sulfide product inhibition kinetics. Inhibition coefficient (Ki) for maximum specific growth rate (μinhmax) was 251 mg l−1 S in a batch experiment. Cell yield determined in a chemostat was reduced in half by a sulfide concentration of about 250 mg l−1 S, which was very close to the Ki value for the batch growth. Maximum specific growth rate (μinhmax) and cell yield (YcLac) were strongly inhibited by high levels of sulfide concentrations, whereas specific lactate utilization rate increased with increasing sulfide concentrations. The results indicated an increase in the relative energy needed for maintenance to overcome sulfide inhibition and uncoupling growth from energy production. However, D. desulfuricans to some extent could recover from the shock of high sulfide concentrations. Stoichiometry for catabolic reactions (energy producing) did not change at high sulfide concentrations, while anabolic reactions (cellular synthesis) were strongly inhibited by high sulfide concentrations. These results suggested that separation of sulfide product inhibition into growth (cell yield) and activity (substrate utilization rate) was important to incorporate the sulfide product inhibition kinetics in a variety of applications.Item Kinetic analysis of microbial sulfate reduction by desulfovibrio desulfuricans in an anaerobic upflow porous media biofilm reactor(1994-02) Chen, Ching-I; Mueller, Robert Franz; Griebe, ThomasAn anaerobic upflow porous media biofilm reactor was designed to study the kinetics and stoichiometry of hydrogen sulfide production by the sulfate-reducing bacterium (SRB) Desulfovibrio desulfuricans (ATCC 5575) as the first step for the modeling and control of formation souring (H2S) in oil field porous media. The reactor was a packed bed (50 × 5.5 cm) tubular reactor. Sea sand (140 to 375 μm) was used as the porous media. The initial indication of souring was the appearance of well-separated black spots (precipitates of iron sulfide) in the sand bed. The blackened zones expanded radially and upward through the column. New spots also appeared and expanded into the cone shapes. Lactate (substrate) was depleted and hydrogen sulfide appeared in the effluent. Analysis of the pseudo–steady state column shows that there were concentration gradients for lactate and hydrogen sulfide along the column. The results indicate that most of the lactate was consumed at the front part of the column. Measurements of SRB biomass on the solid phase (sand) and in the liquid phase indicate that the maximum concentration of SRB biomass resided at the front part of the column while the maximum in the liquid phase occurred further downstream. The stoichiometry regarding lactate consumption and hydrogen sulfide production observed in the porous media reactor was different from that in a chemostat. After analyzing the radial dispersion coefficient for the SRB in porous media and kinetics of microbial growth, it was deduced that transport phenomena dominate the souring process in our porous media reactor system.Item Modeling urban runoff from a planned community(1976-04) Diniz, E. V.; Holloway, David; Characklis, William G.Item Biofilms, biomaterials and device-related infections(2004) Costerton, J. William; Stoodley, Paul; Shirtliff, Mark E.; Pasmore, M.; Cook, Guy S.Item Biocorrosion(2000) Geesey, Gill G.; Beech, Iwona; Bremer, Philip J.; Webster, Barbara J.; Wells, D. BretItem Uranium immobilization by sulfate-reducing biofilms(2004-04) Beyenal, Haluk; Sani, Rajesh K.; Peyton, Brent M.; Dohnalkova, Alice; Amonette, James E.; Lewandowski, ZbigniewHexavalent uranium [U(VI)] was immobilized using biofilms of the sulfate-reducing bacterium (SRB) Desulfovibrio desulfuricans G20. The biofilms were grown in flat-plate continuous-flow reactors using lactate as the electron donor and sulfate as the electron acceptor. U(VI)was continuously fed into the reactor for 32 weeks at a concentration of 126 microM. During this time, the soluble U(VI) was removed (between 88 and 96% of feed) from solution and immobilized in the biofilms. The dynamics of U immobilization in the sulfate-reducing biofilms were quantified by estimating: (1) microbial activity in the SRB biofilm, defined as the hydrogen sulfide (H2S) production rate and estimated from the H2S concentration profiles measured using microelectrodes