@article {3085, title = {Potential for Methanosarcina to Contribute to Uranium Reduction during Acetate-Promoted Groundwater Bioremediation.}, journal = {Microb Ecol}, volume = {76}, year = {2018}, month = {2018 Oct}, pages = {660-667}, abstract = {

Previous studies of acetate-promoted bioremediation of uranium-contaminated aquifers focused on Geobacter because no other microorganisms that can couple the oxidation of acetate with U(VI) reduction had been detected in situ. Monitoring the levels of methyl CoM reductase subunit A (mcrA) transcripts during an acetate-injection field experiment demonstrated that acetoclastic methanogens from the genus Methanosarcina were enriched after 40~days of acetate amendment. The increased abundance of Methanosarcina corresponded with an accumulation of methane in the groundwater. In order to determine whether Methanosarcina species could be participating in U(VI) reduction in the subsurface, cell suspensions of Methanosarcina barkeri were incubated in the presence of U(VI) with acetate provided as the electron donor. U(VI) was reduced by metabolically active M. barkeri cells; however, no U(VI) reduction was observed in inactive controls. These results demonstrate that Methanosarcina species could play an important role in the long-term bioremediation of uranium-contaminated aquifers after depletion of Fe(III) oxides limits the growth of Geobacter species. The results also suggest that Methanosarcina have the potential to influence uranium geochemistry in a diversity of anaerobic sedimentary environments.

}, keywords = {Acetates, Biodegradation, Environmental, Geobacter, Groundwater, Methane, Methanosarcina, Oxidation-Reduction, Uranium, Water Pollutants, Chemical}, issn = {1432-184X}, doi = {10.1007/s00248-018-1165-5}, author = {Holmes, Dawn E and Orelana, Roberto and Giloteaux, Ludovic and Wang, Li-Ying and Shrestha, Pravin and Williams, Kenneth and Lovley, Derek R and Rotaru, Amelia-Elena} } @article {3106, title = {Functional environmental proteomics: elucidating the role of a c-type cytochrome abundant during uranium bioremediation.}, journal = {ISME J}, volume = {10}, year = {2016}, month = {2016 Feb}, pages = {310-20}, abstract = {

Studies with pure cultures of dissimilatory metal-reducing microorganisms have demonstrated that outer-surface c-type cytochromes are important electron transfer agents for the reduction of metals, but previous environmental proteomic studies have typically not recovered cytochrome sequences from subsurface environments in which metal reduction is important. Gel-separation, heme-staining and mass spectrometry of proteins in groundwater from in situ uranium bioremediation experiments identified a putative c-type cytochrome, designated Geobacter subsurface c-type cytochrome A (GscA), encoded within the genome of strain M18, a Geobacter isolate previously recovered from the site. Homologs of GscA were identified in the genomes of other Geobacter isolates in the phylogenetic cluster known as subsurface clade 1, which predominates in a diversity of Fe(III)-reducing subsurface environments. Most of the gscA sequences recovered from groundwater genomic DNA clustered in a tight phylogenetic group closely related to strain M18. GscA was most abundant in groundwater samples in which Geobacter sp. predominated. Expression of gscA in a strain of Geobacter sulfurreducens that lacked the gene for the c-type cytochrome OmcS, thought to facilitate electron transfer from conductive pili to Fe(III) oxide, restored the capacity for Fe(III) oxide reduction. Atomic force microscopy provided evidence that GscA was associated with the pili. These results demonstrate that a c-type cytochrome with an apparent function similar to that of OmcS is abundant when Geobacter sp. are abundant in the subsurface, providing insight into the mechanisms for the growth of subsurface Geobacter sp. on Fe(III) oxide and suggesting an approach for functional analysis of other Geobacter proteins found in the subsurface.

}, keywords = {Amino Acid Sequence, Bacterial Proteins, Biodegradation, Environmental, Cytochrome c Group, Electron Transport, Ferric Compounds, Geobacter, Groundwater, Molecular Sequence Data, Oxidation-Reduction, Phylogeny, Proteomics, Sequence Alignment, Uranium}, issn = {1751-7370}, doi = {10.1038/ismej.2015.113}, author = {Yun, Jiae and Malvankar, Nikhil S and Ueki, Toshiyuki and Lovley, Derek R} } @article {3120, title = {Evidence of Geobacter-associated phage in a uranium-contaminated aquifer.}, journal = {ISME J}, volume = {9}, year = {2015}, month = {2015 Feb}, pages = {333-46}, abstract = {

Geobacter species may be important agents in the bioremediation of organic and metal contaminants in the subsurface, but as yet unknown factors limit the in situ growth of subsurface Geobacter well below rates predicted by analysis of gene expression or in silico metabolic modeling. Analysis of the genomes of five different Geobacter species recovered from contaminated subsurface sites indicated that each of the isolates had been infected with phage. Geobacter-associated phage sequences were also detected by metagenomic and proteomic analysis of samples from a uranium-contaminated aquifer undergoing in situ bioremediation, and phage particles were detected by microscopic analysis in groundwater collected from sediment enrichment cultures. Transcript abundance for genes from the Geobacter-associated phage structural proteins, tail tube Gp19 and baseplate J, increased in the groundwater in response to the growth of Geobacter species when acetate was added, and then declined as the number of Geobacter decreased. Western blot analysis of a Geobacter-associated tail tube protein Gp19 in the groundwater demonstrated that its abundance tracked with the abundance of Geobacter species. These results suggest that the enhanced growth of Geobacter species in the subsurface associated with in situ uranium bioremediation increased the abundance and activity of Geobacter-associated phage and show that future studies should focus on how these phages might be influencing the ecology of this site.

}, keywords = {Bacteriophages, Biodegradation, Environmental, Genes, Viral, Geobacter, Groundwater, Metagenome, Proteomics, Transcriptome, Uranium, Viral Proteins, Water Pollutants, Radioactive}, issn = {1751-7370}, doi = {10.1038/ismej.2014.128}, author = {Holmes, Dawn E and Giloteaux, Ludovic and Chaurasia, Akhilesh K and Williams, Kenneth H and Luef, Birgit and Wilkins, Michael J and Wrighton, Kelly C and Thompson, Courtney A and Comolli, Luis R and Lovley, Derek R} } @article {3153, title = {Bioremediation of uranium-contaminated groundwater: a systems approach to subsurface biogeochemistry.}, journal = {Curr Opin Biotechnol}, volume = {24}, year = {2013}, month = {2013 Jun}, pages = {489-97}, abstract = {

Adding organic electron donors to stimulate microbial reduction of highly soluble U(VI) to less soluble U(IV) is a promising strategy for immobilizing uranium in contaminated subsurface environments. Studies suggest that diagnosing the in situ physiological status of the subsurface community during uranium bioremediation with environmental transcriptomic and proteomic techniques can identify factors potentially limiting U(VI) reduction activity. Models which couple genome-scale in silico representations of the metabolism of key microbial populations with geochemical and hydrological models may be able to predict the outcome of bioremediation strategies and aid in the development of new approaches. Concerns remain about the long-term stability of sequestered U(IV) minerals and the release of co-contaminants associated with Fe(III) oxides, which might be overcome through targeted delivery of electrons to select microorganisms using in situ electrodes.

}, keywords = {Biodegradation, Environmental, Electrodes, Ferric Compounds, Genomics, Groundwater, Proteomics, Uranium, Water Pollutants, Radioactive}, issn = {1879-0429}, doi = {10.1016/j.copbio.2012.10.008}, author = {Williams, Kenneth H and Bargar, John R and Lloyd, Jonathan R and Lovley, Derek R} } @article {3154, title = {Characterization and transcription of arsenic respiration and resistance genes during in situ uranium bioremediation.}, journal = {ISME J}, volume = {7}, year = {2013}, month = {2013 Feb}, pages = {370-83}, abstract = {

The possibility of arsenic release and the potential role of Geobacter in arsenic biogeochemistry during in situ uranium bioremediation was investigated because increased availability of organic matter has been associated with substantial releases of arsenic in other subsurface environments. In a field experiment conducted at the Rifle, CO study site, groundwater arsenic concentrations increased when acetate was added. The number of transcripts from arrA, which codes for the α-subunit of dissimilatory As(V) reductase, and acr3, which codes for the arsenic pump protein Acr3, were determined with quantitative reverse transcription-PCR. Most of the arrA (>60\%) and acr3-1 (>90\%) sequences that were recovered were most similar to Geobacter species, while the majority of acr3-2 (>50\%) sequences were most closely related to Rhodoferax ferrireducens. Analysis of transcript abundance demonstrated that transcription of acr3-1 by the subsurface Geobacter community was correlated with arsenic concentrations in the groundwater. In contrast, Geobacter arrA transcript numbers lagged behind the major arsenic release and remained high even after arsenic concentrations declined. This suggested that factors other than As(V) availability regulated the transcription of arrA in situ, even though the presence of As(V) increased the transcription of arrA in cultures of Geobacter lovleyi, which was capable of As(V) reduction. These results demonstrate that subsurface Geobacter species can tightly regulate their physiological response to changes in groundwater arsenic concentrations. The transcriptomic approach developed here should be useful for the study of a diversity of other environments in which Geobacter species are considered to have an important influence on arsenic biogeochemistry.

}, keywords = {Acetates, Arsenate Reductases, Arsenic, Biodegradation, Environmental, Colorado, Gene Expression Regulation, Bacterial, Genes, Bacterial, Geobacter, Groundwater, Transcriptome, Uranium}, issn = {1751-7370}, doi = {10.1038/ismej.2012.109}, author = {Giloteaux, Ludovic and Holmes, Dawn E and Williams, Kenneth H and Wrighton, Kelly C and Wilkins, Michael J and Montgomery, Alison P and Smith, Jessica A and Orellana, Roberto and Thompson, Courtney A and Roper, Thomas J and Long, Philip E and Lovley, Derek R} } @article {3145, title = {Enrichment of specific protozoan populations during in situ bioremediation of uranium-contaminated groundwater.}, journal = {ISME J}, volume = {7}, year = {2013}, month = {2013 Jul}, pages = {1286-98}, abstract = {

The importance of bacteria in the anaerobic bioremediation of groundwater polluted with organic and/or metal contaminants is well recognized and in some instances so well understood that modeling of the in situ metabolic activity of the relevant subsurface microorganisms in response to changes in subsurface geochemistry is feasible. However, a potentially significant factor influencing bacterial growth and activity in the subsurface that has not been adequately addressed is protozoan predation of the microorganisms responsible for bioremediation. In field experiments at a uranium-contaminated aquifer located in Rifle, CO, USA, acetate amendments initially promoted the growth of metal-reducing Geobacter species, followed by the growth of sulfate reducers, as observed previously. Analysis of 18S rRNA gene sequences revealed a broad diversity of sequences closely related to known bacteriovorous protozoa in the groundwater before the addition of acetate. The bloom of Geobacter species was accompanied by a specific enrichment of sequences most closely related to the ameboid flagellate, Breviata anathema, which at their peak accounted for over 80\% of the sequences recovered. The abundance of Geobacter species declined following the rapid emergence of B. anathema. The subsequent growth of sulfate-reducing Peptococcaceae was accompanied by another specific enrichment of protozoa, but with sequences most similar to diplomonadid flagellates from the family Hexamitidae, which accounted for up to 100\% of the sequences recovered during this phase of the bioremediation. These results suggest a prey-predator response with specific protozoa responding to increased availability of preferred prey bacteria. Thus, quantifying the influence of protozoan predation on the growth, activity and composition of the subsurface bacterial community is essential for predictive modeling of in situ uranium bioremediation strategies.

