EMSL: Alice Dohnalkova's Publications

Kimball BE, R Mathur, A Dohnalkova, AJ Wall, RL Runkel, and SL Brantley. 2009. "Copper isotope fractionation in acid mine drainage." Geochimica et Cosmochimica Acta 73(5):1247-1263. Abstract We surveyed the Cu isotopic composition of primary minerals and stream water affected by acid mine drainage in a mineralized watershed located in southwestern Colorado, USA. The 65Cu values (based on 65Cu/63Cu) of local enargite (65Cu = -0.01 ± 0.10‰) and chalcopyrite (65Cu = 0.16 ± 0.10‰) are within the general range of previously reported values for terrestrial primary Cu sulfides (-1 < 65Cu < 1). These mineral samples show lower 65Cu values than stream waters (65Cu = 1.36 - 1.74 ± 0.10‰), with an average isotopic fractionation (quantified as ∆aq-mino = 65Cuaq – 65Cu mino, where Cuaq is leached Cu and Cu mino is the original mineral) of 1.60 ± 0.14‰ and 1.43 ± 0.14‰ for enargite and chalcopyrite, respectively. To interpret this field survey, we simulated enargite and chalcopyrite leaching in batch experiments and found that, as in the field, leached Cuaq is isotopically enriched relative to primary minerals when microorganisms are absent (average ∆aq-min = 0.94 ± 0.14‰ for enargite, 1.18 ± 0.14‰ for chalcopyrite). Leaching of minerals in the presence of A. ferrooxidans results in smaller average fractionation in the opposite direction for chalcopyrite (∆aq-min = -0.57 ± 0.14‰) and no apparent fractionation for enargite (∆aq-min = 0.10 ± 0.14‰). The isotope effect during release of Cu from leaching minerals is inferred to be the same under both abiotic and biotic conditions. However, preferential association of isotopically enriched Cuaq with A. ferrooxidans cells, observed under TEM to occur as both localized precipitates around cells and Cu inside cells, is inferred to cause isotopic depletion of Cuaq in biotic experiments relative to abiotic experiments. Our results show indications of isotopic signatures of both abiotic chalcopyrite and enargite dissolution. Such signatures will be useful for AMD remediation and ore prospecting purposes.

Marshall MJ, A Dohnalkova, DW Kennedy, AE Plymale, SH Thomas, FE Loffler, R Sanford, JM Zachara, JK Fredrickson, and AS Beliaev. 2009. "Electron donor-dependent radionuclide reduction and nanoparticle formation by Anaeromyxobacter dehalogenans strain 2CP-C." Environmental Microbiology 11(2):534-543. Abstract Anaeromyxobacter dehalogenans strain 2CP-C can rapidly reduce U(VI) or Tc(VII) to U(IV)O2(s) or Tc(IV)O2(S) using either acetate or H2 as an electron donor source. Kinetic studies reveal that the H2-driven reduction of either U(VI) or Tc(VII) is faster than the acetate-driven reduction. The sub-cellular localization of reduced UO2 is extracellular while TcO2 nanoparticles are both periplasmic and extracellular. While electron donor-specific differences in UO2 nanoparticle aggregate size were observed, the association of UO2 nanoparticles with an exopolymeric substance (EPS) was observed and found to be independent of electron donor source. Electron donor-specific localization differences were not observed in cells incubated with Tc(VII). These finding have direct implications on immobilization strategies for highly soluble radionuclide contaminants in subsurface waters and the development of microbially assisted biostimulation efforts.

