Scholarly Work - Center for Biofilm Engineering
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Item Challenges and Opportunities for Tribal Waters: Addressing Disparities in Safe Public Drinking Water on the Crow Reservation in Montana, US(2018-03) Doyle, John T.; Kindness, L.; Real Bird, James; Eggers, Margaret J.; Camper, Anne K.Disparities in access to safe public drinking water are increasingly being recognized as contributing to health disparities and environmental injustice for vulnerable communities in the United States. As the Co-Directors of the Apsaálooke Water and Wastewater Authority (AWWWA) for the Crow Tribe, with our academic partners, we present here the multiple and complex challenges we have addressed in improving and maintaining tribal water and wastewater infrastructure, including the identification of diverse funding sources for infrastructure construction, the need for many kinds of specialized expertise and long-term stability of project personnel, ratepayer difficulty in paying for services, an ongoing legacy of inadequate infrastructure planning, and lack of water quality research capacity. As a tribal entity, the AWWWA faces additional challenges, including the complex jurisdictional issues affecting all phases of our work, lack of authority to create water districts, and additional legal and regulatory gaps—especially with regards to environmental protection. Despite these obstacles, the AWWWA and Crow Tribe have successfully upgraded much of the local water and wastewater infrastructure. We find that ensuring safe public drinking water for tribal and other disadvantaged U.S. communities will require comprehensive, community-engaged approaches across a broad range of stakeholders to successfully address these complex legal, regulatory, policy, community capacity, and financial challenges.Item Cr(VI) reduction and physiological toxicity are impacted by resource ratio in Desulfovibrio vulgaris(2018-03) Franco, Lauren C.; Steinbeisser, Sadie; Zane, Grant M.; Wall, Judy D.; Fields, Matthew W.Desulfovibrio spp. are capable of heavy metal reduction and are well-studied systems for understanding metal fate and transport in anaerobic environments. Desulfovibrio vulgaris Hildenborough was grown under environmentally relevant conditions (i.e., temperature, nutrient limitation) to elucidate the impacts on Cr(VI) reduction on cellular physiology. Growth at 20 °C was slower than 30 °C and the presence of 50 μM Cr(VI) caused extended lag times for all conditions, but once growth resumed the growth rate was similar to that without Cr(VI). Cr(VI) reduction rates were greatly diminished at 20 °C for both 50 and 100 μM Cr(VI), particularly for the electron acceptor limited (EAL) condition in which Cr(VI) reduction was much slower, the growth lag much longer (200 h), and viability decreased compared to balanced (BAL) and electron donor limited (EDL) conditions. When sulfate levels were increased in the presence of Cr(VI), cellular responses improved via a shorter lag time to growth. Similar results were observed between the different resource (donor/acceptor) ratio conditions when the sulfate levels were normalized (10 mM), and these results indicated that resource ratio (donor/acceptor) impacted D. vulgaris response to Cr(VI) and not merely sulfate limitation. The results suggest that temperature and resource ratios greatly impacted the extent of Cr(VI) toxicity, Cr(VI) reduction, and the subsequent cellular health via Cr(VI) influx and overall metabolic rate. The results also emphasized the need to perform experiments at lower temperatures with nutrient limitation to make accurate predictions of heavy metal reduction rates as well as physiological states in the environment.Item Systems biology guided by XCMS Online metabolomics(2017-04) Huan, Tao; Forsberg, Erica M.; Rinehart, Duane; Johnson, Caroline H.; Ivanisevic, Julijana; Benton, H. Paul; Fang, Mingliang; Aisporna, Aries E.; Hilmers, Brian; Poole, Farris L.; Thorgersen, Michael P.; Adams, Michael W. W.; Krantz, Gregory; Fields, Matthew W.; Robbins, Paul D.; Niedernhofer, Laura J.; Ideker, Trey; Majumder, Erica L.; Wall, Judy D.; Rattray, Nicholas J. W.; Goodacre, Royston; Lairson, Luke L.; Siuzdak, GaryItem Resuscitation of Pseudomonas aeruginosa from dormancy requires hibernation promoting factor (PA4463) for ribosome preservation(2017-03) Akiyama, Tatsuya; Williamson, Kerry S.; Schaefer, Robert; Pratt, Shawna; Chang, Connie B.; Franklin, Michael J.Pseudomonas aeruginosa biofilm infections are difficult to treat with antibiotic therapy in part because the biofilms contain subpopulations of dormant antibiotic-tolerant cells. The dormant cells can repopulate the biofilms following alleviation of antibiotic treatments. While dormant, the bacteria must maintain cellular integrity, including ribosome abundance, to reinitiate the de