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Item Genome sequence, phylogenetic analysis, and structure-based annotation reveal metabolic potential of Chlorella sp. SLA-04(Elsevier BV, 2023-01) Goemann, Calvin L.C.; Wilkinson, Royce; Henriques, William; Bui, Huyen; Goemann, Hannah M.; Carlson, Ross P.; Viamajala, Sridhar; Gerlach, Robin; Wiedenheft, BlakeAlgae are a broad class of photosynthetic eukaryotes that are phylogenetically and physiologically diverse. Most of the phylogenetic diversity has been inferred from 18S rDNA sequencing since there are only a few complete genomes available in public databases. Here we use ultra-long-read Nanopore sequencing to determine a gapless, telomere-to-telomere complete genome sequence of Chlorella sp. SLA-04, previously described as Chlorella sorokiniana SLA-04. Chlorella sp. SLA-04 is a green alga that grows to high cell density in a wide variety of environments – high and neutral pH, high and low alkalinity, and high and low salinity. SLA-04's ability to grow in high pH and high alkalinity media without external CO2 supply is favorable for large-scale algal biomass production. Phylogenetic analysis performed using ribosomal DNA and conserved protein sequences consistently reveal that Chlorella sp. SLA-04 forms a distinct lineage from other strains of Chlorella sorokiniana. We complement traditional genome annotation methods with high throughput structural predictions and demonstrate that this approach expands functional prediction of the SLA-04 proteome. Genomic analysis of the SLA-04 genome identifies the genes capable of utilizing TCA cycle intermediates to replenish cytosolic acetyl-CoA pools for lipid production. We also identify a complete metabolic pathway for sphingolipid anabolism that may allow SLA-04 to readily adapt to changing environmental conditions and facilitate robust cultivation in mass production systems. Collectively, this work clarifies the phylogeny of Chlorella sp. SLA-04 within Trebouxiophyceae and demonstrates how structural predictions can be used to improve annotation beyond sequence-based 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 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 Identification and characterization of a novel member of the radical AdoMet enzyme superfamily and implications for the biosynthesis of the Hmd hydrogenase active site cofactor(2009-11) McGlynn, Shawn E.; Boyd, Eric S.; Shepard, Eric M.; Lange, Rachel K.; Gerlach, Robin; Broderick, Joan B.; Peters, John W.The genetic context, phylogeny, and biochemistry of a gene flanking the H2-forming methylene-H4-methanopterin dehydrogenase gene (hmdA), here designated hmdB, indicate that it is a new member of the radical S-adenosylmethionine enzyme superfamily. In contrast to the characteristic CX3CX2C or CX2CX4C motif defining this family, HmdB contains a unique CX5CX2C motif.Item Aerobic degradation of 2,4,6-trinitrotoluene by the yeast strain Geotrichum candidum AN-Z4(2010-04) Ziganshin, Ayrat M.; Gerlach, Robin; Naumenko, E. A.; Naumova, R. P.The yeast strain Geotrichum candidum AN-Z4 isolated from an anthropogenically polluted site was able to transform 2,4,6-trinitrotoluene (TNT) via the formation of unstable intermediate hydride Meisenheimer complexes with their subsequent destruction and accumulation of nitrite and nitrate ions as the end mineral forms of nitrogen. Aeration of the medium promoted more profound destruction of this xenobiotic by the strain G. candidum AN-Z4 than static conditions. The yeast strain was shown to produce citrate, succinate, and isocitrate, which sharply acidified the medium and influenced the TNT destruction. Two possible pathways of TNT biodegradation were confirmed experimentally: (1) via the destruction of the TNT-monohydride complex (3-H−-TNT) and (2) via the destruction of one protonated TNT-dihydride complex (3,5-2H−-TNT · H+). The strain G. candidum AN-Z4, due to its ability for TNT degradation, may be promising for bioremediation of TNT-contaminated soil and water.Item Microbially enhanced carbon capture and storage by mineral-trapping and solubility-trapping(2010-07) Mitchell, Andrew C.; Dideriksen, K.; Spangler, Lee H.; Cunningham, Alfred B.; Gerlach, RobinThe potential of microorganisms for enhancing carbon capture and storage (CCS) via mineral-trapping (where dissolved CO2 is precipitated in carbonate minerals) and solubility trapping (as dissolved carbonate species in solution) was investigated. The bacterial hydrolysis of urea (ureolysis) was investigated in microcosms including synthetic brine (SB) mimicking a prospective deep subsurface CCS site with variable headspace pressures [p(CO2)] of 13C-CO2. Dissolved Ca2+ in the SB was completely precipitated as calcite during microbially induced hydrolysis of 5-20 g L-1 urea. The incorporation of carbonate ions from 13C-CO2 (13C-CO32-) into calcite increased with increasing p(13CO2) and increasing urea concentrations: from 8.3% of total carbon in CaCO3 at 1 g L-1 to 31% at 5 g L-1, and 37% at 20 g L-1. This demonstrated that ureolysis was effective at precipitating initially gaseous [CO2(g)] originating from the headspace over the brine. Modeling the change in brine chemistry and carbonate precipitation after equilibration with the initial p(CO2) demonstrated that no net precipitation of CO2(g) via mineral-trapping occurred, since urea hydrolysis results in the production of dissolved inorganic carbon. However, the pH increase induced by bacterial ureolysis generated a net flux of CO2(g) into the brine. This