Burgos WD, J McDonough, JM Senko, G Zhang, A Dohnalkova, SD Kelly, YA Gorby, and KM Kemner. 2008. "Characterization of uraninite nanoparticles produced by Shewanella oneidensis MR-1 ." Geochimica et Cosmochimica Acta 72(20):4901-4915. doi:10.1016/j.gca.2008.07.016 Abstract The reduction of uranium(VI) by Shewanella oneidensis MR-1 was studied to examine the effects of bioreduction kinetics and background electrolyte on the physical properties and reactivity to re-oxidation of the biogenic uraninite, UO2(s). Bioreduction experiments were conducted with uranyl acetate as the electron acceptor and sodium lactate as the electron donor under resting cell conditions in a 30 mM NaHCO3 buffer, and in a PIPES-buffered artificial groundwater (PBAGW). MR-1 was cultured in batch mode in a defined minimal medium with a specified air-to-medium volume ratio such that electron acceptor (O2) limiting conditions were reached just when cells were harvested for subsequent experiments. The rate of U(VI) bioreduction was manipulated by varying the cell density and the incubation temperature (1.0 _ 108 cell ml_1 at 20 _C or 2.0 _ 108 cell ml_1 at 37 _C) to generate U(IV) solids at ‘‘fast” and ‘‘slow” rates in the two different buffers. The presence of Ca in PBAGW buffer altered U(VI) speciation and solubility, and significantly decreased U(VI) bioreduction kinetics. High resolution transmission electron microscopy was used to measure uraninite particle size distributions produced under the four different conditions. The most common primary particle size was 2.9–3.0 nm regardless of U(VI) bioreduction rate or background electrolyte. Extended X-ray absorption fine-structure spectroscopy was also used to estimate uraninite particle size and was consistent with TEM results. The reactivity of the biogenic uraninite products with dissolved oxygen was tested, and neither U(VI) bioreduction rate nor background electrolyte had any statistical effect on oxidation rates. With MR-1, uraninite particle size was not controlled by the bioreduction rate of U(VI) or the background electrolyte. These results for MR-1, where U(VI) bioreduction rate had no discernible effect on uraninite particle size or oxidation rate, contrast with our recent research with Shewanella putrefaciens CN32, where U(VI) bioreduction rate strongly influenced both uraninite particle size and oxidation rate. These two studies with Shewanella species can be viewed as consistent if one assumes that particle size controls oxidation rates, so the similar uraninite particle sizes produced by MR-1 regardless of U(VI) bioreduction rate would result in similar oxidation rates. Factors that might explain why U(VI) bioreduction rate was an important control on uraninite particle size for CN32 but not for MR-1 are discussed.

Marshall MJ, AE Plymale, DW Kennedy, L Shi, Z Wang, SB Reed, A Dohnalkova, CJ Simonson, C Liu, D Saffarini, MF Romine, JM Zachara, AS Beliaev, and JK Fredrickson. 2008. "Hydrogenase- and Outer Membrane c-Type Cytochrome-Facilitated Reduction of Technetium(VII) by Shewanella oneidensis MR-1." Environmental Microbiology 10(1):125-136. doi:10.1111/j.1462-2920.2007.01438.x Abstract Pertechnetate, 99Tc(VII)O4-, is a highly mobile radionuclide contaminant at U.S. Department of Energy sites that can be enzymatically reduced by a range of anaerobic and facultatively anaerobic microorganisms, including Shewanella oneidensis MR-1, to poorly soluble Tc(IV)O2(s). In other microorganisms, Tc(VII)O4- reduction is generally considered to be catalyzed by hydrogenase. Here, we provide evidence that although the NiFe hydrogenase of MR-1 was involved in the H2-driven reduction of Tc(VII)O4- (presumably through a direct coupling of H2 oxidation and Tc(VII) reduction), the deletion of both hydrogenase genes did not completely eliminate the ability of MR-1 to reduce Tc(VII). With lactate as the electron donor, mutants lacking the outer membrane c-type cytochromes MtrC and OmcA or the proteins required for the maturation of c-type cytochromes were defective in reducing Tc(VII) to nanoparticulate TcO2·nH2O(s) relative to MR-1 or a NiFe hydrogenase mutant. In addition, reduced MtrC and OmcA were oxidized by Tc(VII)O4-, confirming the capacity for direct electron transfer from these OMCs to TcO4-. c-Type cytochrome-catalyzed Tc(VII) reduction could be a potentially important mechanism in environments where organic electron donor concentrations are sufficient to allow this reaction to dominate.

Puzon GJ, YC Huang, A Dohnalkova, and L Xun. 2008. "Isolation and characterization of an NAD+-degrading bacterium PTX1 and its role in chromium biogeochemical cycle." Biodegradation 19(3):417-424. doi:10.1007/s10532-007-9147-1 Abstract Microorganisms can reduce toxic chromate to less toxic trivalent chromium [Cr(III)]. Besides Cr(OH)3 precipitates, some soluble organo-Cr(III) complexes are readily formed upon microbial, enzymatic, and chemical reduction of chromate. However, the biotransformation of the organo-Cr(III) complexes has not been characterized. We have previously reported the formation of a nicotinamide adenine dinucleotide (NAD+)-Cr(III) complex after enzymatic reduction of chromate. Although the NAD+-Cr(III) complex was stable under sterile conditions, microbial cells were identified as precipitates in a non-sterile NAD+-Cr(III) solution after extended incubation. The most dominant bacterium PTX1 was isolated and assigned to Leifsonia genus by phylogenetic analysis of 16S rRNA gene sequence. PTX1 grew slowly on NAD+ with a doubling time of 17 h, and even more slowly on the NAD+-Cr(III) complex with an estimated doubling time of 35 days. The slow growth suggests that PTX1 passively grew on trace NAD+ dissociated from the NAD+-Cr(III) complex, facilitating further dissociation of the complex and formation of Cr(III) precipitates. Thus, organo-Cr(III) complexes might be an intrinsic link of the chromium biogeochemical cycle; they can be produced during chromate reduction and then further mineralized by microorganisms.