}, keywords = {Acetates, Biodegradation, Environmental, Eukaryota, Geobacter, Groundwater, Molecular Sequence Data, Oxidation-Reduction, Phylogeny, RNA, Ribosomal, 16S, RNA, Ribosomal, 18S, Uranium}, issn = {1751-7370}, doi = {10.1038/ismej.2013.20}, author = {Holmes, Dawn E and Giloteaux, Ludovic and Williams, Kenneth H and Wrighton, Kelly C and Wilkins, Michael J and Thompson, Courtney A and Roper, Thomas J and Long, Philip E and Lovley, Derek R} } @article {3138, title = {Field evidence of selenium bioreduction in a uranium-contaminated aquifer.}, journal = {Environ Microbiol Rep}, volume = {5}, year = {2013}, month = {2013 Jun}, pages = {444-52}, abstract = {

Removal of selenium from groundwater was documented during injection of acetate into a uranium-contaminated aquifer near Rifle, Colorado (USA). Bioreduction of aqueous selenium to its elemental form (Se0) concentrated it within mineralized biofilms affixed to tubing used to circulate acetate-amended groundwater. Scanning and transmission electron microscopy revealed close association between Se0 precipitates and cell surfaces, with Se0 aggregates having a diameter of 50-60 nm. Accumulation of Se0 within biofilms occurred over a three-week interval at a rate of c. 9 mg Se0 m(-2) tubing day(-1). Removal was inferred to result from the activity of a mixed microbial community within the biofilms capable of coupling acetate oxidation to the reduction of oxygen, nitrate and selenate. Phylogenetic analysis of the biofilm revealed a community dominated by strains of Dechloromonas sp. and Thauera sp., with isolates exhibiting genetic similarity to the latter known to reduce selenate to Se0. Enrichment cultures of selenate-respiring microorganisms were readily established using Rifle site groundwater and acetate, with cultures dominated by strains closely related to D. aromatica (96-99\% similarity). Predominance of Dechloromonas sp. in recovered biofilms and enrichments suggests this microorganism may play a role in the removal of selenium oxyanions present in Se-impacted groundwaters and sediments.

}, keywords = {Acetates, Betaproteobacteria, Biodegradation, Environmental, Biofilms, Colorado, Groundwater, Humans, Microbial Consortia, Oxidation-Reduction, Oxygen, Phylogeny, RNA, Ribosomal, 16S, Selenic Acid, Selenium, Selenium Compounds, Thauera, Uranium, Water Pollutants, Chemical}, issn = {1758-2229}, doi = {10.1111/1758-2229.12032}, author = {Williams, Kenneth H and Wilkins, Michael J and N{\textquoteright}Guessan, A Lucie and Arey, Bruce and Dodova, Elena and Dohnalkova, Alice and Holmes, Dawn and Lovley, Derek R and Long, Philip E} } @article {3143, title = {Fluctuations in species-level protein expression occur during element and nutrient cycling in the subsurface.}, journal = {PLoS One}, volume = {8}, year = {2013}, month = {2013}, pages = {e57819}, abstract = {

While microbial activities in environmental systems play a key role in the utilization and cycling of essential elements and compounds, microbial activity and growth frequently fluctuates in response to environmental stimuli and perturbations. To investigate these fluctuations within a saturated aquifer system, we monitored a carbon-stimulated in situ Geobacter population while iron reduction was occurring, using 16S rRNA abundances and high-resolution tandem mass spectrometry proteome measurements. Following carbon amendment, 16S rRNA analysis of temporally separated samples revealed the rapid enrichment of Geobacter-like environmental strains with strong similarity to G. bemidjiensis. Tandem mass spectrometry proteomics measurements suggest high carbon flux through Geobacter respiratory pathways, and the synthesis of anapleurotic four carbon compounds from acetyl-CoA via pyruvate ferredoxin oxidoreductase activity. Across a 40-day period where Fe(III) reduction was occurring, fluctuations in protein expression reflected changes in anabolic versus catabolic reactions, with increased levels of biosynthesis occurring soon after acetate arrival in the aquifer. In addition, localized shifts in nutrient limitation were inferred based on expression of nitrogenase enzymes and phosphate uptake proteins. These temporal data offer the first example of differing microbial protein expression associated with changing geochemical conditions in a subsurface environment.

}, keywords = {Biomass, Carbon, Environment, Gene Expression Regulation, Bacterial, Geobacter, Groundwater, Humic Substances, Iron, Oxidation-Reduction, Phosphates, Plankton, Proteomics, RNA, Ribosomal, 16S, Tandem Mass Spectrometry, Uranium, Vanadium, Water Microbiology}, issn = {1932-6203}, doi = {10.1371/journal.pone.0057819}, author = {Wilkins, Michael J and Wrighton, Kelly C and Nicora, Carrie D and Williams, Kenneth H and McCue, Lee Ann and Handley, Kim M and Miller, Chris S and Giloteaux, Ludovic and Montgomery, Alison P and Lovley, Derek R and Banfield, Jillian F and Long, Philip E and Lipton, Mary S} } @article {3147, title = {Molecular analysis of the in situ growth rates of subsurface Geobacter species.}, journal = {Appl Environ Microbiol}, volume = {79}, year = {2013}, month = {2013 Mar}, pages = {1646-53}, abstract = {

Molecular tools that can provide an estimate of the in situ growth rate of Geobacter species could improve understanding of dissimilatory metal reduction in a diversity of environments. Whole-genome microarray analyses of a subsurface isolate of Geobacter uraniireducens, grown under a variety of conditions, identified a number of genes that are differentially expressed at different specific growth rates. Expression of two genes encoding ribosomal proteins, rpsC and rplL, was further evaluated with quantitative reverse transcription-PCR (qRT-PCR) in cells with doubling times ranging from 6.56 h to 89.28 h. Transcript abundance of rpsC correlated best (r(2) = 0.90) with specific growth rates. Therefore, expression patterns of rpsC were used to estimate specific growth rates of Geobacter species during an in situ uranium bioremediation field experiment in which acetate was added to the groundwater to promote dissimilatory metal reduction. Initially, increased availability of acetate in the groundwater resulted in higher expression of Geobacter rpsC, and the increase in the number of Geobacter cells estimated with fluorescent in situ hybridization compared well with specific growth rates estimated from levels of in situ rpsC expression. However, in later phases, cell number increases were substantially lower than predicted from rpsC transcript abundance. This change coincided with a bloom of protozoa and increased attachment of Geobacter species to solid phases. These results suggest that monitoring rpsC expression may better reflect the actual rate that Geobacter species are metabolizing and growing during in situ uranium bioremediation than changes in cell abundance.

}, keywords = {Acetates, Biodegradation, Environmental, DNA, Bacterial, Gene Expression Profiling, Geobacter, Groundwater, In Situ Hybridization, Fluorescence, Molecular Sequence Data, Ribosomal Proteins, Sequence Analysis, DNA, Uranium}, issn = {1098-5336}, doi = {10.1128/AEM.03263-12}, author = {Holmes, Dawn E and Giloteaux, Ludovic and Barlett, Melissa and Chavan, Milind A and Smith, Jessica A and Williams, Kenneth H and Wilkins, Michael and Long, Philip and Lovley, Derek R} } @article {3137, title = {U(VI) reduction by diverse outer surface c-type cytochromes of Geobacter sulfurreducens.}, journal = {Appl Environ Microbiol}, volume = {79}, year = {2013}, month = {2013 Oct}, pages = {6369-74}, abstract = {

Early studies with Geobacter sulfurreducens suggested that outer-surface c-type cytochromes might play a role in U(VI) reduction, but it has recently been suggested that there is substantial U(VI) reduction at the surface of the electrically conductive pili known as microbial nanowires. This phenomenon was further investigated. A strain of G. sulfurreducens, known as Aro-5, which produces pili with substantially reduced conductivity reduced U(VI) nearly as well as the wild type, as did a strain in which the gene for PilA, the structural pilin protein, was deleted. In order to reduce rates of U(VI) reduction to levels less than 20\% of the wild-type rates, it was necessary to delete the genes for the five most abundant outer surface c-type cytochromes of G. sulfurreducens. X-ray absorption near-edge structure spectroscopy demonstrated that whereas 83\% {\textpm} 10\% of the uranium associated with wild-type cells correspond to U(IV) after 4 h of incubation, with the quintuple mutant, 89\% {\textpm} 10\% of uranium was U(VI). Transmission electron microscopy and X-ray energy dispersion spectroscopy revealed that wild-type cells did not precipitate uranium along pili as previously reported, but U(IV) was precipitated at the outer cell surface. These findings are consistent with those of previous studies, which have suggested that G. sulfurreducens requires outer-surface c-type cytochromes but not pili for the reduction of soluble extracellular electron acceptors.

