Scholarly Work - Center for Biofilm Engineering
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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 UO2+2 speciation determines uranium toxicity and bioaccumulation in an environmental Pseudomonas sp. isolate(2010-04) VanEngelen, Michael R.; Field, E. K.; Gerlach, Robin; Lee, Brady D.; Apel, William A.; Peyton, Brent M.In the present study, experiments were performed to investigate how representative cellulosic breakdown products, when serving as growth substrates under aerobic conditions, affect hexavalent uranyl cation (UO2+2 ) toxicity and bioaccumulation within a Pseudomonas sp. isolate (designated isolate A). Isolate A taken from the Cold Test Pit South (CTPS) region of the Idaho National Laboratory (INL), Idaho Falls, ID, USA. The INL houses low-level uranium-contaminated cellulosic material and understanding how this material, and specifically its breakdown products, affect U-bacterial interactions is important for understanding UO2+2 fate and mobility. Toxicity was modeled using a generalized Monod expression. Butyrate, dextrose, ethanol, and lactate served as growth substrates. The potential contribution of bicarbonate species present in high concentrations was also investigated and compared with toxicity and bioaccumulation patterns seen in low-bicarbonate conditions. Isolate A was significantly more sensitive to UO2+2 and accumulated significantly more UO2+2 in low-bicarbonate concentrations. In addition, UO2+2 growth inhibition and bioaccumulation varied depending on the growth substrate. In the presence of high bicarbonate concentrations, sensitivity to UO2+2 inhibition was greatly mitigated, and did not vary between the four substrates tested. The extent of UO2+2 accumulation was also diminished. The observed patterns were related to UO2+2 aqueous complexation, as predicted by MINTEQ (ver. 2.52) (Easton, PA, USA). In the low- bicarbonate medium, the presence of positively charged and unstable UO2+2 -hydroxide complexes explained both the greater sensitivity of isolate A to UO2+2, and the ability of isolate A to accumulate significant amounts of UO2+2 . The exclusive presence of negatively charged and stable UO2+2 -carbonate complexes in the high bi-carbonate medium explained the diminished sensitivity of isolate A to UO2+2 toxicity, and limited ability of isolate A to accumulate UO2+2 .Item Multiple mechanisms of uranium immobilization by Cellulomonas sp. strain ES6(2011-02) Sivaswamy, V.; Boyanov, M. I.; Peyton, Brent M.; Viamajala, Sridhar; Gerlach, Robin; Apel, William A.; Sani, Rajesh K.; Dohnalkova, Alice; Kemner, K. M.; Borch, ThomasRemoval of hexavalent uranium (U(VI)) from aqueous solution was studied using a gram-positive facultative anaerobe, Cellulomonas sp. strain ES6, under anaerobic, non-growth conditions in bicarbonate and PIPES buffers.Inorganic phosphate was released by cells during the experiments providing ligands for formation of insoluble U(VI) phosphates. Phosphate release was most probably the result of anaerobic hydrolysis of intracellular polyphosphates accumulated by ES6 during aerobic growth. Microbial reduction of U(VI) to U(IV) was also observed. However, the relative magnitudes of U(VI) removal by abiotic (phosphate-based) precipitation and microbial reduction depended on the buffer chemistry. In bicarbonate buffer, X-ray absorption fine structure (XAFS) spectroscopy showed that U in the solid phase was present primarily as a non-uraninite U(IV) phase, whereas in PIPES buffer, U precipitates consisted primarily of U(VI)-phosphate. In both bicarbonate and PIPES buffer, net release of cellular phosphate was measured to be lower than that observed in U-free controls suggesting simultaneous precipitation of U and PO₄³⠻. In PIPES, U(VI) phosphates formed a significant portion of U precipitates and mass balance estimates of U and P along with XAFS data corroborate this hypothesis. High-resolution transmission electron microscopy (HR-TEM) and energy dispersive X-ray spectroscopy (EDS) of samples from PIPES treatments indeed showed both extracellular and intracellular accumulation of U solids with nanometer sized lath structures that contained U and P. In bicarbonate, however, more phosphate was removed than required to stoichiometrically balance the U(VI)/U(IV) fraction determined by XAFS, suggesting that U(IV) precipitated together with phosphate in this system. When anthraquinone-2,6-disulfonate (AQDS), a known electron shuttle, was added to the