Scholarly Work - Center for Biofilm Engineering
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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 Biofilm formation in Desulfovibrio vulgaris Hildenborough is dependent upon protein filaments(2007-11) Clark, M. E.; Edelmann, Richard E.; Duley, Matt L.; Wall, Judy D.; Fields, Matthew W.Desulfovibrio vulgaris Hildenborough is a gram-negative sulfate-reducing bacterium (SRB), and the physiology of SRBs can impact many anaerobic environments including radionuclide waste sites, oil reservoirs and metal pipelines. In an attempt to understand D. vulgaris as a population that can adhere to surfaces, D. vulgaris cultures were grown in a defined medium and analysed for carbohydrate production, motility and biofilm formation. Desulfovibrio vulgaris wild-type cells had increasing amounts of carbohydrate into stationary phase and approximately half of the carbohydrate remained internal. In comparison, a mutant that lacked the 200 kb megaplasmid, strain DeltaMP, produced less carbohydrate and the majority of carbohydrate remained internal of the cell proper. To assess the possibility of carbohydrate re-allocation, biofilm formation was investigated. Wild-type cells produced approximately threefold more biofilm on glass slides compared with DeltaMP; however, wild-type biofilm did not contain significant levels of exopolysaccharide. In addition, stains specific for extracellular carbohydrate did not reveal polysaccharide material within the biofilm. Desulfovibrio vulgaris wild-type biofilms contained long filaments as observed with scanning electron microscopy (SEM), and the biofilm-deficient DeltaMP strain was also deficient in motility. Biofilms grown directly on silica oxide transmission electron microscopy (TEM) grids did not contain significant levels of an exopolysaccharide matrix when viewed with TEM and SEM, and samples stained with ammonium molybdate also showed long filaments that resembled flagella. Biofilms subjected to protease treatments were degraded, and different proteases that were added at the time of inoculation inhibited biofilm formation. The data indicated that D. vulgaris did not produce an extensive exopolysaccharide matrix, used protein filaments to form biofilm between cells and silica oxide surfaces, and the filaments appeared to be flagella. It is likely that D. vulgaris used flagella for more than a means of locomotion to a surface, but also used flagella, or modified flagella, to establish and/or maintain biofilm structure.Item In situ bioreduction of uranium (VI) in situ and stability of immobilized uranium: Impact of dissolved oxygen(2007-08) Wu, Wei-Min; Carley, Jack; Luo, Jian; Ginder-Vogel, Matthew A.; Cardenas, Erick; Leigh, Mary Beth; Hwang, Chiachi; Kelly, Shelly D.; Ruan, Chuanmin; Wu, Liyou; Nostrand, Joy V.; Gentry, Terry J.; Lowe, K. A.; Mehlhorn, T. L.; Carroll, Sue L.; Luo, Wensui; Fields, Matthew W.; Gu, Baohua; Watson, David B.; Kemner, K. M.; Marsh, Terence; Tiedje, J. M.; Zhou, Jizhong; Fendorf, Scott; Kitanidis, Peter K.; Jardine, Phil M.; Criddle, Craig S.Groundwater within Area 3 of the U.S. Department of Energy (DOE) Environmental Remediation Sciences Program (ERSP) Field Research Center at Oak Ridge, TN (ORFRC) contains up to 135 microM uranium as U(VI). Through a series of experiments at a pilot scale test facility, we explored the lower limits of groundwater U(VI) that can be achieved by in-situ biostimulation and the effects of dissolved oxygen on immobilized uranium. Weekly 2-day additions of ethanol over a 2-year period stimulated growth of denitrifying, Fe(III)-reducing, and sulfate-reducing bacteria, and immobilization of uranium as U(IV), with dissolved uranium concentrations decreasing to low levels. Following sulfite addition to remove dissolved oxygen, aqueous U(VI) concentrations fell below the U.S. Environmental Protection Agengy maximum contaminant limit (MCL) for drinking water (< 30/microg L(-1) or 0.126 microM). Under anaerobic conditions, these low concentrations were stable, even in the absence of added ethanol. However, when sulfite additions stopped, and dissolved oxygen (4.0-5.5 mg L(-1)) entered the injection well, spatially variable changes in aqueous U(VI) occurred over a 60 day period, with concentrations increasing rapidly from < 0.13 to 2.0 microM at a multilevel sampling (MLS) well located close to the injection well, but changing little at an MLS well located further away. Resumption of ethanol addition restored reduction of Fe(III), sulfate, and U(VI) within 36 h. After 2 years of ethanol addition, X-ray absorption near-edge structure spectroscopy (XANES) analyses indicated that U(IV) comprised 60-80% of the total uranium