}, keywords = {Cytochromes, Fimbriae, Bacterial, Gene Deletion, Geobacter, Microscopy, Electron, Transmission, Oxidation-Reduction, Uranium, X-Ray Absorption Spectroscopy}, issn = {1098-5336}, doi = {10.1128/AEM.02551-13}, author = {Orellana, Roberto and Leavitt, Janet J and Comolli, Luis R and Csencsits, Roseann and Janot, Noemie and Flanagan, Kelly A and Gray, Arianna S and Leang, Ching and Izallalen, Mounir and Mester, T{\"u}nde and Lovley, Derek R} } @article {413, title = {Microbial functional gene diversity with a shift of subsurface redox conditions during In Situ uranium reduction.}, journal = {Appl Environ Microbiol}, volume = {78}, year = {2012}, month = {2012 Apr}, pages = {2966-72}, abstract = {To better understand the microbial functional diversity changes with subsurface redox conditions during in situ uranium bioremediation, key functional genes were studied with GeoChip, a comprehensive functional gene microarray, in field experiments at a uranium mill tailings remedial action (UMTRA) site (Rifle, CO). The results indicated that functional microbial communities altered with a shift in the dominant metabolic process, as documented by hierarchical cluster and ordination analyses of all detected functional genes. The abundance of dsrAB genes (dissimilatory sulfite reductase genes) and methane generation-related mcr genes (methyl coenzyme M reductase coding genes) increased when redox conditions shifted from Fe-reducing to sulfate-reducing conditions. The cytochrome genes detected were primarily from Geobacter sp. and decreased with lower subsurface redox conditions. Statistical analysis of environmental parameters and functional genes indicated that acetate, U(VI), and redox potential (E(h)) were the most significant geochemical variables linked to microbial functional gene structures, and changes in microbial functional diversity were strongly related to the dominant terminal electron-accepting process following acetate addition. The study indicates that the microbial functional genes clearly reflect the in situ redox conditions and the dominant microbial processes, which in turn influence uranium bioreduction. Microbial functional genes thus could be very useful for tracking microbial community structure and dynamics during bioremediation.}, keywords = {Biodegradation, Environmental, Biota, Environmental Microbiology, Environmental Pollutants, Genetic Variation, Microarray Analysis, Oxidation-Reduction, Uranium}, issn = {1098-5336}, doi = {10.1128/AEM.06528-11}, author = {Liang, Yuting and Van Nostrand, Joy D and N{\textquoteright}guessan, Lucie A and Peacock, Aaron D and Deng, Ye and Long, Philip E and Resch, C Tom and Wu, Liyou and He, Zhili and Li, Guanghe and Hazen, Terry C and Lovley, Derek R and Zhou, Jizhong} } @article {426, title = {Development of a biomarker for Geobacter activity and strain composition; proteogenomic analysis of the citrate synthase protein during bioremediation of U(VI).}, journal = {Microb Biotechnol}, volume = {4}, year = {2011}, month = {2011 Jan}, pages = {55-63}, abstract = {Monitoring the activity of target microorganisms during stimulated bioremediation is a key problem for the development of effective remediation strategies. At the US Department of Energy{\textquoteright}s Integrated Field Research Challenge (IFRC) site in Rifle, CO, the stimulation of Geobacter growth and activity via subsurface acetate addition leads to precipitation of U(VI) from groundwater as U(IV). Citrate synthase (gltA) is a key enzyme in Geobacter central metabolism that controls flux into the TCA cycle. Here, we utilize shotgun proteomic methods to demonstrate that the measurement of gltA peptides can be used to track Geobacter activity and strain evolution during in situ biostimulation. Abundances of conserved gltA peptides tracked Fe(III) reduction and changes in U(VI) concentrations during biostimulation, whereas changing patterns of unique peptide abundances between samples suggested sample-specific strain shifts within the Geobacter population. Abundances of unique peptides indicated potential differences at the strain level between Fe(III)-reducing populations stimulated during in situ biostimulation experiments conducted a year apart at the Rifle IFRC. These results offer a novel technique for the rapid screening of large numbers of proteomic samples for Geobacter species and will aid monitoring of subsurface bioremediation efforts that rely on metal reduction for desired outcomes.}, keywords = {Amino Acid Sequence, Bacterial Proteins, Biodegradation, Environmental, Biological Markers, Citrate (si)-Synthase, Geobacter, Groundwater, Molecular Sequence Data, Phylogeny, Proteomics, Sequence Alignment, Uranium}, issn = {1751-7915}, doi = {10.1111/j.1751-7915.2010.00194.x}, author = {Wilkins, Michael J and Callister, Stephen J and Miletto, Marzia and Williams, Kenneth H and Nicora, Carrie D and Lovley, Derek R and Long, Philip E and Lipton, Mary S} } @article {429, title = {Direct coupling of a genome-scale microbial in silico model and a groundwater reactive transport model.}, journal = {J Contam Hydrol}, volume = {122}, year = {2011}, month = {2011 Mar 25}, pages = {96-103}, abstract = {The activity of microorganisms often plays an important role in dynamic natural attenuation or engineered bioremediation of subsurface contaminants, such as chlorinated solvents, metals, and radionuclides. To evaluate and/or design bioremediated systems, quantitative reactive transport models are needed. State-of-the-art reactive transport models often ignore the microbial effects or simulate the microbial effects with static growth yield and constant reaction rate parameters over simulated conditions, while in reality microorganisms can dynamically modify their functionality (such as utilization of alternative respiratory pathways) in response to spatial and temporal variations in environmental conditions. Constraint-based genome-scale microbial in silico models, using genomic data and multiple-pathway reaction networks, have been shown to be able to simulate transient metabolism of some well studied microorganisms and identify growth rate, substrate uptake rates, and byproduct rates under different growth conditions. These rates can be identified and used to replace specific microbially-mediated reaction rates in a reactive transport model using local geochemical conditions as constraints. We previously demonstrated the potential utility of integrating a constraint-based microbial metabolism model with a reactive transport simulator as applied to bioremediation of uranium in groundwater. However, that work relied on an indirect coupling approach that was effective for initial demonstration but may not be extensible to more complex problems that are of significant interest (e.g., communities of microbial species and multiple constraining variables). Here, we extend that work by presenting and demonstrating a method of directly integrating a reactive transport model (FORTRAN code) with constraint-based in silico models solved with IBM ILOG CPLEX linear optimizer base system (C library). The models were integrated with BABEL, a language interoperability tool. The modeling system is designed in such a way that constraint-based models targeting different microorganisms or competing organism communities can be easily plugged into the system. Constraint-based modeling is very costly given the size of a genome-scale reaction network. To save computation time, a binary tree is traversed to examine the concentration and solution pool generated during the simulation in order to decide whether the constraint-based model should be called. We also show preliminary results from the integrated model including a comparison of the direct and indirect coupling approaches and evaluated the ability of the approach to simulate field experiment.}, keywords = {Biodegradation, Environmental, Biological Transport, Colorado, Computer Simulation, Genome, Bacterial, Geobacter, Models, Biological, Uranium}, issn = {1873-6009}, doi = {10.1016/j.jconhyd.2010.11.007}, author = {Fang, Yilin and Scheibe, Timothy D and Mahadevan, Radhakrishnan and Garg, Srinath and Long, Philip E and Lovley, Derek R} } @article {438, title = {Genome-scale dynamic modeling of the competition between Rhodoferax and Geobacter in anoxic subsurface environments.}, journal = {ISME J}, volume = {5}, year = {2011}, month = {2011 Feb}, pages = {305-16}, abstract = {The advent of rapid complete genome sequencing, and the potential to capture this information in genome-scale metabolic models, provide the possibility of comprehensively modeling microbial community interactions. For example, Rhodoferax and Geobacter species are acetate-oxidizing Fe(III)-reducers that compete in anoxic subsurface environments and this competition may have an influence on the in situ bioremediation of uranium-contaminated groundwater. Therefore, genome-scale models of Geobacter sulfurreducens and Rhodoferax ferrireducens were used to evaluate how Geobacter and Rhodoferax species might compete under diverse conditions found in a uranium-contaminated aquifer in Rifle, CO. The model predicted that at the low rates of acetate flux expected under natural conditions at the site, Rhodoferax will outcompete Geobacter as long as sufficient ammonium is available. The model also predicted that when high concentrations of acetate are added during in situ bioremediation, Geobacter species would predominate, consistent with field-scale observations. This can be attributed to the higher expected growth yields of Rhodoferax and the ability of Geobacter to fix nitrogen. The modeling predicted relative proportions of Geobacter and Rhodoferax in geochemically distinct zones of the Rifle site that were comparable to those that were previously documented with molecular techniques. The model also predicted that under nitrogen fixation, higher carbon and electron fluxes would be diverted toward respiration rather than biomass formation in Geobacter, providing a potential explanation for enhanced in situ U(VI) reduction in low-ammonium zones. These results show that genome-scale modeling can be a useful tool for predicting microbial interactions in subsurface environments and shows promise for designing bioremediation strategies.}, keywords = {Acetates, Anaerobiosis, Biodegradation, Environmental, Biomass, Comamonadaceae, Genome, Genome, Bacterial, Geobacter, Models, Biological, Nitrogen Fixation, Quaternary Ammonium Compounds, RNA, Ribosomal, 16S, Uranium, Water Microbiology, Water Pollutants, Radioactive}, issn = {1751-7370}, doi = {10.1038/ismej.2010.117}, author = {Zhuang, Kai and Izallalen, Mounir and Mouser, Paula and Richter, Hanno and Risso, Carla and Mahadevan, Radhakrishnan and Lovley, Derek R} } @article {444, title = {Expression of acetate permease-like (apl ) genes in subsurface communities of Geobacter species under fluctuating acetate concentrations.}, journal = {FEMS Microbiol Ecol}, volume = {73}, year = {2010}, month = {2010 Sep}, pages = {441-9}, abstract = {The addition of acetate to uranium-contaminated aquifers in order to stimulate the growth and activity of Geobacter species that reduce uranium is a promising in situ bioremediation option. Optimizing this bioremediation strategy requires that sufficient acetate be added to promote Geobacter species growth. We hypothesized that under acetate-limiting conditions, subsurface Geobacter species would increase the expression of either putative acetate symporters genes (aplI and aplII). Acetate was added to a uranium-contaminated aquifer (Rifle, CO) in two continuous amendments separated by 5 days of groundwater flush to create changing acetate concentrations. While the expression of aplI in monitoring well D04 (high acetate) weakly correlated with the acetate concentration over time, the transcript levels for this gene were relatively constant in well D08 (low acetate). At the lowest acetate concentrations during the groundwater flush, the transcript levels of aplII were the highest. The expression of aplII decreased 2-10-fold upon acetate reintroduction. However, the overall instability of acetate concentrations throughout the experiment could not support a robust conclusion regarding the role of apl genes in response to acetate limitation under field conditions, in contrast to previous chemostat studies, suggesting that the function of a microbial community cannot be inferred based on lab experiments alone.