Final Technical Report

The biochemistry of bacterial proteins involved in redox transformations of metals and minerals is, without dispute, an important area of research. Nevertheless, most studies on bacterial metal transformation have focused not on biochemistry but on genetics and genomics. The objective of this research is to better understand the role of conformation change in electron transfer from cytochromes to minerals, a process that underpins respiratory metal reduction by bacteria in nature and in bioremediation strategies, including reductive immobilization of radioactive contaminants. Our DOE-funded work is specifically focused on answering long-standing questions about the biochemical behavior of these very interesting proteins, and our findings thus far have already made impacts in the fields of environmental microbiology and biogeochemistry. Among the key findings from the project are 1) Successful large-scale production of biomass for protein isolation; 2) Purification of several c-type cytochromes for biochemical study; 3) Characterization of these proteins using spectrophotometric and electrochemical techniques; 4) Examination of protein conformational change and redox activity towards metal oxides using a small mass cytochrome c from Acidiphilium cryptum; 5) Proteomic characterization of A. cryptum biofilms; 6) Training of 2 undergraduate research assistants; 7) Publications and several meeting presentations

Metal Transformation by a Novel Pelosinus Isolate From a Subsurface Environment

The capability of microorganisms to alter metal speciation offers potential for the development of new strategies for immobilization of toxic metals in the environment. A metal-reducing microbe, “Pelosinus lilae” strain UFO1, was isolated under strictly anaerobic conditions from an Fe(III)-reducing enrichment established with uncontaminated soil from the Department of Energy Oak Ridge Field Research Center, Tennessee. “P. lilae” UFO1 is a rod-shaped, spore-forming, and Gram-variable anaerobe with a fermentative metabolism. It is capable of reducing the humic acid analog anthraquinone-2,6-disulfonate (AQDS) using a variety of fermentable substrates and H2. Reduction of Fe(III)-nitrilotriacetic acid occurred in the presence of lactate as carbon and electron donor. Ferrihydrite was not reduced in the absence of AQDS. Nearly complete reduction of 1, 3, and 5 ppm Cr(VI) occurred within 24 h in suspensions containing 108 cells mL−1 when provided with 10 mM lactate; when 1 mM AQDS was added, 3 and 5 ppm Cr(VI) were reduced to 0.1 ppm within 2 h. Strain UFO1 is a novel species within the bacterial genus Pelosinus, having 98.16% 16S rRNA gene sequence similarity with the most closely related described species, Pelosinus fermentans R7T. The G+C content of the genomic DNA was 38 mol%, and DNA-DNA hybridization of “P. lilae” UFO1 against P. fermentans R7T indicated an average 16.8% DNA-DNA similarity. The unique phylogenetic, physiologic, and metal-transforming characteristics of “P. lilae” UFO1 reveal it is a novel isolate of the described genus Pelosinus

Influence of ocean–atmospheric oscillations on lake ice phenology in eastern North America

Our results reveal long-term trends in ice out dates (1836–2013) for twelve lakes in Maine, New Brunswick and New Hampshire, in eastern North America. The trends are remarkably coherent between lakes (rs = 0.462–0.933, p < 0.01) and correlate closely with the March–April (MA) instrumental temperature records from the region (rs = 0.488–0.816, p < 0.01). This correlation permits use of ice out dates as a proxy to extend the shorter MA instrumental record (1876–2013). Mean ice out dates trended progressively earlier during the recovery from the Little Ice Age through to the 1940s, and gradually became later again through to the late 1970s, when ice out dates had returned to values more typical of the late nineteenth century. Post-1970’s ice out dates resumed trending toward earlier dates, with the twenty-first century being characterized by the earliest ice out dates on record. Spectral and wavelet time series analysis indicate that ice out is influenced by several teleconnections including the Quasi-biennial Oscillation, El Niño-Southern Oscillation, North Atlantic Oscillation, as well as a significant correlation between inland lake records and the Atlantic Multidecadal Oscillation. The relative influence of these teleconnections is variable with notable shifts occurring after ~1870, ~1925, and ~1980–2000. The intermittent expression of these cycles in the ice out and MA instrumental record is not only influenced by absolute changes in the intensity of the various teleconnections and other climate drivers, but through phase interference between teleconnections, which periodically damps the various signals

Review of the algal biology program within the National Alliance for Advanced Biofuels and Bioproducts

In 2010,when the National Alliance for Advanced Biofuels and Bioproducts (NAABB) consortiumbegan, littlewas known about themolecular basis of algal biomass or oil production. Very fewalgal genome sequenceswere available and efforts to identify the best-producing wild species through bioprospecting approaches had largely stalled after the U.S. Department of Energy\u27s Aquatic Species Program. This lack of knowledge included how reduced carbon was partitioned into storage products like triglycerides or starch and the role played bymetabolite remodeling in the accumulation of energy-dense storage products. Furthermore, genetic transformation and metabolic engineering approaches to improve algal biomass and oil yields were in their infancy. Genome sequencing and transcriptional profiling were becoming less expensive, however; and the tools to annotate gene expression profiles under various growth and engineered conditions were just starting to be developed for algae. It was in this context that an integrated algal biology program was introduced in the NAABB to address the greatest constraints limiting algal biomass yield. This review describes the NAABB algal biology program, including hypotheses, research objectives, and strategies to move algal biology research into the twenty-first century and to realize the greatest potential of algae biomass systems to produce biofuels