across the biofilms; (2) concentration of dissolved U in the solution; and (3) the mass of U precipitated in the biofilm. Results suggest that U was immobilized in the biofilms as a result of two processes: (1) enzymatically and (2) chemically, by reacting with microbially generated H2S. Visual inspection showed that the dissolved sulfide species reacted with U(VI) to produce a black precipitate. Synchrotron-based U L3-edge X-ray absorption near edge structure (XANES) spectroscopy analysis of U precipitated abiotically by sodium sulfide indicated that U(VI) had been reduced to U(IV). Selected-area electron diffraction pattern and crystallographic analysis of transmission electron microscope lattice-fringe images confirmed the structure of precipitated U as being that of uraninite.Item Resolving biogeochemical phenomena at high spatial resolution through electron microscopy(2008-06) Geesey, Gill G.; Borch, Thomas; Reardon, Catherine L.Our understanding of microbe-metal interactions has advanced dramatically since the mid-1970s when little was known about the reactivity of bacterial cell wall components toward metal ions in the extracellular milieu. Although certain metals such as and Pb+ were known to react with components of bacterial cell walls and used to visualize their structure by electron microscopy (Garland et al., 1975), little physicochemical data were available on the specificity and sites of interactions (Humphrey & Vincent, 1966; Heptinstall et al., 1970; Irvin et al., 1975; Lambert et al., 1975; Raymond & MacLeod, 1975). Furthermore, there were no model systems to explorethe mechanisms of these interactions. This began to change when Beveridge and Murray used isolated cell walls of Bacillus subtilis to quantify metal ion binding to wall components. Beveridge demonstrated that cell walls concentrated cations such as Mg++, Na+, K+, Cu++ and Fe+++, but not Ba++, Li+ or Al+++ (Beveridge & Murray, 1976). Since these initial studies, Beveridge and his students and collaborators have contributed greatly to our understanding of the complex interactions between microbial cell surface polymers and metals in the environment. As fellow scientists working in this research area, we have developed a deep admiration of Beveridge’s scientific insight, technical skills and collegial demeanor. Not surprisingly, Beveridge’s research has had a significant impact on our research, as well as on the research of our collaborators and colleagues, and will likely influence the work of future generations of scientists working in the field of geobiology. Some examples are cited below.Item Measurements of accumulation and displacement at the single cell cluster level in Pseudomonas aeruginosa biofilms(2008-09) Klayman, Benjamin J.; Klapper, Isaac; Stewart, Philip S.; Camper, Anne K.Quantitative descriptions of biofilm growth and dynamics at the individual cell level are largely missing from the literature. To fill this gap, research was done to describe growth, accumulation and displacement patterns in developing Pseudomonas aeruginosa biofilms. A parent strain of PAO1 was labelled with either a cyan or yellow fluorescent protein. These were then grown in a flow cell biofilm together so that pockets of dividing cells could be identified and their accumulation and displacement tracked. This analysis revealed a pattern of exponential accumulation for all clusters followed by a stationary accumulation phase. A background ‘carpet’ layer of cells uniformly colonizing the surface exhibited zero net accumulation of bio-volume. The individual clusters were found to have a mean accumulation rate of 0.34 h-1 with a range of 0.28–0.41 h-1. Cluster accumulation rates were negatively correlated with cluster size; larger clusters accumulated volume at a slower rate (P < 0.001). Pockets of cells on the inside of clusters initially accumulated at a comparable rate to the cluster within which they resided, but later invariably exhibited zero to slightly negative accumulation despite continued exponential (positive) accumulation of the cluster. Expanding clusters were able to displace neighbouring cells from the surface, and larger clusters displaced smaller clusters. This work provides a more detailed quantitative experimental observation of biofilm behaviour than has been described previously.