experimental reactors, the dominant removal mechanism in both buffers was reduction to a non-uraninite U(IV) phase.Uranium immobilization by abiotic precipitation or microbial reduction has been extensively reported; however, the present work suggests that strain ES6 can remove U(VI) from solution simultaneously through precipitation with phosphate ligands and microbial reduction, depending on the environmental conditions. Cellulomonadaceae are environmentally relevant subsurface bacteria and here, for the first time, the presence of multiple U immobilization mechanisms within one organism is reported using Cellulomonas sp. strain ES6Item Influence of carbon sources and electron shuttles on ferric iron reduction by Cellulomonas sp. strain ES6(2011-09) Gerlach, Robin; Field, E. K.; Viamajala, Sridhar; Peyton, Brent M.; Apel, William A.; Cunningham, Alfred B.Microbially reduced iron minerals can reductively transform a variety of contaminants including heavy metals, radionuclides, chlorinated aliphatics, and nitroaromatics. A number of Cellulomonas spp. strains, including strain ES6, isolated from aquifer samples obtained at the U.S. Department of Energy’s Hanford site in Washington, have been shown to be capable of reducing Cr(VI), TNT, natural organic matter, and soluble ferric iron [Fe(III)]. This research investigated the ability of Cellulomonas sp. strain ES6 to reduce solid phase and dissolved Fe(III) utilizing different carbon sources and various electron shuttling compounds. Results suggest that Fe(III) reduction by and growth of strain ES6 was dependent upon the type of electron donor, the form of iron present, and the presence of synthetic or natural organic matter, such as anthraquinone-2,6-disulfonate (AQDS) or humic substances. This research suggests that Cellulomonas sp. strain ES6 could play a significant role in metal reduction in the Hanford subsurface and that the choice of carbon source and organic matter addition can allow for independent control of growth and iron reduction activity.Item Use of sodium bicarbonate to stimulate triacylglycerol accumulation in the chlorophyte Scenedesmus sp. and the diatom Phaeodactylum tricornutum(2012-10) Gardner, Robert D.; Cooksey, Keith E.; Mus, Florence; Macur, Richard E.; Moll, Karen M.; Eustance, E. O.; Carlson, Ross P.; Gerlach, Robin; Fields, Matthew W.; Peyton, Brent M.There is potential for algal-derived biofuel to help alleviate part of the world’s dependency on petroleum based fuels. However, research must still be done on strain selection, induction of triacylglycerol (TAG) accumulation, and fundamental algal metabolic studies, along with large-scale culturing techniques, harvesting, and biofuel/biomass processing. Here, we have advanced the knowledge on Scenedesmus sp. strain WC-1 by monitoring growth, pH, and TAG accumulation on a 14:10 light–dark cycle with atmospheric air or 5% CO2 in air (v/v) aeration. Under ambient aeration, there was a loss of pH-induced TAG accumulation, presumably due to TAG consumption during the lower culture pH observed during dark hours (pH 9.4). Under 5% CO2 aeration, the growth rate nearly doubled from 0.78 to 1.53 d−1, but the pH was circumneutral (pH 6.9) and TAG accumulation was minimal. Experiments were also performed with 5% CO2 during the exponential growth phase, which was then switched to aeration with atmospheric air when nitrate was close to depletion. These tests were run with and without the addition of 50 mM sodium bicarbonate. Cultures without added bicarbonate showed decreased growth rates with the aeration change, but there was no immediate TAG accumulation. The cultures with bicarbonate added immediately ceased cellular replication and rapid TAG accumulation was observed, as monitored by Nile Red fluorescence which has previously been correlated by gas chromatography to cellular TAG levels. Sodium bicarbonate addition (25 mM final concentration) was also tested with the marine diatom Phaeodactylum tricornutum strain Pt-1 and this organism also accumulated TAG.