Final Technical Report

The biochemistry of bacterial proteins involved in redox transformations of metals and minerals is, without dispute, an important area of research. Nevertheless, most studies on bacterial metal transformation have focused not on biochemistry but on genetics and genomics. The objective of this research is to better understand the role of conformation change in electron transfer from cytochromes to minerals, a process that underpins respiratory metal reduction by bacteria in nature and in bioremediation strategies, including reductive immobilization of radioactive contaminants. Our DOE-funded work is specifically focused on answering long-standing questions about the biochemical behavior of these very interesting proteins, and our findings thus far have already made impacts in the fields of environmental microbiology and biogeochemistry. Among the key findings from the project are 1) Successful large-scale production of biomass for protein isolation; 2) Purification of several c-type cytochromes for biochemical study; 3) Characterization of these proteins using spectrophotometric and electrochemical techniques; 4) Examination of protein conformational change and redox activity towards metal oxides using a small mass cytochrome c from Acidiphilium cryptum; 5) Proteomic characterization of A. cryptum biofilms; 6) Training of 2 undergraduate research assistants; 7) Publications and several meeting presentations

Evidence for multiple modes of uranium immobilization by an anaerobic bacterium

Microbial reduction of hexavalent uranium has been studied widely for its potential role in bioremediation and immobilization of soluble U(VI) in contaminated groundwater. More recently, some microorganisms have been examined for their role in immobilization of U(VI) via precipitation of uranyl phosphate minerals mediated by microbial phosphate release, alleviating the requirement for long-term redox control. Here, we investigated the mechanism of U(VI) removal mediated by an environmental isolate, strain UFO1, that is indigenous to the Field Research Center (FRC) in Oak Ridge, TN and has been detected in U(VI)- contaminated sediments. Changes in U(VI) speciation were examined in the presence and absence of the electron-shuttling moiety, anthraquinone-2,6-disulfonate (AQDS). Cell suspensions were capable of nearly complete removal of 100 µM U(VI) from solution within 48 h; U(VI) removal was not dependent on the presence of an exogenous electron donor or AQDS, although AQDS increased the rate of U(VI) removal. X-ray Absorption Near Edge Structure (XANES) and Extended X-ray Absorption Fine Structure (EXAFS) spectroscopic measurements indicated that U(IV) was the predominant oxidation state of uranium in cell suspensions in both the absence and presence of 100 µM AQDS. Interestingly, 17% of the cell-associated precipitates in a U(VI)-treated suspension that lacked AQDS had spectral characteristics consistent with a uranyl phosphate solid phase. The potential involvement of phosphate was consistent with observed increases in soluble phosphate concentrations over time in UFO1 cell suspensions, which suggested phosphate liberation from the cells. TEM-EDS confirmed the presence of uranyl phosphate with a U:P ratio consistent with autunite (1:1). EXAFS analyses further suggested that U (IV) was bound to low-Z neighbors such asC or P, inferred to be present as functional groups on biomass. These results suggest that strain UFO1 has the ability to facilitate U (VI) removal from solution via reductive and phosphate precipitation mechanisms. Both mechanisms offer potential for the remediation of U-contaminated sediments at the FRC or elsewhere

Biogenic Mineral Production by a Novel Arsenic-Metabolizing Thermophilic Bacterium from the Alvord Basin, Oregon▿

The Alvord Basin in southeast Oregon contains a variety of hydrothermal features which have never been microbiologically characterized. A sampling of Murky Pot (61°C; pH 7.1) led to the isolation of a novel arsenic-metabolizing organism (YeAs) which produces an arsenic sulfide mineral known as β-realgar, a mineral that has not previously been observed as a product of bacterial arsenic metabolism. YeAs was grown on a freshwater medium and utilized a variety of organic substrates, particularly carbohydrates and organic acids. The temperature range for growth was 37 to 75°C (optimum, 55°C), and the pH range for growth was 6.0 to 8.0 (optimum, pH 7.0 to 7.5). No growth was observed when YeAs was grown under aerobic conditions. The doubling time when the organism was grown with yeast extract and As(V) was 0.71 h. Microscopic examination revealed Gram stain-indeterminate, non-spore-forming, nonmotile, rod-shaped cells, with dimensions ranging from 0.1 to 0.2 μm wide by 3 to 10 μm long. Arsenic sulfide mineralization of cell walls and extracellular arsenic sulfide particulate deposition were observed with electron microscopy and elemental analysis. 16S rRNA gene analysis placed YeAs in the family Clostridiaceae and indicated that the organism is most closely related to the Caloramator and Thermobrachium species. The G+C content was 35%. YeAs showed no detectable respiratory arsenate reductase but 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 mineral signatures