novo protein synthesis required for resuscitation. Here, we demonstrate that the P. aeruginosa gene PA4463 [hibernation promoting factor (HPF)], but not the ribosome modulation factor (PA3049), is required for ribosomal NA preservation during prolonged nutrient starvation conditions. Single-cell–level studies using fluorescence in situ hybridization (FISH) and growth in microfluidic drops demonstrate that, in the absence of hpf, the rRNA abundances of starved cells decrease to levels that cause them to lose their ability to resuscitate from starvation, leaving intact nondividing cells. P. aeruginosa defective in the stringent response also had reduced ability to resuscitate from dormancy. However, FISH analysis of the starved stringent response mutant showed a bimodal response where the individual cells contained either abundant or low ribosome content, compared with the wild-type strain. The results indicate that ribosome maintenance is key for maintaining the ability of P. aeruginosa to resuscitate from starvation-induced dormancy and that HPF is the major factor associated with P. aeruginosa ribosome preservation.Item Cultivation of a native alga for biomass and biofuel accumulation in coal bed methane production water(2016-11) Hodgkiss, Logan H.; Nagy, J.; Barnhart, Elliott P.; Cunningham, Alfred B.; Fields, Matthew W.Coal bed methane (CBM) production has resulted in thousands of ponds in the Powder River Basin of low-quality water in a water-challenged region. A green alga isolate, PW95, was isolated from a CBM production pond, and analysis of a partial ribosomal gene sequence indicated the isolate belongs to the Chlorococcaceae family. Different combinations of macro- and micronutrients were evaluated for PW95 growth in CBM water compared to a defined medium. A small level of growth was observed in unamended CBM water (0.15 g/l), and biomass increased (2-fold) in amended CBM water or defined growth medium. The highest growth rate was observed in CBM water amended with both N and P, and the unamended CBM water displayed the lowest growth rate. The highest lipid content (27%) was observed in CBM water with nitrate, and a significant level of lipid accumulation was not observed in the defined growth medium. Growth analysis indicated that nitrate deprivation coincided with lipid accumulation in CBM production water, and lipid accumulation did not increase with additional phosphorus limitation. The presented results show that CBM production wastewater can be minimally amended and used for the cultivation of a native, lipid-accumulating alga.Item Use of Alternative Carrier Materials in AOAC Official Method SM2008.05, Efficacy of Liquid Sporicides Against Spores of Bacillus subtilis on a Hard, Nonporous Surface, Quantitative Three-Step Method(2010-01) Tomasino, S. F.; Rastogi, Vipin K.; Wallace, Lalena; Smith, Lisa S.; Hamilton, Martin A.; Pines, R. M.The quantitative Three-Step Method (TSM) for testing the efficacy of liquid sporicides against spores of Bacillus subtilis on a hard, nonporous surface (glass) was adopted as AOAC Official MethodSM 2008.05 in May 2008. The TSM uses 5x5x1 mm coupons (carriers) upon which spores have been inoculated and which are introduced into liquid sporicidal agent contained in a microcentrifuge tube. Following exposure of inoculated carriers and neutralization, spores are removed from carriers in three fractions (gentle washing, fraction A; sonication, fraction B; and gentle agitation, fraction C). Liquid from each fraction is serially diluted and plated on a recovery medium for spore enumeration. The counts are summed over the three fractions to provide the density (viable spores per carrier), which is log10-transformed to arrive at the log density. The log reduction is calculated by subtracting the mean log density for treated carriers from the mean log density for control carriers. This paper presents a single-laboratory investigation conducted to evaluate the applicability of using two porous carrier materials (ceramic tile and untreated pine wood) and one alternative nonporous material (stainless steel). Glass carriers were included in the study as the reference material. Inoculated carriers were evaluated against three commercially available liquid sporicides (sodium hypochlorite, a combination of peracetic acid and hydrogen peroxide, and glutaraldehyde), each at two levels of presumed efficacy (medium and high) to provide data for assessing the responsiveness of the TSM. Three coupons of each material were evaluated across three replications at each level; three replications of a control were required. Even though all carriers were inoculated with approximately the same number of spores, the observed counts of recovered spores were consistently higher for the nonporous carriers. For control carriers, the mean log densities for the four materials ranged from 