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.; Peyton, Brent M.; Fields, Matthew W.BACKGROUND: Phaeodactylum tricornutum is a unicellular diatom in the class Bacillariophyceae. The full genome hasbeen sequenced (<30 Mb), and approximately 20 to 30% triacylglyceride (TAG) accumulation on a dry cell basis hasbeen reported under different growth conditions. To elucidate P. tricornutum gene expression profiles duringnutrient-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, theincrease in NR fluorescence was initiated before external nitrate was completely exhausted. Exogenous phosphatewas depleted before nitrate, and these results indicated that the depletion of exogenous phosphate might be anearly trigger for lipid accumulation that is magnified upon nitrate depletion. As expected, many of the genesassociated with nitrate and phosphate utilization were up-expressed. The diatom-specific cyclins cyc7 and cyc10were down-expressed during the nutrient-deplete state, and cyclin B1 was up-expressed during lipid-accumulationafter growth cessation. While many of the genes associated with the C3 pathway for photosynthetic carbonreduction were not significantly altered, genes involved in a putative C4 pathway for photosynthetic carbonassimilation 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 HCO-3 were also suggested by the gene expression profiles (e.g., putativepropionyl-CoA and methylmalonyl-CoA decarboxylases).CONCLUSION: The results indicate that P. tricornutum continued carbon dioxide reduction when population growthwas 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 ofprecursors to the acetyl-CoA carboxylases may play a more significant role in TAG synthesis rather than the actualenzyme levels of acetyl-CoA carboxylases per se. The presented insights into the types and timing of cellularresponses to inorganic carbon will help maximize photoautotrophic carbon flow to lipid accumulation.Item Comparison of CO2 and bicarbonate as inorganic carbon sources for triacylglycerol and starch accumulation in Chlamydomonas reinhardtii(2013-01) Gardner, Robert D.; Lohman, Egan J.; Gerlach, Robin; Cooksey, Keith E.; Peyton, Brent M.Microalgae are capable of accumulating high levels of lipids and starch as carbon storage compounds. Investigation into the metabolic activities involved in the synthesis of these compounds has escalated because these compounds can be used as precursors for food and fuel. Here, we detail the results of a comprehensive analysis of Chlamydomonas reinhardtii using high or low inorganic carbon concentrations and speciation between carbon dioxide and bicarbonate, and the effects these have on inducing lipid and starch accumulation during nitrogen depletion. High concentrations of CO2 (5%;v/v) produced the highest amount of biofuel precursors, transesterified to fatty acid methyl esters, but exhibited rapid accumulation and degradation characteristics. Low CO2 (0.04%;v/v) caused carbon limitation and minimized triacylglycerol (TAG) and starch accumulation. High bicarbonate caused a cessation of cell cycling and accumulation of both TAG and starch that was more stable than the other experimental conditions. Starch accumulated prior to TAG and then degraded as maximum TAG was reached. This suggests carbon reallocation from starch-based to TAG-based carbon storage.Item Physiological and molecular analysis of carbon source supplementation and pH stress-induced lipid accumulation in the marine diatom Phaeodactylum tricornutum(2013-03) Mus, Florence; Toussaint, Jean-Paul; Cooksey, Keith E.; Fields, Matthew W.; Gerlach, Robin; Peyton, Brent M.; Carlson, Ross P.A detailed physiological and molecular analysis of lipid accumulation under a suite of conditions including nitrogen limitation, alkaline pH stress, bicarbonate supplementation, and organic acid supplementation was performed on the marine diatom Phaeodactylum tricornutum. For all tested conditions, nitrogen limitation was a prerequisite for lipid accumulation and the other culturing strategies only enhanced accumulation highlighting the importance of compounded stresses on lipid metabolism. Volumetric lipid levels varied depending on condition; the observed rankings from highest to lowest were for inorganic carbon addition (15 mM bicarbonate), organic acid addition (15 carbon mM acetate), and alkaline pH stress (pH9.0). For all lipidaccumulating cultures except acetate supplementation, a common series of physiological steps were observed. Upon extracellular nitrogen exhaustion, culture growth continued for approximately 1.5 cell doublings with decreases in specific protein and photosynthetic pigment content. As nitrogen limitation arrested cell growth, carbohydrate content decreased with a corresponding increase in lipid content. Addition of the organic carbon source acetate appeared to activate alternative metabolic pathways for lipid accumulation. Molecular level data on more than 50 central metabolism transcripts were measured using