}, keywords = {Acetates, Bacterial Proteins, Biodegradation, Environmental, Fresh Water, Gene Expression Regulation, Bacterial, Gene Library, Geobacter, Membrane Transport Proteins, Multigene Family, RNA, Bacterial, Uranium, Water Pollutants, Radioactive}, issn = {1574-6941}, doi = {10.1111/j.1574-6941.2010.00907.x}, author = {Elifantz, Hila and N{\textquoteright}guessan, Lucie A and Mouser, Paula J and Williams, Kenneth H and Wilkins, Michael J and Risso, Carla and Holmes, Dawn E and Long, Philip E and Lovley, Derek R} } @article {454, title = {Modeling and sensitivity analysis of electron capacitance for Geobacter in sedimentary environments.}, journal = {J Contam Hydrol}, volume = {112}, year = {2010}, month = {2010 Mar 1}, pages = {30-44}, abstract = {In situ stimulation of the metabolic activity of Geobacter species through acetate amendment has been shown to be a promising bioremediation strategy to reduce and immobilize hexavalent uranium [U(VI)] as insoluble U(IV). Although Geobacter species are reducing U(VI), they primarily grow via Fe(III) reduction. Unfortunately, the biogeochemistry and the physiology of simultaneous reduction of multiple metals are still poorly understood. A detailed model is therefore required to better understand the pathways leading to U(VI) and Fe(III) reduction by Geobacter species. Based on recent experimental evidence of temporary electron capacitors in Geobacter we propose a novel kinetic model that physically distinguishes planktonic cells into electron-loaded and -unloaded states. Incorporation of an electron load-unload cycle into the model provides insight into U(VI) reduction efficiency, and elucidates the relationship between U(VI)- and Fe(III)-reducing activity and further explains the correlation of high U(VI) removal with high fractions of planktonic cells in subsurface environments. Global sensitivity analysis was used to determine the level of importance of geochemical and microbial processes controlling Geobacter growth and U(VI) reduction, suggesting that the electron load-unload cycle and the resulting repartition of the microbes between aqueous and attached phases are critical for U(VI) reduction. As compared with conventional Monod modeling approaches without inclusion of the electron capacitance, the new model attempts to incorporate a novel cellular mechanism that has a significant impact on the outcome of in situ bioremediation.}, keywords = {Biodegradation, Environmental, Electric Capacitance, Geobacter, Kinetics, Models, Biological, Uranium}, issn = {1873-6009}, doi = {10.1016/j.jconhyd.2009.10.002}, author = {Zhao, Jiao and Fang, Yilin and Scheibe, Timothy D and Lovley, Derek R and Mahadevan, R} } @article {451, title = {Molecular analysis of phosphate limitation in Geobacteraceae during the bioremediation of a uranium-contaminated aquifer.}, journal = {ISME J}, volume = {4}, year = {2010}, month = {2010 Feb}, pages = {253-66}, abstract = {Nutrient limitation is an environmental stress that may reduce the effectiveness of bioremediation strategies, especially when the contaminants are organic compounds or when organic compounds are added to promote microbial activities such as metal reduction. Genes indicative of phosphate-limitation were identified by microarray analysis of chemostat cultures of Geobacter sulfureducens. This analysis revealed that genes in the pst-pho operon, which is associated with a high-affinity phosphate uptake system in other microorganisms, had significantly higher transcript abundance under phosphate-limiting conditions, with the genes pstB and phoU upregulated the most. Quantitative PCR analysis of pstB and phoU transcript levels in G. sulfurreducens grown in chemostats demonstrated that the expression of these genes increased when phosphate was removed from the culture medium. Transcripts of pstB and phoU within the subsurface Geobacter species predominating during an in situ uranium-bioremediation field experiment were more abundant than in chemostat cultures of G. sulfurreducens that were not limited for phosphate. Addition of phosphate to incubations of subsurface sediments did not stimulate dissimilatory metal reduction. The added phosphate was rapidly adsorbed onto the sediments. The results demonstrate that Geobacter species can effectively reduce U(VI) even when experiencing suboptimal phosphate concentrations and that increasing phosphate availability with phosphate additions is difficult to achieve because of the high reactivity of this compound. This transcript-based approach developed for diagnosing phosphate limitation should be applicable to assessing the potential need for additional phosphate in other bioremediation processes.}, keywords = {Biodegradation, Environmental, Fresh Water, Gene Expression Regulation, Bacterial, Geobacter, Phosphates, Uranium, Water Pollutants}, issn = {1751-7370}, doi = {10.1038/ismej.2009.115}, author = {N{\textquoteright}guessan, Lucie A and Elifantz, Hila and Nevin, Kelly P and Mouser, Paula J and Meth{\'e}, Barbara and Woodard, Trevor L and Manley, Kimberly and Williams, Kenneth H and Wilkins, Michael J and Larsen, Joern T and Long, Philip E and Lovley, Derek R} } @article {464, title = {Coupling a genome-scale metabolic model with a reactive transport model to describe in situ uranium bioremediation.}, journal = {Microb Biotechnol}, volume = {2}, year = {2009}, month = {2009 Mar}, pages = {274-86}, abstract = {The increasing availability of the genome sequences of microorganisms involved in important bioremediation processes makes it feasible to consider developing genome-scale models that can aid in predicting the likely outcome of potential subsurface bioremediation strategies. Previous studies of the in situ bioremediation of uranium-contaminated groundwater have demonstrated that Geobacter species are often the dominant members of the groundwater community during active bioremediation and the primary organisms catalysing U(VI) reduction. Therefore, a genome-scale, constraint-based model of the metabolism of Geobacter sulfurreducens was coupled with the reactive transport model HYDROGEOCHEM in an attempt to model in situ uranium bioremediation. In order to simplify the modelling, the influence of only three growth factors was considered: acetate, the electron donor added to stimulate U(VI) reduction; Fe(III), the electron acceptor primarily supporting growth of Geobacter; and ammonium, a key nutrient. The constraint-based model predicted that growth yields of Geobacter varied significantly based on the availability of these three growth factors and that there are minimum thresholds of acetate and Fe(III) below which growth and activity are not possible. This contrasts with typical, empirical microbial models that assume fixed growth yields and the possibility for complete metabolism of the substrates. The coupled genome-scale and reactive transport model predicted acetate concentrations and U(VI) reduction rates in a field trial of in situ uranium bioremediation that were comparable to the predictions of a calibrated conventional model, but without the need for empirical calibration, other than specifying the initial biomass of Geobacter. These results suggest that coupling genome-scale metabolic models with reactive transport models may be a good approach to developing models that can be truly predictive, without empirical calibration, for evaluating the probable response of subsurface microorganisms to possible bioremediation approaches prior to implementation.}, keywords = {Acetates, Biodegradation, Environmental, Biological Transport, Genome, Bacterial, Geobacter, Iron, Models, Biological, Uranium}, issn = {1751-7915}, doi = {10.1111/j.1751-7915.2009.00087.x}, author = {Scheibe, Timothy D and Mahadevan, Radhakrishnan and Fang, Yilin and Garg, Srinath and Long, Philip E and Lovley, Derek R} } @article {459, title = {Influence of heterogeneous ammonium availability on bacterial community structure and the expression of nitrogen fixation and ammonium transporter genes during in situ bioremediation of uranium-contaminated groundwater.}, journal = {Environ Sci Technol}, volume = {43}, year = {2009}, month = {2009 Jun 15}, pages = {4386-92}, abstract = {The influence of ammonium availability on bacterial community structure and the physiological status of Geobacter species during in situ bioremediation of uranium-contaminated groundwater was evaluated. Ammonium concentrations varied by 2 orders of magnitude (< 4 to 400 microM) across th study site. Analysis of 16S rRNA sequences suggested that ammonium may have been one factor influencing the community composition prior to acetate amendment with Rhodoferax species predominating over Geobacter species with higher ammonium and Dechloromonas species dominating at the site with lowest ammonium. However, once acetate was added and dissimilatory metal reduction was stimulated, Geobacter species became the predominant organisms at all locations. Rates of U(VI) reduction appeared to be more related to acetate concentrations rather than ammonium levels. In situ mRNA transcript abundance of the nitrogen fixation gene, nifD, and the ammonium transporter gene, amtB, in Geobacter species indicated that ammonium was the primary source of nitrogen during uranium reduction. The abundance of amtB was inversely correlated to ammonium levels, whereas nifD transcript levels were similar across all sites examined. These results suggest that nifD and amtB expression are closely regulated in response to ammonium availability to ensure an adequate supply of nitrogen while conserving cell resources. Thus, quantifying nifD and amtB transcript expression appears to be a useful approach for monitoring the nitrogen-related physiological status of subsurface Geobacter species. This study also emphasizes the need for more detailed analysis of geochemical and physiological interactions at the field scale in order to adequately model subsurface microbial processes during bioremediation.}, keywords = {Carrier Proteins, DNA, Bacterial, Environmental Remediation, Gene Expression Regulation, Bacterial, Gene Library, Geobacter, Nitrogen Fixation, Quaternary Ammonium Compounds, Time Factors, Uranium, Water, Water Pollutants, Radioactive}, issn = {0013-936X}, author = {Mouser, Paula J and N{\textquoteright}guessan, Lucie A and Elifantz, Hila and Holmes, Dawn E and Williams, Kenneth H and Wilkins, Michael J and Long, Philip E and Lovley, Derek R} } @article {457, title = {Proteogenomic monitoring of Geobacter physiology during stimulated uranium bioremediation.}, journal = {Appl Environ Microbiol}, volume = {75}, year = {2009}, month = {2009 Oct}, pages = {6591-9}, abstract = {Implementation of uranium bioremediation requires methods for monitoring the membership and activities of the subsurface microbial communities that are responsible for reduction of soluble U(VI) to insoluble U(IV). Here, we report a proteomics-based approach for simultaneously documenting the strain membership and microbial physiology of the dominant Geobacter community members during in situ acetate amendment of the U-contaminated Rifle, CO, aquifer. Three planktonic Geobacter-dominated samples were obtained from two wells down-gradient of acetate addition. Over 2,500 proteins from each of these samples were identified by matching liquid chromatography-tandem mass spectrometry spectra to peptides predicted from seven isolate Geobacter genomes. Genome-specific peptides indicate early proliferation of multiple M21 and Geobacter bemidjiensis-like strains and later possible emergence of M21 and G. bemidjiensis-like strains more closely related to Geobacter lovleyi. Throughout biostimulation, the proteome is dominated by enzymes that convert acetate to acetyl-coenzyme A and pyruvate for central metabolism, while abundant peptides matching tricarboxylic acid cycle proteins and ATP synthase subunits were also detected, indicating the importance of energy generation during the period of rapid growth following the start of biostimulation. Evolving Geobacter strain composition may be linked to changes in protein abundance over the course of biostimulation and may reflect changes in metabolic functioning. Thus, metagenomics-independent community proteogenomics can be used to diagnose the status of the subsurface consortia upon which remediation biotechnology relies.