reduced the headspace concentration of CO2 by up to 32 mM per 100 mM urea hydrolyzed because the capacity of the brine for carbonate ions was increased, thus enhancing the solubility-trapping capacity of the brine. Together with the previously demonstrated permeability reduction of rock cores at high pressure by microbial biofilms and resilience of biofilms to supercritical CO2, this suggests that engineered biomineralizing biofilms may enhance CCS via solubility-trapping, mineral formation, and CO2(g) leakage reduction.Item Modeling biofilm growth in the presence of carbon dioxide and water flow in the subsurface(2010-07) Ebigbo, Anozie; Helmig, Rainer; Cunningham, Alfred B.; Class, Holger; Gerlach, RobinThe concentration of greenhouse gases—particularly carbon dioxide (CO2)—in the atmosphere has been on the rise in the past decades. One of the methods which have been proposed to help reduce anthropogenic CO2 emissions is the capture of CO2 from large, stationary point sources and storage in deep geological formations. The caprock is an impermeable geological layer which prevents the leakage of stored CO2, and its integrity is of utmost importance for storage security. Due to the high pressure build-up during injection, the caprock in the vicinity of the well is particularly at risk of fracturing. Biofilms could be used as biobarriers which help prevent the leakage of CO2 through the caprock in injection well vicinity by blocking leakage pathways. The biofilm could also protect well cement from corrosion by CO2-rich brine.The goal of this paper is to develop and test a numerical model which is capable of simulating the development of a biofilm in a CO2 storage reservoir. This involves the description of the growth of the biofilm, flow and transport in the geological formation, and the interaction between the biofilm and the flow processes. Important processes which are accounted for in the model include the effect of biofilm growth on the permeability of the formation, the hazardous effect of supercritical CO2 on suspended and attached bacteria, attachment and detachment of biomass, and two-phase fluid flow processes. The model is tested by comparing simulation results to experimental data.Item Imaging biologically induced mineralization in fully hydrated flow systems(2011) Schultz, Logan N.; Pitts, Betsey; Mitchell, Andrew C.; Cunningham, Alfred B.; Gerlach, RobinA number of proposed technologies involve the controlled implementation of biologically induced carbonate mineral precipitation in the geologic subsurface. Examples include the enhancement of soil stability [1], immobilization of groundwater contaminants such as strontium and uranium [2], and the enhancement of oil recovery and geologic carbon sequestration via controlled permeability reduction [3]. The most significant challenge in these technologies remains to identify and better understand an industrially, environmentally, and economically viable carbonate precipitation route.One of the most promising routes is ureolytic biomineralization, because of the ample availability of urea and the controllable reaction rate. In this process, ureolytic bacteria hydrolyze urea, leading to an increase in pH. In the presence of calcium, this process favors the formation of solid calcium carbonate, as illustrated in the following equations:CO(NH2)2 + H2O → NH2COOH + NH3→ 2 NH3 + CO2 (Urea hydrolysis) (1)2 NH3 + 2 H2O ↔ 2NH4+ + 2OH– (pH increase) (2)CO2 + 2 OH– ↔ CO32– + H2O(Carbonate ion formation) (3)CO32– + Ca2+ ↔ CaCO3 (solid)(Precipitation is favored at high pH) (4)This process relies on molecular-level chemical and biological processes that must be better understood for large-scale implementation.Researchers at the Center for Biofilm Engineering at Montana State University (USA) and Aberystwyth University (UK) have conducted several biomineralization experiments in simulated porous media reactors. Microscopy has proven to be one of the most useful analytical tools in these studies, providing the ability to non-invasively visualize, differentiate, and quantify the various components, including the cells, cell matrix, and mineral precipitates. Because of the possibility of real-time observation and the lack of dehydration artifacts, microscopy has been tremendously useful for elucidating the temporal and spatial relationships of these components.Item Uranium exerts acute toxicity by binding to pyrroloquinoline quinone cofactor(2010-12) VanEngelen, Michael R.; Szilagyi, Robert K.; Gerlach, Robin; Lee, Brady D.; Apel, William A.; Peyton, Brent M.Uranium as an environmental contaminant has been shown to be toxic to eukaryotes and prokaryotes; however, no specific mechanisms of uranium toxicity have been proposed so far. Here a combination of in vivo, in vitro, and in silico studies are presented describing direct inhibition of pyrroloquinoline quinone (PQQ)-dependent growth and metabolism by uranyl cations. Electrospray-ionization mass spectroscopy, UV-vis optical spectroscopy, competitive Ca2+/uranyl binding studies, relevant crystal structures, and molecular modeling unequivocally indicate the preferred binding of uranyl simultaneously to the carboxyl oxygen, pyridine nitrogen, and quinone oxygen of the PQQmolecule. The observed toxicity patterns are consistent with the biotic ligand model of acute metal toxicity. In addition to the environmental implications, this work represents the first proposed molecular mechanism of uranium toxicity in bacteria, and has relevance for uranium toxicity in many living systems.