real-time PCR. Analysis of transcripts suggested the central metabolism pathways associated with bicarbonate transport, carbonic anhydrases, and C4 carbon fixations were important for lipid accumulation. Transcriptomic data also suggested that repurposing of phospholipids may play a role in lipid accumulation. This study provides a detailed physiological and molecular-level foundation for improved understanding of diatom nutrient cycling and contributes to a metabolic blueprint for controlling lipid accumulation in diatoms.Item Hexavalent chromium reduction by Cellulomonas sp. strain ES6: the influence of carbon source, iron minerals, and electron shuttling compounds(2013-06) Field, E. K.; Gerlach, Robin; Viamajala, Sridhar; Jennings, Laura K.; Peyton, Brent M.; Apel, William A.The reduction of hexavalent chromium, Cr(VI), to trivalent chromium, Cr(III), can be an important aspect of remediation processes at contaminated sites. Cellulomonas species are found at several Cr(VI) contaminated and uncontaminated locations at the Department of Energy site in Hanford, Washington. Members of this genus have demonstrated the ability to effectively reduce Cr(VI) to Cr(III) fermentatively and therefore play a potential role in Cr(VI) remediation at this site. Batch studies were conducted with Cellulomonas sp. strain ES6 to assess the influence of various carbon sources, iron minerals, and electron shuttling compounds on Cr(VI) reduction rates as these chemical species are likely to be present in, or added to, the environment during in situ bioremediation. Results indicated that the type of carbon source as well as the type of electron shuttle present influenced Cr(VI) reduction rates. Molasses stimulated Cr(VI) reduction more effectively than pure sucrose, presumably due to presence of more easily utilizable sugars, electron shuttling compounds or compounds with direct Cr(VI) reduction capabilities. Cr(VI) reduction rates increased with increasing concentration of anthraquinone-2,6-disulfonate (AQDS) regardless of the carbon source. The presence of iron minerals and their concentrations did not significantly influence Cr(VI) reduction rates. However, strain ES6 or AQDS could directly reduce surface-associated Fe(III) to Fe(II), which was capable of reducing Cr(VI) at a near instantaneous rate. These results suggest the rate limiting step in these systems was the transfer of electrons from strain ES6 to the intermediate or terminal electron acceptor whether that was Cr(VI), Fe(III), or AQDS.Item Nutrient resupplementation arrests bio-oil accumulation in Phaeodactylum tricornutum(2013-08) Valenzuela, Jacob J.; Carlson, Ross P.; Gerlach, Robin; Cooksey, Keith E.; Peyton, Brent M.; Bothner, Brian; Fields, Matthew W.Phaeodactylum tricornutum is a marine diatom in the class Bacillariophyceae and is important ecologically and industrially with regards to ocean primary production and lipid accumulation for biofuel production, respectively. Triacylglyceride (TAG) accumulation has been reported in P. tricornutum under different nutrient stresses, and our results show that lipid accumulation can occur with nitrate or phosphate depletion. However, greater lipid accumulation was observed when both nutrients were depleted as observed using a Nile Red assay and fatty acid methyl ester (FAME) profiles. Nitrate depletion had a greater effect on lipid accumulation than phosphate depletion. Lipid accumulation in P. tricornutum was arrested upon resupplementation with the depleted nutrient. Cells depleted of nitrogen showed a distinct shift from a lipid accumulation mode to cellular growth post resupplementation with nitrate, as observed through increased cell numbers and consumption of accumulated lipid. Phosphate depletion caused lipid accumulation that was arrested upon phosphate resupplementation. The cessation of lipid accumulation was followed by lipid consumption without an increase in cell numbers. Cells depleted in both nitrate and phosphate displayed cell growth upon the addition of both nitrate and phosphate and had the largest observed lipid consumption upon resupplementation. These results indicate that phosphate resupplementation can shut down lipid accumulation but does not cause cells to shift into cellular growth, unlike nitrate resupplementation. These data suggest that nutrient resupplementation will arrest lipid accumulation and that switching between cellular growth and lipid accumulation can be regulated upon the availability of nitrogen and phosphorus.