}, keywords = {Amino Acid Sequence, Bacterial Proteins, Biodegradation, Environmental, Genomics, Geobacter, Molecular Sequence Data, Oxidation-Reduction, Peptide Mapping, Plankton, Proteomics, Uranium, Water Microbiology, Water Pollutants, Radioactive}, issn = {1098-5336}, doi = {10.1128/AEM.01064-09}, author = {Wilkins, Michael J and VerBerkmoes, Nathan C and Williams, Kenneth H and Callister, Stephen J and Mouser, Paula J and Elifantz, Hila and N{\textquoteright}guessan, Lucie A and Thomas, Brian C and Nicora, Carrie D and Shah, Manesh B and Abraham, Paul and Lipton, Mary S and Lovley, Derek R and Hettich, Robert L and Long, Philip E and Banfield, Jillian F} } @article {467, title = {Quantifying expression of Geobacter spp. oxidative stress genes in pure culture and during in situ uranium bioremediation.}, journal = {ISME J}, volume = {3}, year = {2009}, month = {2009 Apr}, pages = {454-65}, abstract = {As part of an effort to diagnose the physiological status of Geobacter species during in situ bioremediation of uranium-contaminated groundwater, transcript levels for two genes potentially associated with oxidative stress, cydA and sodA, were quantified throughout a bioremediation field study in Rifle, CO, USA. Despite the accumulation of Fe(II) in the groundwater, which is inconsistent with the presence of dissolved oxygen, both genes were highly expressed during the bioremediation process. Therefore, the response to oxidative stress was further evaluated with Geobacter uraniireducens, an isolate from the Rifle site. When G. uraniireducens cultured with fumarate as the electron acceptor was exposed to 5\% oxygen for 8 h, there was a significant increase in cydA and sodA transcripts as well as other genes associated with oxygen respiration or oxidative stress. Oxygen-exposed cells had lower transcript abundance for genes associated with anaerobic respiration, metabolism and motility. Short-term oxygen exposure had little impact on cydA transcript levels, as more than 1 h was required for increases to levels comparable to the subsurface. Abundance of cydA and sodA transcripts for the isolate G. sulfurreducens were always higher in cells cultured with Fe(III) compared with fumarate as an electron acceptor, even when fumarate-grown cells were exposed to oxygen, and Fe(III)-grown cells were grown anaerobically. These results suggest that the apparently high Geobacter cydA and sodA expression during bioremediation cannot necessarily be attributed to oxidative stress and demonstrate that diagnosis of the metabolic status of subsurface microorganisms through transcript analysis should be coupled with appropriate geochemical analyses.}, keywords = {Anaerobiosis, Bacterial Proteins, Biodegradation, Environmental, Colorado, Ferric Compounds, Fumarates, Gene Expression Profiling, Geobacter, Oxidative Stress, Soil Microbiology, Uranium}, issn = {1751-7370}, doi = {10.1038/ismej.2008.126}, author = {Mouser, Paula J and Holmes, Dawn E and Perpetua, Lorrie A and DiDonato, Raymond and Postier, Brad and Liu, Anna and Lovley, Derek R} } @article {471, title = {Transcriptome of Geobacter uraniireducens growing in uranium-contaminated subsurface sediments.}, journal = {ISME J}, volume = {3}, year = {2009}, month = {2009 Feb}, pages = {216-30}, abstract = {To learn more about the physiological state of Geobacter species living in subsurface sediments, heat-sterilized sediments from a uranium-contaminated aquifer in Rifle, Colorado, were inoculated with Geobacter uraniireducens, a pure culture representative of the Geobacter species that predominates during in situ uranium bioremediation at this site. Whole-genome microarray analysis comparing sediment-grown G. uraniireducens with cells grown in defined culture medium indicated that there were 1084 genes that had higher transcript levels during growth in sediments. Thirty-four c-type cytochrome genes were upregulated in the sediment-grown cells, including several genes that are homologous to cytochromes that are required for optimal Fe(III) and U(VI) reduction by G. sulfurreducens. Sediment-grown cells also had higher levels of transcripts, indicative of such physiological states as nitrogen limitation, phosphate limitation and heavy metal stress. Quantitative reverse transcription PCR showed that many of the metabolic indicator genes that appeared to be upregulated in sediment-grown G. uraniireducens also showed an increase in expression in the natural community of Geobacter species present during an in situ uranium bioremediation field experiment at the Rifle site. These results demonstrate that it is feasible to monitor gene expression of a microorganism growing in sediments on a genome scale and that analysis of the physiological status of a pure culture growing in subsurface sediments can provide insights into the factors controlling the physiology of natural subsurface communities.}, keywords = {Colorado, DNA, Bacterial, Environmental Microbiology, Gene Expression Profiling, Geobacter, Geologic Sediments, Molecular Sequence Data, Oligonucleotide Array Sequence Analysis, Sequence Analysis, DNA, Uranium}, issn = {1751-7370}, doi = {10.1038/ismej.2008.89}, author = {Holmes, Dawn E and O{\textquoteright}Neil, Regina A and Chavan, Milind A and N{\textquoteright}guessan, Lucie A and Vrionis, Helen A and Perpetua, Lorrie A and Larrahondo, M Juliana and DiDonato, Raymond and Liu, Anna and Lovley, Derek R} } @article {486, title = {Characterization of extracellular minerals produced during dissimilatory Fe(III) and U(VI) reduction at 100 degrees C by Pyrobaculum islandicum.}, journal = {Geobiology}, volume = {6}, year = {2008}, month = {2008 Mar}, pages = {147-54}, abstract = {In order to gain insight into the significance of biotic metal reduction and mineral formation in hyperthermophilic environments, metal mineralization as a result of the dissimilatory reduction of poorly crystalline Fe(III) oxide, and U(VI) reduction at 100 degrees C by Pyrobaculum islandicum was investigated. When P. islandicum was grown in a medium with poorly crystalline Fe(III) oxide as an electron acceptor and hydrogen as an electron donor, the Fe(III) oxide was reduced to an extracellular, ultrafine-grained magnetite with characteristics similar to that found in some hot environments and that was previously thought to be of abiotic origin. Furthermore, cell suspensions of P. islandicum rapidly reduced the soluble and oxidized form of uranium, U(VI), to extracellular precipitates of the highly insoluble U(IV) mineral, uraninite (UO(2)). The reduction of U(VI) was dependent on the presence of hydrogen as the electron donor. These findings suggest that microbes may play a key role in metal deposition in hyperthermophilic environments and provide a plausible explanation for such phenomena as magnetite accumulation and formation of uranium deposits at ca. 100 degrees C.}, keywords = {Ferric Compounds, Ferumoxytol, Hot Temperature, Hydrogen, Minerals, Oxidation-Reduction, Pyrobaculum, Uranium, X-Ray Diffraction}, issn = {1472-4669}, doi = {10.1111/j.1472-4669.2007.00142.x}, author = {Kashefi, K and Moskowitz, B M and Lovley, D R} } @article {488, title = {Gene transcript analysis of assimilatory iron limitation in Geobacteraceae during groundwater bioremediation.}, journal = {Environ Microbiol}, volume = {10}, year = {2008}, month = {2008 May}, pages = {1218-30}, abstract = {Limitations on the availability of Fe(III) as an electron acceptor are thought to play an important role in restricting the growth and activity of Geobacter species during bioremediation of contaminated subsurface environments, but the possibility that these organisms might also be limited in the subsurface by the availability of iron for assimilatory purposes was not previously considered because copious quantities of Fe(II) are produced as the result of Fe(III) reduction. Analysis of multiple Geobacteraceae genomes revealed the presence of a three-gene cluster consisting of homologues of two iron-dependent regulators, fur and dtxR (ideR), separated by a homologue of feoB, which encodes an Fe(II) uptake protein. This cluster appears to be conserved among members of the Geobacteraceae and was detected in several environments. Expression of the fur-feoB-ideR cluster decreased as Fe(II) concentrations increased in chemostat cultures. The number of Geobacteraceae feoB transcripts in groundwater samples from a site undergoing in situ uranium bioremediation was relatively high until the concentration of dissolved Fe(II) increased near the end of the field experiment. These results suggest that, because much of the Fe(II) is sequestered in solid phases, Geobacter species, which have a high requirement for iron for iron-sulfur proteins, may be limited by the amount of iron available for assimilatory purposes. These results demonstrate the ability of transcript analysis to reveal previously unsuspected aspects of the in situ physiology of microorganisms in subsurface environments.}, keywords = {Bacterial Proteins, Biodegradation, Environmental, Culture Media, Ferric Compounds, Ferrous Compounds, Fresh Water, Gene Expression Regulation, Bacterial, Geobacter, Iron, Multigene Family, Phylogeny, Polymerase Chain Reaction, Repressor Proteins, Reverse Transcriptase Polymerase Chain Reaction, Transcription, Genetic, Uranium, Water Pollution, Radioactive}, issn = {1462-2920}, doi = {10.1111/j.1462-2920.2007.01537.x}, author = {O{\textquoteright}Neil, Regina A and Holmes, Dawn E and Coppi, Maddalena V and Adams, Lorrie A and Larrahondo, M Juliana and Ward, Joy E and Nevin, Kelly P and Woodard, Trevor L and Vrionis, Helen A and N{\textquoteright}guessan, Lucie A and Lovley, Derek R} } @article {483, title = {Genes for two multicopper proteins required for Fe(III) oxide reduction in Geobacter sulfurreducens have different expression patterns both in the subsurface and on energy-harvesting electrodes.}, journal = {Microbiology}, volume = {154}, year = {2008}, month = {2008 May}, pages = {1422-35}, abstract = {Previous studies have shown that Geobacter sulfurreducens requires the outer-membrane, multicopper protein OmpB for Fe(III) oxide reduction. A homologue of OmpB, designated OmpC, which is 36 \% similar to OmpB, has been discovered in the G. sulfurreducens genome. Deletion of ompC inhibited reduction of insoluble, but not soluble Fe(III). Analysis of multiple Geobacter and Pelobacter genomes, as well as in situ Geobacter, indicated that genes encoding multicopper proteins are conserved in Geobacter species but are not found in Pelobacter species. Levels of ompB transcripts were similar in G. sulfurreducens at different growth rates in chemostats and during growth on a microbial fuel cell anode. In contrast, ompC transcript levels increased at higher growth rates in chemostats and with increasing current production in fuel cells. Constant levels of Geobacter ompB transcripts were detected in groundwater during a field experiment in which acetate was added to the subsurface to promote in situ uranium bioremediation. In contrast, ompC transcript levels increased during the rapid phase of growth of Geobacter species following addition of acetate to the groundwater and then rapidly declined. These results demonstrate that more than one multicopper protein is required for optimal Fe(III) oxide reduction in G. sulfurreducens and suggest that, in environmental studies, quantifying OmpB/OmpC-related genes could help alleviate the problem that Pelobacter genes may be inadvertently quantified via quantitative analysis of 16S rRNA genes. Furthermore, comparison of differential expression of ompB and ompC may provide insight into the in situ metabolic state of Geobacter species in environments of interest.}, keywords = {Acetates, Amino Acid Sequence, Bacterial Outer Membrane Proteins, Electrodes, Ferric Compounds, Gene Deletion, Gene Expression Profiling, Geobacter, Molecular Sequence Data, Oxidation-Reduction, Phylogeny, Sequence Alignment, Sequence Homology, Nucleic Acid, Soil Microbiology, Uranium}, issn = {1350-0872}, doi = {10.1099/mic.0.2007/014365-0}, author = {Holmes, Dawn E and Mester, T{\"u}nde and O{\textquoteright}Neil, Regina A and Perpetua, Lorrie A and Larrahondo, M Juliana and Glaven, Richard and Sharma, Manju L and Ward, Joy E and Nevin, Kelly P and Lovley, Derek R} } @article {484, title = {Geobacter uraniireducens sp. nov., isolated from subsurface sediment undergoing uranium bioremediation.