Scholarly Work - Center for Biofilm Engineering
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Item Microbial community changes during a toxic cyanobacterial bloom in an alkaline Hungarian lake(2018-08) Bell, Tisza A. S.; Feldoldi, Tamas; Sen-Kilic, Emel; Vasas, Gabor; Fields, Matthew W.; Peyton, Brent M.The Carpathian Basin is a lowland plain located mainly in Hungary. Due to the nature of the bedrock, alluvial deposits, and a bowl shape, many lakes and ponds of the area are characterized by high alkalinity. In this study, we characterized temporal changes in eukaryal and bacterial community dynamics with high throughput sequencing and relate the changes to environmental conditions in Lake Velence located in Fejer county, Hungary. The sampled Lake Velence microbial populations (algal and bacterial) were analyzed to identify potential correlations with other community members and environmental parameters at six timepoints over 6weeks in the Spring of 2012. Correlations between community members suggest a positive relationship between certain algal and bacterial populations (e.g. Chlamydomondaceae with Actinobacteria and Acidobacteria), while other correlations allude to changes in these relationships over time. During the study, high nitrogen availability may have favored non-nitrogen fixing cyanobacteria, such as the toxin-producing Microcystis aeruginosa, and the eutrophic effect may have been exacerbated by high phosphorus availability as well as the high calcium and magnesium content of the Carpathian Basin bedrock, potentially fostering exopolymer production and cell aggregation. Cyanobacterial bloom formation could have a negative environmental impact on other community members and potentially affect overall water quality as well as recreational activities. To our knowledge, this is the first prediction for relationships between photoautotrophic eukaryotes and bacteria from an alkaline, Hungarian lake.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 Influence of pH and inorganic phosphate on toxicity of zinc to Arthrobacter sp. isolated from heavy-metal-contaminated sediments(2010-10) Moberly, James G.; Staven, A.; Sani, Rajesh K.; Peyton, Brent M.Because of its high solubility over a wide range of pH conditions, zinc is found in many natural and human-impacted systems. Zinc speciation is critical in assessing zinc toxicity to microorganisms because it varies considerably with pH and is dependent on other aqueous constituents. Combined results of thermodynamic modeling, statistical analysis, and batch culture studies using Arthrobacter sp. JM018 suggest that the toxic species may not be solely limited to the free ion, but also includes ZnHPO40(aq). Cellular uptake of ZnHPO40(aq) through the inorganic phosphate transporter (Pit family), which requires a neutral metal phosphate complex for phosphate transport, may explain the observed toxicity. Based on visual MINTEQ (v3.0) modeling, at 50 μM total zinc, ZnHPO40(aq) constitutes 33, 70, and 76% of the neutral metal phosphate pool at pH 6, 7, and 8, respectively. At 50 μM total zinc, cultures supplied with organic phosphate (glycerol-3-phosphate) show no significant response to pH (p=0.13) while inhibition of inorganic phosphate-supplemented cultures, whose neutral metal phosphates are increasingly dominated by ZnHPO40(aq), show significant pH dependence (p=9.45 x 10-7). Using sodium to decrease the distribution of ZnHPO40(aq) in the neutral metal phosphate pool also decreased the pH dependent toxicity, further supporting this mechanism.These findings show the important role of minor zinc species in organism toxicity and have wider implications because the Pit inorganic phosphate transport system is widely distributed in Bacteria, Archaea, and Eukarya.Item Medium pH and nitrate concentration effects on accumulation of triacylglycerol in two members of the chlorophyta(2010-12) Gardner, Robert D.; Peters, P.; Peyton, Brent M.; Cooksey, Keith E.Algal-derived biodiesel is of particular interest because of several factors including: the potential for a near-carbon-neutral life cycle, the prospective ability for algae to capture carbon dioxide generated from coal, and algae’s high per acre yield potential. Our group and others have shown that in nitrogen limitation, and for a single species of Chlorella, a rise in culture medium pH yields triacylglycerol (TAG) accumulation. To solidify and expand on these triggers, the influence and interaction of pH and nitrogen concentration on lipid production was further investigated on Chlorophyceae Scenedesmus sp. and Coelastrella sp. Growth was monitored optically and TAG accumulation was monitored by Nile red fluorescence and confirmed by gas chromatography. Both organisms grew in all treatments and TAG accumulation was observed by two distinct conditions: high pH and nitrogen limitation. The Scenedesmus sp. was shown to grow and produce lipids to a larger degree in alkaliphilic conditions (pH >9) and was used to further investigate the interplay between TAG accumulation from high pH and/or nitrate depletion. Results given here indicate that TAG accumulation per cell, monitored by Nile red fluorescence, correlates with pH at the time of nitrate depletion.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 Growth effects and assimilation of organic acids in chemostat and batch cultures of Acidithiobacillus caldus(2011-01) Aston, John E.; Apel, William A.; Lee, Brady D.; Peyton, Brent M.The ability of Acidithiobacillus caldus to grow aerobically using pyruvate, acetate, citrate, 2-ketoglutarate, succinate, and malate as either an electron donor and carbon source (heterotrophic growth), or as a carbon source when potassium tetrathionate was added as an electron donor (mixotrophic growth), was tested in chemostat cultures. Under both heterotrophic and mixotrophic conditions, organic acids were added to a sub-lethal concentration (50 μM). Under mixotrophic conditions, potassium tetrathionate was added to an excess concentration (10 mM). No cell growth was observed under heterotrophic conditions; however, effluent cell concentrations increased over threefold when pyruvate was coupled with potassium tetrathionate. Under these conditions, the effluent pyruvate concentration was reduced to below the detection limit (2 μM), and oxygen consumption increased by approximately 100%. Although pyruvate provided a carbon source in these experiments, ambient carbon dioxide was also available to the cells. To test whether At. caldus could grow mixotrophically using pyruvate as a sole carbon source and potassium tetrathionate as an electron donor, cells were batch cultured in a medium free of dissolved inorganic carbon, and with no carbon dioxide in the headspace. These experiments showed that At. caldus was able to convert between 65 ± 8 and 82 ± 15% of the pyruvate carbon to cellular biomass, depending on the initial pyruvate concentrations. This work is the first to identify a defined organic-carbon source, other than glucose, that At. caldus can assimilate. This has important implications, as mixotrophic and heterotrophic activity has been shown to increase mineral leaching in acidic systems.Item 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 Comparison of single and joint effects of Zn and Cu in continuous flow and batch reactors(2012-03) Sengor, S. S.; Gikas, P.; Moberly, James G.; Peyton, Brent M.; Ginn, Timothy R.BACKGROUND: Microbial behavior in batch reactors may be different from that in continuous flow reactors, which is expected to affect microbial response to heavy metal exposure. Four parallel continuous flow reactors and batch growth tests were used to investigate the single and joint toxicity of Zn and Cu to Artrobacter sp. JM018.RESULTS: The results indicated that Cu is more toxic than Zn under all conditions. In the batch reactors, all Zn concentrations showed a stimulatory effect on microbial growth. However in the continuous system, 125 µmol L−1 Zn exposure produced inhibition. In the case of mixed Zn and Cu exposures in the batch system, the presence of Zn reduced the severity of Cu inhibition, with a net impact of reduced growth in all cases, whereas in the continuous system microbial growth and substrate utilization rates sharply decreased and ceased.CONCLUSION: The results clearly showed that growth in batch reactors underestimated significantly the heavy metal inhibition, compared with the continuous system. Therefore, the results of batch reactor tests should not be used directly when heavy metal inhibition is to be interpreted for continuous flow systems.
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