Sani R, BM Peyton, and A Dohnalkova. 2008. "Comparison of uranium(VI) removal by Shewanella oneidensis MR-1 in flow and batch reactors." Water Research 42(12):2993-3002. doi:10.1016/j.watres.2008.04.003 Abstract To better understand the interactions among metal contaminants, nutrients, and microorganisms in subsurface under fracture-flow conditions, iron-reducing biofilms (pure cultures of Shewanella oneidensis MR-1) were grown in six fracture flow reactors (FFRs) of different geometries. The spatial and temporal distribution of nutrients, contaminant, and bacteria were examined using a tracer dye (brilliant blue FCF) and microscopy. The results showed that plugging by bacterial cells depended on the geometry of the reactor; and iron-reducing biofilms grown in FFRs had a definite U(VI)-reduction capacity. To find out the U(VI)-reduction capacity of iron-reducing biofilms, batch experiments of U(VI) reduction were performed in repetitive addition mode. U(VI)-reduction rates of stationary phase grown iron-reducing cultures with and without spent medium decreased after each U(VI) addition. At the end of the fourth U(VI)-addition, stationary phase iron-reducing cultures treated with U(VI) with and without spent medium yielded grey and black precipitates, respectively. These grey and black U precipitates were analyzed using High Resolution-Transmission Electron Microscopy, Energy-dispersive X-ray spectroscopy, and X-ray diffraction. Data for randomly selected area of black and grey U precipitates showed that reduced U particles (3-6 nm) were crystalline and amorphous in nature, respectively. This information obtained in this study could be used to develop substrate addition strategies for metal immobilization in subsurface fracture flow systems.

Shi L, S Deng, MJ Marshall, Z Wang, DW Kennedy, A Dohnalkova, HM Mottaz, EA Hill, YA Gorby, AS Beliaev, DJ Richardson, JM Zachara, and JK Fredrickson. 2008. " Direct Involvement of Type II Secretion System in Extracellular Translocation of Shewanella Oneidensis Outer Membrane Cytochromes MtrC and OmcA." Journal of Bacteriology 190(15):5512-5516. doi:10.1128/JB.00514-08 Abstract Outer membrane decaheme c-type cytochromes MtrC and OmcA of Shewanella oneidensis MR-1 are extracellular lipoproteins important for dissimilatory reduction of solid metal (hydr)oxides during anaerobic respiration. To investigate the roles of type II secretion system (T2S) in translocation of MtrC and OmcA across outer membrane, we measured the effects of deleting two T2S genes, gspD and gspG, on the secretion of MtrC and OmcA when cells were grown under anaerobic conditions. Deletion of gspD or gspG resulted in slightly yellowish supernatants, different from the pink supernatant of wild type (wt). Comparative proteomic analyses revealed that, although MtrC, OmcA and NrfA, a periplasmic nitrite reductase, were present the supernatants of wt and ΔgspD mutant, their peptides counts were much lower in ΔgspD than in wt. Subsequent analyses with heme-staining and Western blot not only confirmed that deletion of gspD or gspG reduced the abundances of MtrC and OmcA in the supernatants, but also revealed that the deletions consequently increased their abundances inside the cells. Complementation of ΔgspG mutant with functional GspG could reverse the effects of deleting gspG on the colors of the supernatants and the abundances of MtrC and OmcA. In contrast, Western results showed that the abundance of NrfA was reduced in the supernatant and the cells of ΔgspD mutant, suggesting that reduced NrfA in the periplasm, where MtrC and OmcA were accumulated, contributed to its reduction in the supernatant. Thus, our results demonstrate at the first time that T2S facilitates translocation of MtrC and OmcA across outer membrane.