in sediment samples. Atthe completion of the project (day 1260), U concentrations in MLS wells were less than 0.1 microM. The microbial community at MLS wells with low U(VI) contained bacteria that are known to reduce uranium, including Desulfovibrio spp. and Geobacter spp., in both sediment and groundwater. The dominant Fe(III)-reducing species were Geothrix spp.Item Expression profiling of hypothetical genes in Desulfovibrio vulgaris leads to improved functional annotation(2009-03) Elias, Dwayne A.; Mukhopadhyay, A.; Joachimiak, M. P.; Drury, Elliott C.; Redding, Alyssa M.; Yen, Huei-Che B.; Fields, Matthew W.; Hazen, Terry C.; Arkin, Adam P.; Keasling, J. D.; Wall, Judy D.Hypothetical (HyP) and conserved HyP genes account for >30% of sequenced bacterial genomes. For the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough, 347 of the 3634 genes were annotated as conserved HyP (9.5%) along with 887 HyP genes (24.4%). Given the large fraction of the genome, it is plausible that some of these genes serve critical cellular roles. The study goals were to determine which genes were expressed and provide a more functionally based annotation. To accomplish this, expression profiles of 1234 HyP and conserved genes were used from transcriptomic datasets of 11 environmental stresses, complemented with shotgun LC–MS/MS and AMT tag proteomic data. Genes were divided into putatively polycistronic operons and those predicted to be monocistronic, then classified by basal expression levels and grouped according to changes in expression for one or multiple stresses. One thousand two hundred and twelve of these genes were transcribed with 786 producing detectable proteins. There was no evidence for expression of 17 predicted genes. Except for the latter, monocistronic gene annotation was expanded using the above criteria along with matching Clusters of Orthologous Groups. Polycistronic genes were annotated in the same manner with inferences from their proximity to more confidently annotated genes. Two targeted deletion mutants were used as test cases to determine the relevance of the inferred functional annotations.Item Real-time molecular monitoring of chemical environment in obligate anaerobes during oxygen adaptive response(2009-06) Holman, Hoi-Ying N.; Wozei, Eleanor; Lin, Zhang; Comolli, Luis R.; Ball, David A.; Borglin, Sharon; Fields, Matthew W.; Hazen, Terry C.; Downing, Kenneth H.Determining the transient chemical properties of the intracellular environment can elucidate the paths through which a biological system adapts to changes in its environment, for example, the mechanisms that enable some obligate anaerobic bacteria to survive a sudden exposure to oxygen. Here we used high-resolution Fourier transform infrared (FTIR) spectromicroscopy to continuously follow cellular chemistry within living obligate anaerobes by monitoring hydrogen bond structures in their cellular water. We observed a sequence of well orchestrated molecular events that correspond to changes in cellular processes in those cells that survive, but only accumulation of radicals in those that do not. We thereby can interpret the adaptive response in terms of transient intracellular chemistry and link it to oxygen stress and survival. This ability to monitor chemical changes at the molecular level can yield important insights into a wide range of adaptive responses.Item Bacterial community succession during in situ uranium bioremediation: spatial similarities along controlled flow paths(2009-01) Hwang, Chiachi; Wu, Wei-Min; Gentry, Terry J.; Carley, Jack; Corbin, Gail A.; Carroll, Sue L.; Watson, David B.; Jardine, Phil M.; Zhou, Jizhong; Criddle, Craig S.; Fields, Matthew W.Bacterial community succession was investigated in a field-scale subsurface reactor formed by a series of wells that received weekly ethanol additions to re-circulating groundwater. Ethanol additions stimulated denitrification, metal reduction, sulfate reduction and U(VI) reduction to sparingly soluble U(IV). Clone libraries of SSU rRNA gene sequences from groundwater samples enabled tracking of spatial and temporal changes over a 1.5-year period. Analyses showed that the communities changed in a manner consistent with geochemical variations that occurred along temporal and spatial scales. Canonical correspondence analysis revealed that the levels of nitrate, uranium, sulfide, sulfate and ethanol were strongly correlated with particular bacterial populations. As sulfate and U(VI) levels declined, sequences representative of sulfate reducers and metal reducers were detected at high levels. Ultimately, sequences associated with sulfate-reducing populations predominated, and sulfate levels declined as U(VI) remained at low levels. When engineering controls were compared with the population variation through canonical ordination, changes could be related to dissolved oxygen control and ethanol addition. The data also indicated that the indigenous populations responded differently to stimulation for bioreduction; however, the two biostimulated communities became more similar after different transitions in an idiosyncratic manner. The strong associations between particular environmental variables and certain populations provide insight into the establishment of practical and successful remediation strategies in radionuclide-contaminated environments with respect to engineering controls and microbial ecology.