}, journal = {Int J Syst Evol Microbiol}, volume = {58}, year = {2008}, month = {2008 May}, pages = {1075-8}, abstract = {A Gram-negative, rod-shaped, motile bacterium, strain Rf4T, which conserves energy from dissimilatory Fe(III) reduction concomitant with acetate oxidation, was isolated from subsurface sediment undergoing uranium bioremediation. The 16S rRNA gene sequence of strain Rf4T matched sequences recovered in 16S rRNA gene clone libraries constructed from DNA extracted from groundwater sampled at the same time as the source sediment. Cells of strain Rf4T were regular, motile rods, 1.2-2.0 microm long and 0.5-0.6 microm in diameter, with rounded ends. Cells had one lateral flagellum. Growth was optimal at pH 6.5-7.0 and 32 degrees C. With acetate as the electron donor, strain Rf4T used Fe(III), Mn(IV), anthraquinone-2,6-disulfonate, malate and fumarate as electron acceptors and reduced U(VI) in cell suspensions. With poorly crystalline Fe(III) oxide as the electron acceptor, strain Rf4T oxidized the following electron donors: acetate, lactate, pyruvate and ethanol. Phylogenetic analysis of the 16S rRNA gene sequence of strain Rf4T placed it in the genus Geobacter. Strain Rf4T was most closely related to {\textquoteright}Geobacter humireducens{\textquoteright} JW3 (95.9 \% sequence similarity), Geobacter bremensis Dfr1T (95.4 \%) and Geobacter bemidjiensis BemT (95.1 \%). Based on phylogenetic analysis and phenotypic differences between strain Rf4T and closely related Geobacter species, this strain is described as a representative of a novel species, Geobacter uraniireducens sp. nov. The type strain is Rf4T (=ATCC BAA-1134T =JCM 13001T).}, keywords = {Bacterial Typing Techniques, DNA, Bacterial, Genes, rRNA, Genotype, Geobacter, Geologic Sediments, Molecular Sequence Data, Oxidation-Reduction, Phenotype, Phylogeny, RNA, Ribosomal, 16S, Sequence Analysis, DNA, Species Specificity, Uranium}, issn = {1466-5026}, doi = {10.1099/ijs.0.65377-0}, author = {Shelobolina, Evgenya S and Vrionis, Helen A and Findlay, Robert H and Lovley, Derek R} } @article {472, title = {Highly conserved genes in Geobacter species with expression patterns indicative of acetate limitation.}, journal = {Microbiology}, volume = {154}, year = {2008}, month = {2008 Sep}, pages = {2589-99}, abstract = {Analysis of the genome of Geobacter sulfurreducens revealed four genes encoding putative symporters with homology to ActP, an acetate transporter in Escherichia coli. Three of these genes, aplA, aplB and aplC, are highly similar (over 90 \% identical) and fell within a tight phylogenetic cluster (Group I) consisting entirely of Geobacter homologues. Transcript levels for all three genes increased in response to acetate limitation. The fourth gene, aplD, is phylogenetically distinct (Group II) and its expression was not influenced by acetate availability. Deletion of any one of the three genes in Group I did not significantly affect acetate-dependent growth, suggesting functional redundancy. Attempts to recover mutants in which various combinations of two of these genes were deleted were unsuccessful, suggesting that at least two of these three transporter genes are required to support growth. Closely related Group I apl genes were found in the genomes of other Geobacter species whose genome sequences are available. Furthermore, related genes could be detected in genomic DNA extracted from a subsurface environment undergoing in situ uranium bioremediation. The transporter genes recovered from the subsurface were most closely related to Group I apl genes found in the genomes of cultured Geobacter species that were isolated from contaminated subsurface environments. The increased expression of these genes in response to acetate limitation, their high degree of conservation among Geobacter species and the ease with which they can be detected in environmental samples suggest that Group I apl genes of the Geobacteraceae may be suitable biomarkers for acetate limitation. Monitoring the expression of these genes could aid in the design of strategies for acetate-mediated in situ bioremediation of uranium-contaminated groundwater.}, keywords = {Acetates, Biodegradation, Environmental, DNA, Bacterial, Escherichia coli, Escherichia coli Proteins, Gene Deletion, Gene Expression, Genes, Bacterial, Genome, Bacterial, Geobacter, Membrane Transport Proteins, Phylogeny, Uranium}, issn = {1350-0872}, doi = {10.1099/mic.0.2008/017244-0}, author = {Risso, Carla and Meth{\'e}, Barbara A and Elifantz, Hila and Holmes, Dawn E and Lovley, Derek R} } @article {482, title = {Sustained removal of uranium from contaminated groundwater following stimulation of dissimilatory metal reduction.}, journal = {Environ Sci Technol}, volume = {42}, year = {2008}, month = {2008 Apr 15}, pages = {2999-3004}, abstract = {Previous field studies on in situ bioremediation of uranium-contaminated groundwater in an aquifer in Rifle, Colorado identified two distinct phases following the addition of acetate to stimulate microbial respiration. In phase I, Geobacter species are the predominant organisms, Fe(III) is reduced, and microbial reduction of soluble U(VI) to insoluble U(IV) removes uranium from the groundwater. In phase II, Fe(III) is depleted, sulfate is reduced, and sulfate-reducing bacteria predominate. Long-term monitoring revealed an unexpected third phase during which U(VI) removal continues even after acetate additions are stopped. All three of these phases were successfully reproduced in flow-through sediment columns. When sediments from the third phase were heat sterilized, the capacity for U(VI) removal was lost. In the live sediments U(VI) removed from the groundwater was recovered as U(VI) in the sediments. This contrasts to the recovery of U(IV) in sediments resulting from the reduction of U(VI) to U(IV) during the Fe(III) reduction phase in acetate-amended sediments. Analysis of 16S rRNA gene sequences in the sediments in which U(VI) was being adsorbed indicated that members of the Firmicutes were the predominant organisms whereas no Firmicutes sequences were detected in background sediments which did not have the capacity to sorb U(VI), suggesting that the U(VI) adsorption might be due to the presence of these living organisms or at least their intact cell components. This unexpected enhanced adsorption of U(VI) onto sediments following the stimulation of microbial growth in the subsurface may potentially enhance the cost effectiveness of in situ uranium bioremediation.}, keywords = {Acetates, Adsorption, Bacteria, Colorado, Geologic Sediments, Oxidation-Reduction, RNA, Ribosomal, 16S, Sulfates, Uranium, Water Pollutants, Radioactive, Water Supply}, issn = {0013-936X}, author = {N{\textquoteright}guessan, Lucie A and Vrionis, Helen A and Resch, Charles T and Long, Philip E and Lovley, Derek R} } @article {505, title = {Importance of c-Type cytochromes for U(VI) reduction by Geobacter sulfurreducens.}, journal = {BMC Microbiol}, volume = {7}, year = {2007}, month = {2007}, pages = {16}, abstract = {BACKGROUND: In order to study the mechanism of U(VI) reduction, the effect of deleting c-type cytochrome genes on the capacity of Geobacter sulfurreducens to reduce U(VI) with acetate serving as the electron donor was investigated. RESULTS: The ability of several c-type cytochrome deficient mutants to reduce U(VI) was lower than that of the wild type strain. Elimination of two confirmed outer membrane cytochromes and two putative outer membrane cytochromes significantly decreased (ca. 50-60\%) the ability of G. sulfurreducens to reduce U(VI). Involvement in U(VI) reduction did not appear to be a general property of outer membrane cytochromes, as elimination of two other confirmed outer membrane cytochromes, OmcB and OmcC, had very little impact on U(VI) reduction. Among the periplasmic cytochromes, only MacA, proposed to transfer electrons from the inner membrane to the periplasm, appeared to play a significant role in U(VI) reduction. A subpopulation of both wild type and U(VI) reduction-impaired cells, 24-30\%, accumulated amorphous uranium in the periplasm. Comparison of uranium-accumulating cells demonstrated a similar amount of periplasmic uranium accumulation in U(VI) reduction-impaired and wild type G. sulfurreducens. Assessment of the ability of the various suspensions to reduce Fe(III) revealed no correlation between the impact of cytochrome deletion on U(VI) reduction and reduction of Fe(III) hydroxide and chelated Fe(III). CONCLUSION: This study indicates that c-type cytochromes are involved in U(VI) reduction by Geobacter sulfurreducens. The data provide new evidence for extracellular uranium reduction by G. sulfurreducens but do not rule out the possibility of periplasmic uranium reduction. Occurrence of U(VI) reduction at the cell surface is supported by the significant impact of elimination of outer membrane cytochromes on U(VI) reduction and the lack of correlation between periplasmic uranium accumulation and the capacity for uranium reduction. Periplasmic uranium accumulation may reflect the ability of uranium to penetrate the outer membrane rather than the occurrence of enzymatic U(VI) reduction. Elimination of cytochromes rarely had a similar impact on both Fe(III) and U(VI) reduction, suggesting that there are differences in the routes of electron transfer to U(VI) and Fe(III). Further studies are required to clarify the pathways leading to U(VI) reduction in G. sulfurreducens.}, keywords = {Biodegradation, Environmental, Cytochrome c Group, Ferric Compounds, Geobacter, Microscopy, Electron, Transmission, Mutation, Oxidation-Reduction, Periplasm, Uranium}, issn = {1471-2180}, doi = {10.1186/1471-2180-7-16}, author = {Shelobolina, Evgenya S and Coppi, Maddalena V and Korenevsky, Anton A and DiDonato, Laurie N and Sullivan, Sara A and Konishi, Hiromi and Xu, Huifang and Leang, Ching and Butler, Jessica E and Kim, Byoung-Chan and Lovley, Derek R} } @article {493, title = {Subsurface clade of Geobacteraceae that predominates in a diversity of Fe(III)-reducing subsurface environments.}, journal = {ISME J}, volume = {1}, year = {2007}, month = {2007 Dec}, pages = {663-77}, abstract = {There are distinct differences in the physiology of Geobacter species available in pure culture. Therefore, to understand the ecology of Geobacter species in subsurface environments, it is important to know which species predominate. Clone libraries were assembled with 16S rRNA genes and transcripts amplified from three subsurface environments in which Geobacter species are known to be important members of the microbial community: (1) a uranium-contaminated aquifer located in Rifle, CO, USA undergoing in situ bioremediation; (2) an acetate-impacted aquifer that serves as an analog for the long-term acetate amendments proposed for in situ uranium bioremediation and (3) a petroleum-contaminated aquifer in which Geobacter species play a role in the oxidation of aromatic hydrocarbons coupled with the reduction of Fe(III). The majority of Geobacteraceae 16S rRNA sequences found in these environments clustered in a phylogenetically coherent subsurface clade, which also contains a number of Geobacter species isolated from subsurface environments. Concatamers constructed with 43 Geobacter genes amplified from these sites also clustered within this subsurface clade. 16S rRNA transcript and gene sequences in the sediments and groundwater at the Rifle site were highly similar, suggesting that sampling groundwater via monitoring wells can recover the most active Geobacter species. These results suggest that further study of Geobacter species in the subsurface clade is necessary to accurately model the behavior of Geobacter species during subsurface bioremediation of metal and organic contaminants.}, keywords = {Biodegradation, Environmental, Ecosystem, Ferric Compounds, Geobacter, Hydrocarbons, Aromatic, Molecular Sequence Data, Oxidation-Reduction, Petroleum, Phylogeny, Polymerase Chain Reaction, RNA, Ribosomal, 16S, Sequence Analysis, DNA, Uranium}, issn = {1751-7362}, doi = {10.1038/ismej.2007.85}, author = {Holmes, Dawn E and O{\textquoteright}Neil, Regina A and Vrionis, Helen A and N{\textquoteright}guessan, Lucie A and Ortiz-Bernad, Irene and Larrahondo, Maria J and Adams, Lorrie A and Ward, Joy A and Nicoll, Julie S and Nevin, Kelly P and Chavan, Milind A and Johnson, Jessica P and Long, Philip E and Lovley, Derek R} } @article {526, title = {Microbial incorporation of 13C-labeled acetate at the field scale: detection of microbes responsible for reduction of U(VI).