Flaherty DW, Z Dohnalek, A Dohnalkova, BW Arey, DE McCready, N Ponnusany, CB Mullins, and BD Kay. 2007. "Reactive Ballistic Deposition of Porous TiO2 Films: Growth and Characterization." Journal of Physical Chemistry C 111(12):4765-4773. doi:10.1021/jp067641m Abstract Nanoporous, high-surface area films of TiO2 are synthesized by reactive ballistic deposition of titanium metal in an oxygen ambient. Auger electron spectroscopy (AES) is used to investigate the stoichiometric dependence of the films on growth conditions (surface temperature and partial pressure of oxygen). Scanning and transmission electron microscopy show that the films consist of arrays of separated filaments. The surface area and the distribution of binding site energies of the films are measured as functions of growth temperature, deposition angle, and annealing conditions using temperature programmed desorption (TPD) of N2. TiO2 films deposited at 50 K at 70º from substrate normal display the greatest specific surface area of ~100 m2/g. In addition, the films retain greater than 70% of their original surface area after annealing to 600 K. The combination of high surface area and thermal stability suggest that these films could serve as supports for applications in heterogeneous catalysis.

Kim MI, J Kim, J Lee, H Jia, HB Na, J Youn, JH Kwak, A Dohnalkova, JW Grate, P Wang, T Hyeon, HG Park, and HN Chang. 2007. "Crosslinked Enzyme Aggregates in Hierarchically-Ordered Mesoporous Silica: A Simple and Effective Method for Enzyme Stabilization." Biotechnology and Bioenegineering 96(2):210-218. doi:10.1002/bit.21107 Abstract alpha-chymotrypsin (CT) and lipase (LP) were immobilized in hierarchically-ordered mesocellular mesoporous silica (HMMS) in a simple but effective way for the enzyme stabilization, which was achieved by the enzyme adsorption followed by glutaraldehyde (GA) crosslinking. This resulted in the formation of nanometer scale crosslinked enzyme aggregates (CLEAs) entrapped in the mesocellular pores of HMMS (37 nm), which did not leach out of HMMS through narrow mesoporous channels (13 nm). CLEA of alpha-chymotrypsin (CLEA-CT) in HMMS showed a high enzyme loading capacity and significantly increased enzyme stability. No activity decrease of CLEA-CT was observed for two weeks under even rigorously shaking condition, while adsorbed CT in HMMS and free CT showed a rapid inactivation due to the enzyme leaching and presumably autolysis, respectively. With the CLEA-CT in HMMS, however, there was no tryptic digestion observed suggesting that the CLEA-CT is not susceptible to autolysis. Moreover, CLEA of lipase (CLEA-LP) in HMMS retained 30% specific activity of free lipase with greatly enhanced stability. This work demonstrates that HMMS can be efficiently employed as host materials for enzyme immobilization leading to highly enhanced stability of the immobilized enzymes with high enzyme loading and activity.

Ledbetter RN, SA Connon, AL Neal, A Dohnalkova, and TS Magnuson. 2007. "Biogenic mineral production by a novel arsenic-metabolizing thermophilic bacterium from the Alvord Basin, OR." Applied and Environmental Microbiology 73(18):5928-5936. doi:10.1128/AEM.00371-07 Abstract The Alvord Basin in southeast Oregon, USA contains a variety of hydrothermal features, which have never been microbiologically characterized. Murky Pot (61°C, pH 7.1) was selected for this study. Sampling of Murky Pot led to the isolation of a novel arsenic-metabolizing organism (YeAs), which produces an arsenic sulfide mineral known as beta-realgar, a mineral that has not previously been observed as a product of bacterial arsenic metabolism. Our goal was to characterize and identify YeAs based on its phylogenetic, physiological, and morphological characteristics. 16S rRNA gene analysis revealed that YeAs has 98.9% sequence similarity to that of Thermobrachium celere. YeAs was grown on a freshwater medium and could utilize a variety of organic substrates, particularly carbohydrates and organic acids. Optimum growth of the organism was seen at 55ºC, but showed growth at a range of 37° to 75°C. No growth was observed when YeAs was grown under aerobic conditions. Microscopic examination revealed Gram-indeterminate, non-spore forming, rod shaped cells. Electron microscopy and elemental analysis revealed significant arsenic sulfide mineralization of cell walls, and extracellular particulate deposition of arsenic sulfide minerals. YeAs showed no detectable respiratory arsenate reductase; however, the organism did display significant detoxification arsenate reductase activity. The phylogenetic, physiological, and morphological characteristics of YeAs demonstrate that it is an anaerobic, moderately thermophilic, arsenic-reducing bacterium. This organism and its associated metabolism could have major implications in the search for innovative methods for arsenic waste management and in the search for novel biogenic signatures.