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 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 Global transcriptional, physiological, and metabolite analyses of the responses of Desulfovibrio vulgaris Hildenborough to salt adaptation(2009-12) He, Zhili; Zhou, Aifen; Baidoo, Edward E. K.; He, Q.; Joachimiak, M. P.; Benke, P.; Phan, R.; Mukhopadhyay, A.; Hemme, C. L.; Huang, K.; Alm, E. J.; Fields, Matthew W.; Wall, Judy D.; Stahl, David A.; Hazen, Terry C.; Keasling, J. D.; Arkin, Adam P.; Zhou, JizhongThe response of Desulfovibrio vulgaris Hildenborough to salt adaptation (long-term NaCl exposure) was examined by performing physiological, global transcriptional, and metabolite analyses. Salt adaptation was reflected by increased expression of genes involved in amino acid biosynthesis and transport, electron transfer, hydrogen oxidation, and general stress responses (e.g., heat shock proteins, phage shock proteins, and oxidative stress response proteins). The expression of genes involved in carbon metabolism, cell growth, and phage structures was decreased. Transcriptome profiles of D. vulgaris responses to salt adaptation were compared with transcriptome profiles of D. vulgaris responses to salt shock (short-term NaCl exposure). Metabolite assays showed that glutamate and alanine accumulated under salt adaptation conditions, suggesting that these amino acids may be used as osmoprotectants in D. vulgaris. Addition of amino acids (glutamate, alanine, and tryptophan) or yeast extract to the growth medium relieved salt-related growth inhibition. A conceptual model that links the observed results to currently available knowledge is proposed to increase our understanding of the mechanisms of D. vulgaris adaptation to elevated NaCl levels.Item Impact of elevated nitrate on sulfate-reducing bacteria: A comparative study of Desulfovibrio vulgaris(2010-05) He, Q.; He, Zhili; Joyner, D. C.; Joachimiak, M. P.; Price, M. N.; Yang, Zamin K.; Yen, Huei-Che B.; Hemme, C. L.; Chen, W.; Fields, Matthew W.; Stahl, David A.; Keasling, J. D.; Keller, M.; Arkin, Adam P.; Hazen, Terry C.; Wall, Judy D.; Zhou, JizhongSulfate-reducing bacteria have been extensively studied for their potential in heavy-metal bioremediation. However, the occurrence of elevated nitrate in contaminated environments has been shown to inhibit sulfate reduction activity. Although the inhibition has been suggested to result from the competition with nitrate-reducing bacteria, the possibility of direct inhibition of sulfate reducers by elevated nitrate needs to be explored. Using Desulfovibrio vulgaris as a model sulfate-reducing bacterium, functional genomics analysis reveals that osmotic stress contributed to growth inhibition by nitrate as shown by the upregulation of the glycine/betaine transporter genes and the relief of nitrate inhibition by osmoprotectants. The observation that significant growth inhibition was effected by 70mM NaNO3 but not by 70mM NaCl suggests the presence of inhibitory mechanisms in addition to osmotic stress. The differential expression of genes characteristic of nitrite stress responses, such as the hybrid cluster protein gene, under nitrate stress condition further indicates that nitrate stress response by D. vulgaris was linked to components of both osmotic and nitrite stress responses. The involvement of the oxidative stress response pathway, however, might be the result of a more general stress response. Given the low similarities between the response profiles to nitrate and other stresses, less-defined stress response pathways could also be important in nitrate stress, which might involve the shift in energy metabolism. The involvement of nitrite stress response upon exposure to nitrate may provide detoxification mechanisms for nitrite, which is inhibitory to sulfate-reducing bacteria, produced by microbial nitrate reduction as a metabolic intermediate and may enhance the survival of sulfate-reducing bacteria in environments with elevated nitrate level.