}, journal = {Environ Sci Technol}, volume = {39}, year = {2005}, month = {2005 Dec 1}, pages = {9039-48}, abstract = {A field-scale acetate amendment experiment was performed in a contaminated aquifer at Old Rifle, CO to stimulate in situ microbial reduction of U(VI) in groundwater. To evaluate the microorganisms responsible for microbial uranium reduction during the experiment, 13C-labeled acetate was introduced into well bores via bio-traps containing porous activated carbon beads (Bio-Sep). Incorporation of the 13C from labeled acetate into cellular DNA and phospholipid fatty acid (PLFA) biomarkers was analyzed in parallel with geochemical parameters. An enrichment of active sigma-proteobacteria was demonstrated in downgradient monitoring wells: Geobacter dominated in wells closer to the acetate injection gallery, while various sulfate reducers were prominent in different downgradient wells. These results were consistent with the geochemical evidence of Fe(III), U(VI), and SO(4)2- reduction. PLFA profiling of bio-traps suspended in the monitoring wells also showed the incorporation of 13C into bacterial cellular lipids. Community composition of downgradient monitoring wells based on quinone and PLFA profiling was in general agreement with the 13C-DNA result. The direct application of 13C label to biosystems, coupled with DNA and PLFA analysis,}, keywords = {Acetates, Biodegradation, Environmental, Carbon Isotopes, Electrophoresis, Polyacrylamide Gel, Geobacter, Phylogeny, Polymerase Chain Reaction, Proteobacteria, Uranium}, issn = {0013-936X}, author = {Chang, Yun-Juan and Long, Philip E and Geyer, Roland and Peacock, Aaron D and Resch, Charles T and Sublette, Kerry and Pfiffner, Susan and Smithgall, Amanda and Anderson, Robert T and Vrionis, Helen A and Stephen, John R and Dayvault, Richard and Ortiz-Bernad, Irene and Lovley, Derek R and White, David C} } @article {530, title = {Microbiological and geochemical heterogeneity in an in situ uranium bioremediation field site.}, journal = {Appl Environ Microbiol}, volume = {71}, year = {2005}, month = {2005 Oct}, pages = {6308-18}, abstract = {The geochemistry and microbiology of a uranium-contaminated subsurface environment that had undergone two seasons of acetate addition to stimulate microbial U(VI) reduction was examined. There were distinct horizontal and vertical geochemical gradients that could be attributed in large part to the manner in which acetate was distributed in the aquifer, with more reduction of Fe(III) and sulfate occurring at greater depths and closer to the point of acetate injection. Clone libraries of 16S rRNA genes derived from sediments and groundwater indicated an enrichment of sulfate-reducing bacteria in the order Desulfobacterales in sediment and groundwater samples. These samples were collected nearest the injection gallery where microbially reducible Fe(III) oxides were highly depleted, groundwater sulfate concentrations were low, and increases in acid volatile sulfide were observed in the sediment. Further down-gradient, metal-reducing conditions were present as indicated by intermediate Fe(II)/Fe(total) ratios, lower acid volatile sulfide values, and increased abundance of 16S rRNA gene sequences belonging to the dissimilatory Fe(III)- and U(VI)-reducing family Geobacteraceae. Maximal Fe(III) and U(VI) reduction correlated with maximal recovery of Geobacteraceae 16S rRNA gene sequences in both groundwater and sediment; however, the sites at which these maxima occurred were spatially separated within the aquifer. The substantial microbial and geochemical heterogeneity at this site demonstrates that attempts should be made to deliver acetate in a more uniform manner and that closely spaced sampling intervals, horizontally and vertically, in both sediment and groundwater are necessary in order to obtain a more in-depth understanding of microbial processes and the relative contribution of attached and planktonic populations to in situ uranium bioremediation.}, keywords = {Acetates, Biodegradation, Environmental, Deltaproteobacteria, DNA, Bacterial, DNA, Ribosomal, Ferric Compounds, Fresh Water, Geologic Sediments, Phylogeny, Polymerase Chain Reaction, RNA, Ribosomal, 16S, Sulfates, Uranium, Water Pollution}, issn = {0099-2240}, doi = {10.1128/AEM.71.10.6308-6318.2005}, author = {Vrionis, Helen A and Anderson, Robert T and Ortiz-Bernad, Irene and O{\textquoteright}Neill, Kathleen R and Resch, Charles T and Peacock, Aaron D and Dayvault, Richard and White, David C and Long, Philip E and Lovley, Derek R} } @article {529, title = {Potential for quantifying expression of the Geobacteraceae citrate synthase gene to assess the activity of Geobacteraceae in the subsurface and on current-harvesting electrodes.}, journal = {Appl Environ Microbiol}, volume = {71}, year = {2005}, month = {2005 Nov}, pages = {6870-7}, abstract = {The Geobacteraceae citrate synthase is phylogenetically distinct from those of other prokaryotes and is a key enzyme in the central metabolism of Geobacteraceae. Therefore, the potential for using levels of citrate synthase mRNA to estimate rates of Geobacter metabolism was evaluated in pure culture studies and in four different Geobacteraceae-dominated environments. Quantitative reverse transcription-PCR studies with mRNA extracted from cultures of Geobacter sulfurreducens grown in chemostats with Fe(III) as the electron acceptor or in batch with electrodes as the electron acceptor indicated that transcript levels of the citrate synthase gene, gltA, increased with increased rates of growth/Fe(III) reduction or current production, whereas the expression of the constitutively expressed housekeeping genes recA, rpoD, and proC remained relatively constant. Analysis of mRNA extracted from groundwater collected from a U(VI)-contaminated site undergoing in situ uranium bioremediation revealed a remarkable correspondence between acetate levels in the groundwater and levels of transcripts of gltA. The expression of gltA was also significantly greater in RNA extracted from groundwater beneath a highway runoff recharge pool that was exposed to calcium magnesium acetate in June, when acetate concentrations were high, than in October, when the levels had significantly decreased. It was also possible to detect gltA transcripts on current-harvesting anodes deployed in freshwater sediments. These results suggest that it is possible to monitor the in situ metabolic rate of Geobacteraceae by tracking the expression of the citrate synthase gene.}, keywords = {Acetates, Citrate (si)-Synthase, Deltaproteobacteria, DNA, Ribosomal, Electrodes, Ferric Compounds, Fresh Water, Geobacter, Geologic Sediments, Petroleum, Phylogeny, RNA, Ribosomal, 16S, Uranium, Water Pollutants, Chemical, Water Pollutants, Radioactive}, issn = {0099-2240}, doi = {10.1128/AEM.71.11.6870-6877.2005}, author = {Holmes, Dawn E and Nevin, Kelly P and O{\textquoteright}Neil, Regina A and Ward, Joy E and Adams, Lorrie A and Woodard, Trevor L and Vrionis, Helen A and Lovley, Derek R} } @article {528, title = {Remediation and recovery of uranium from contaminated subsurface environments with electrodes.}, journal = {Environ Sci Technol}, volume = {39}, year = {2005}, month = {2005 Nov 15}, pages = {8943-7}, abstract = {Previous studies have demonstrated that Geobacter species can effectively remove uranium from contaminated groundwater by reducing soluble U(VI) to the relatively insoluble U(IV) with organic compounds serving as the electron donor. Studies were conducted to determine whether electrodes might serve as an alternative electron donor for U(VI) reduction by a pure culture of Geobacter sulfurreducens and microorganisms in uranium-contaminated sediments. Electrodes poised at -500 mV (vs a Ag/AgCl reference) rapidly removed U(VI) from solution in the absence of cells. However, when the poise at the electrode was removed, all of the U(VI) returned to solution, demonstrating that the electrode did not reduce U(VI). If G. sulfurreducens was present on the electrode, U(VI) did not return to solution until the electrode was exposed to dissolved oxygen. This suggeststhat G. sulfurreducens on the electrode reduced U(VI) to U(IV) which was stably precipitated until reoxidized in the presence of oxygen. When an electrode was placed in uranium-contaminated subsurface sediments, U(VI) was removed and recovered from groundwater using poised electrodes. Electrodes emplaced in flow-through columns of uranium-contaminated sediments readily removed U(VI) from the groundwater, and 87\% of the uranium that had been removed was recovered from the electrode surface after the electrode was pulled from the sediments. These results suggest that microorganisms can use electrons derived from electrodes to reduce U(VI) and that it may be possible to remove and recover uranium from contaminated groundwater with poised electrodes.}, keywords = {Biodegradation, Environmental, Electrodes, Geobacter, Graphite, Soil Pollutants, Uranium, Water Pollutants, Chemical}, issn = {0013-936X}, author = {Gregory, Kelvin B and Lovley, Derek R} } @article {554, title = {Isolation, characterization, and U(VI)-reducing potential of a facultatively anaerobic, acid-resistant Bacterium from Low-pH, nitrate- and U(VI)-contaminated subsurface sediment and description of Salmonella subterranea sp. nov.}, journal = {Appl Environ Microbiol}, volume = {70}, year = {2004}, month = {2004 May}, pages = {2959-65}, abstract = {A facultatively anaerobic, acid-resistant bacterium, designated strain FRCl, was isolated from a low-pH, nitrate- and U(VI)-contaminated subsurface sediment at site FW-024 at the Natural and Accelerated Bioremediation Research Field Research Center in Oak Ridge, Tenn. Strain FRCl was enriched at pH 4.5 in minimal medium with nitrate as the electron acceptor, hydrogen as the electron donor, and acetate as the carbon source. Clones with 16S ribosomal DNA (rDNA) sequences identical to the sequence of strain FRCl were also detected in a U(VI)-reducing enrichment culture derived from the same sediment. Cells of strain FRCl were gram-negative motile regular rods 2.0 to 3.4 micro m long and 0.7 to 0.9 microm in diameter. Strain FRCl was positive for indole production, by the methyl red test, and for ornithine decarboxylase; it was negative by the Voges-Proskauer test (for acetylmethylcarbinol production), for urea hydrolysis, for arginine dihydrolase, for lysine decarboxylase, for phenylalanine deaminase, for H(2)S production, and for gelatin hydrolysis. Strain FRCl was capable of using O(2), NO(3)(-), S(2)O(3)(2-), fumarate, and malate as terminal electron acceptors and of reducing U(VI) in the cell suspension. Analysis of the 16S rDNA sequence of the isolate indicated that this strain was 96.4\% similar to Salmonella bongori and 96.3\% similar to Enterobacter cloacae. Physiological and phylogenetic analyses suggested that strain FRCl belongs to the genus Salmonella and represents a new species, Salmonella subterranea sp. nov.}, keywords = {Anaerobiosis, Culture Media, DNA, Ribosomal, Fresh Water, Geologic Sediments, Hydrogen-Ion Concentration, Molecular Sequence Data, Nitrates, Oxidation-Reduction, Phylogeny, RNA, Ribosomal, 16S, Salmonella, Sequence Analysis, DNA, Uranium, Water Pollution, Chemical}, issn = {0099-2240}, author = {Shelobolina, Evgenya S and Sullivan, Sara A and O{\textquoteright}Neill, Kathleen R and Nevin, Kelly P and Lovley, Derek R} } @article {542, title = {Resistance of solid-phase U(VI) to microbial reduction during in situ bioremediation of uranium-contaminated groundwater.}, journal = {Appl Environ Microbiol}, volume = {70}, year = {2004}, month = {2004 Dec}, pages = {7558-60}, abstract = {Speciation of solid-phase uranium in uranium-contaminated subsurface sediments undergoing uranium bioremediation demonstrated that although microbial reduction of soluble U(VI) readily immobilized uranium as U(IV), a substantial portion of the U(VI) in the aquifer was strongly associated with the sediments and was not microbially reducible. These results have important implications for in situ uranium bioremediation strategies.}, keywords = {Acetates, Biodegradation, Environmental, Deltaproteobacteria, Fresh Water, Geologic Sediments, Oxidation-Reduction, Solubility, Uranium, Water Pollutants, Radioactive}, issn = {0099-2240}, doi = {10.1128/AEM.70.12.7558-7560.2004}, author = {Ortiz-Bernad, Irene and Anderson, Robert T and Vrionis, Helen A and Lovley, Derek R} } @article {568, title = {Microorganisms associated with uranium bioremediation in a high-salinity subsurface sediment.