6.63 for wood to 7.14 for steel. The pairwise differences between mean log densities, except for glass minus steel, were statistically significant (P < 0.001). The repeatability standard deviations (Sr) for the mean control log density per test were similar for the four materials, ranging from 0.08 for wood to 0.13 for tile. Spore recovery from the carrier materials ranged from approximately 20 to 70%: 20% (pine wood), 40% (ceramic tile), 55% (glass), and 70% (steel). Although the percent spore recovery from pine wood was significantly lower than that from other materials, the performance data indicate that the TSM provides a repeatable and responsive test for determining the efficacy of liquid sporicides on both porous and nonporous materials.Item Symmetry breaking clusters in soft clustering decoding of neural codes(2010-02) Parker, Albert E.; Dimitrov, Alexander G.; Gedeon, TomasInformation-based distortion methods have been used successfully in the analysis of neural coding problems. These approaches allow the discovery of neural symbols and the corresponding stimulus space of a neuron or neural ensemble quantitatively, while making few assumptions about the nature of either the code or of relevant stimulus features. The neural codebook is derived by quantizing sensory stimuli and neural responses into a small set of clusters, and optimizing the quantization to minimize an information distortion function. The method of annealing has been used to solve the corresponding high-dimensional nonlinear optimization problem. The annealing solutions undergo a series of bifurcations, which we study using bifurcation theory in the presence of symmetries. In this contribution we describe these symmetry breaking bifurcations in detail, and indicate some of the consequences of the form of the bifurcations. In particular, we show that the annealing solutions break symmetry at pitchfork bifurcations, and that subcritical branches can exist. Thus, at a subcritical bifurcation, there are local information distortion solutions which are not found by the method of annealing. Since the annealing procedure is guaranteed to converge to a local solution eventually, the subcritical branch must turn and become optimal at some later saddle-node bifurcation, which we have shown occur generically for this class of problems. This implies that the rate distortion curve, while convex for noninformation-based distortion measures, is not convex for information-based distortion methods.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 Heterogeneous rpoS and rhlR mRNA levels and 16S rRNA/rDNA ratios within Pseudomonas aeruginosa biofilms, sampled by laser capture microdissection(2010-03) Perez-Osorio, Ailyn C.; Williamson, Kerry S.; Franklin, Michael J.The local environmental conditions in biofilms are dependent on the impinging aqueous solution, chemical diffusion, and the metabolic activities of cells within the biofilms. Chemical gradients established in biofilms lead to physiological heterogeneities of bacterial gene expression. Previously, we used laser capture microdissection (LCM) and quantitative RT-PCR to target defined biofilm subpopulations for gene expression studies. Here, we combined that approach with quantitative PCR of bacterial DNA to normalize gene expression per cell. By comparing the ratio of 16S rRNA to 16S rDNA, we demonstrate that cells at the top of thick Pseudomonas aeruginosa biofilms have 16S rRNA/genome ratios similar to cells in a transition between exponential and stationary phase. Cells in the middle and bottom layers of these biofilms have ratios that are not significantly different from stationary phase planktonic cultures. Since much of the biofilm appeared to be in a stationary phase-like state, we analyzed local amounts of the stationary phase sigma factor, rpoS, and a quorum sensing regulator, rhlR, per cell. Surprisingly, the amount of rpoS mRNA was greatest at the top of these biofilms at the air-biofilm interface. Less than one rpoS mRNA transcript per cell was observed in the middle or base of the biofilms. The rhlR mRNA content was also greatest at the top of these biofilms, with little detectable rhlR expression at the middle or bottom of the biofilms. While cell density is slightly greater at the bottom of the biofilms, expression of this quorum sensing regulator occurs primarily at the top of the biofilms, where cell metabolic activity is greatest, as indicated by the local expression of the housekeeping gene, acpP and by expression from a constitutive Ptrc promoter. The results indicate that in thick P. aeruginosa biofilms, cells in the 30 µm adjacent to the air-biofilm interface actively express genes associated with stationary phase, while cells in the interior portions do not express these genes, and therefore are in a late stationary phase-like state and are possibly dormant.