}, journal = {Appl Environ Microbiol}, volume = {69}, year = {2003}, month = {2003 Jun}, pages = {3672-5}, abstract = {Although stimulation of dissimilatory metal reduction to promote the reductive precipitation of uranium has been shown to successfully remove uranium from some aquifer sediments, the organisms in the family Geobacteraceae that have been found to be associated with metal reduction in previous studies are not known to grow at the high salinities found in some uranium-contaminated groundwaters. Studies with a highly saline uranium-contaminated aquifer sediment demonstrated that the addition of acetate could stimulate the removal of U(VI) from the groundwater. This removal was associated with an enrichment in microorganisms most closely related to Pseudomonas and Desulfosporosinus species.}, keywords = {Acetates, Biodegradation, Environmental, DNA, Ribosomal, Fresh Water, Geologic Sediments, Peptococcaceae, Polymerase Chain Reaction, Pseudomonas, RNA, Ribosomal, 16S, Sodium Chloride, Uranium, Water Pollution}, issn = {0099-2240}, author = {Nevin, Kelly P and Finneran, Kevin T and Lovley, Derek R} } @article {563, title = {Stimulating the in situ activity of Geobacter species to remove uranium from the groundwater of a uranium-contaminated aquifer.}, journal = {Appl Environ Microbiol}, volume = {69}, year = {2003}, month = {2003 Oct}, pages = {5884-91}, abstract = {The potential for removing uranium from contaminated groundwater by stimulating the in situ activity of dissimilatory metal-reducing microorganisms was evaluated in a uranium-contaminated aquifer located in Rifle, Colo. Acetate (1 to 3 mM) was injected into the subsurface over a 3-month period via an injection gallery composed of 20 injection wells, which was installed upgradient from a series of 15 monitoring wells. U(VI) concentrations decreased in as little as 9 days after acetate injection was initiated, and within 50 days uranium had declined below the prescribed treatment level of 0.18 micro M in some of the monitoring wells. Analysis of 16S ribosomal DNA (rDNA) sequences and phospholipid fatty acid profiles demonstrated that the initial loss of uranium from the groundwater was associated with an enrichment of Geobacter species in the treatment zone. Fe(II) in the groundwater also increased during this period, suggesting that U(VI) reduction was coincident with Fe(III) reduction. As the acetate injection continued over 50 days there was a loss of sulfate from the groundwater and an accumulation of sulfide and the composition of the microbial community changed. Organisms with 16S rDNA sequences most closely related to those of sulfate reducers became predominant, and Geobacter species became a minor component of the community. This apparent switch from Fe(III) reduction to sulfate reduction as the terminal electron accepting process for the oxidation of the injected acetate was associated with an increase in uranium concentration in the groundwater. These results demonstrate that in situ bioremediation of uranium-contaminated groundwater is feasible but suggest that the strategy should be optimized to better maintain long-term activity of Geobacter species.}, keywords = {Acetates, DNA, Ribosomal, Ecosystem, Fatty Acids, Ferric Compounds, Fresh Water, Geobacter, Mining, Oxidation-Reduction, Phospholipids, RNA, Ribosomal, 16S, Sulfates, Uranium, Water Pollution, Chemical}, issn = {0099-2240}, author = {Anderson, Robert T and Vrionis, Helen A and Ortiz-Bernad, Irene and Resch, Charles T and Long, Philip E and Dayvault, Richard and Karp, Ken and Marutzky, Sam and Metzler, Donald R and Peacock, Aaron and White, David C and Lowe, Mary and Lovley, Derek R} } @article {572, title = {Analysis of the genetic potential and gene expression of microbial communities involved in the in situ bioremediation of uranium and harvesting electrical energy from organic matter.}, journal = {OMICS}, volume = {6}, year = {2002}, month = {2002}, pages = {331-9}, abstract = {The proposed research will investigate two microbial communities that are of direct relevance to Department of Energy interests. One is the microbial community associated with the in situ bioremediation of uranium-contaminated groundwater. The second is a microbial community that harvests energy from waste organic matter in the form of electricity. These studies will address DOE needs for (1) remediation of metals and radionuclides at DOE sites and (2) the development of cleaner forms of energy and biomass conversion to energy. Our previous studies have demonstrated that the microbial communities involved in uranium bioremediation and energy harvesting are both dominated by microorganisms in the family Geobacteraceae and that the organisms in this family are responsible for uranium bioremediation and electron transfer to electrodes. The initial objectives of this study are to (1) describe the genetic potential of the Geobacteraceae that predominate in the environments of interest; (2) identify conserved patterns of gene expression within the Geobacteraceae family in response to a range of environmental conditions; (3) begin to identify mechanisms controlling the expression of key genes related to survival, growth, and activity in subsurface environments and on electrodes; and (4) use the results from subobjectives 1-3 to develop a conceptual model for predicting gene expression of Geobacteraceae in the environments of interest. This will serve as the basis for a subsequent simulation model of the growth and activity of Geobacteraceae in the subsurface and on electrodes.}, keywords = {Biodegradation, Environmental, Deltaproteobacteria, Electricity, Electrodes, Energy Metabolism, Gene Expression Regulation, Bacterial, Genome, Bacterial, Geologic Sediments, Models, Theoretical, Uranium}, issn = {1536-2310}, doi = {10.1089/153623102321112755}, author = {Lovley, Derek R} } @article {583, title = {Enrichment of members of the family Geobacteraceae associated with stimulation of dissimilatory metal reduction in uranium-contaminated aquifer sediments.}, journal = {Appl Environ Microbiol}, volume = {68}, year = {2002}, month = {2002 May}, pages = {2300-6}, abstract = {Stimulating microbial reduction of soluble U(VI) to insoluble U(IV) shows promise as a strategy for immobilizing uranium in uranium-contaminated subsurface environments. In order to learn more about which microorganisms might be involved in U(VI) reduction in situ, the changes in the microbial community when U(VI) reduction was stimulated with the addition of acetate were monitored in sediments from three different uranium-contaminated sites in the floodplain of the San Juan River in Shiprock, N.Mex. In all three sediments U(VI) reduction was accompanied by concurrent Fe(III) reduction and a dramatic enrichment of microorganisms in the family Geobacteraceae, which are known U(VI)- and Fe(III)-reducing microorganisms. At the point when U(VI) reduction and Fe(III) reduction were nearing completion, Geobacteraceae accounted for ca. 40\% of the 16S ribosomal DNA (rDNA) sequences recovered from the sediments with bacterial PCR primers, whereas Geobacteraceae accounted for fewer than 5\% of the 16S rDNA sequences in control sediments that were not amended with acetate and in which U(VI) and Fe(III) reduction were not stimulated. Between 55 and 65\% of these Geobacteraceae sequences were most similar to sequences from Desulfuromonas species, with the remainder being most closely related to Geobacter species. Quantitative analysis of Geobacteraceae sequences with most-probable-number PCR and TaqMan analyses indicated that the number of Geobacteraceae sequences increased from 2 to 4 orders of magnitude over the course of U(VI) and Fe(III) reduction in the acetate-amended sediments from the three sites. No increase in Geobacteraceae sequences was observed in control sediments. In contrast to the predominance of Geobacteraceae sequences, no sequences related to other known Fe(III)-reducing microorganisms were detected in sediments. These results compare favorably with an increasing number of studies which have demonstrated that Geobacteraceae are important components of the microbial community in a diversity of subsurface environments in which Fe(III) reduction is an important process. The combination of these results with the finding that U(VI) reduction takes place during Fe(III) reduction and prior to sulfate reduction suggests that Geobacteraceae will be responsible for much of the Fe(III) and U(VI) reduction during uranium bioremediation in these sediments.}, keywords = {Gene Library, Geologic Sediments, Iron, Oxidation-Reduction, Proteobacteria, RNA, Ribosomal, 16S, Soil Pollutants, Uranium}, issn = {0099-2240}, author = {Holmes, Dawn E and Finneran, Kevin T and O{\textquoteright}Neil, Regina A and Lovley, Derek R} } @article {577, title = {Multiple influences of nitrate on uranium solubility during bioremediation of uranium-contaminated subsurface sediments.}, journal = {Environ Microbiol}, volume = {4}, year = {2002}, month = {2002 Sep}, pages = {510-6}, abstract = {Microbiological reduction of soluble U(VI) to insoluble U(IV) has been proposed as a remediation strategy for uranium-contaminated groundwater. Nitrate is a common co-contaminant with uranium. Nitrate inhibited U(VI) reduction in acetate-amended aquifer sediments collected from a uranium-contaminated site in New Mexico. Once nitrate was depleted, both U(VI) and Fe(III) were reduced concurrently. When nitrate was added to sediments in which U(VI) had been reduced, U(VI) reappeared in solution. Parallel studies with the dissimilatory Fe(III)-, U(VI)- and nitrate-reducing microorganism, Geobacter metallireducens, demonstrated that nitrate inhibited reduction of Fe(III) and U(VI) in cell suspensions of cells that had been grown with nitrate as the electron acceptor, but not in Fe(III)-grown cells. Suspensions of nitrate-grown G. metallireducens oxidized Fe(II) and U(IV) with nitrate as the electron acceptor. U(IV) oxidation was accelerated when Fe(II) was also added, presumably due to the Fe(III) being formed abiotically oxidizing U(IV). These studies demonstrate that although the presence of nitrate is not likely to be an impediment to the bioremediation of uranium contamination with microbial U(VI) reduction, it is necessary to reduce nitrate before U(VI) can be reduced. These results also suggest that anaerobic oxidation of U(IV) to U(VI) with nitrate serving as the electron acceptor may provide a novel strategy for solubilizing and extracting microbial U(IV) precipitates from the subsurface.}, keywords = {Ferric Compounds, Ferrous Compounds, Geologic Sediments, Nitrates, Oxidation-Reduction, Proteobacteria, Soil Pollutants, Solubility, Uranium, Water Pollutants, Chemical}, issn = {1462-2912}, author = {Finneran, Kevin T and Housewright, Meghan E and Lovley, Derek R} } @article {614, title = {Reduction of Fe(III), Mn(IV), and toxic metals at 100 degrees C by Pyrobaculum islandicum.}, journal = {Appl Environ Microbiol}, volume = {66}, year = {2000}, month = {2000 Mar}, pages = {1050-6}, abstract = {It has recently been noted that a diversity of hyperthermophilic microorganisms have the ability to reduce Fe(III) with hydrogen as the electron donor, but the reduction of Fe(III) or other metals by these organisms has not been previously examined in detail. When Pyrobaculum islandicum was grown at 100 degrees C in a medium with hydrogen as the electron donor and Fe(III)-citrate as the electron acceptor, the increase in cell numbers of P. islandicum per mole of Fe(III) reduced was found to be ca. 10-fold higher than previously reported. Poorly crystalline Fe(III) oxide could also serve as the electron acceptor for growth on hydrogen. The stoichiometry of hydrogen uptake and Fe(III) oxide reduction was consistent with the oxidation of 1 mol of hydrogen resulting in the reduction of 2 mol of Fe(III). The poorly crystalline Fe(III) oxide was reduced to extracellular magnetite. P. islandicum could not effectively reduce the crystalline Fe(III) oxide minerals goethite and hematite. In addition to using hydrogen as an electron donor for Fe(III) reduction, P. islandicum grew via Fe(III) reduction in media in which peptone and yeast extract served as potential electron donors. The closely related species P. aerophilum grew via Fe(III) reduction in a similar complex medium. Cell suspensions of P. islandicum reduced the following metals with hydrogen as the electron donor: U(VI), Tc(VII), Cr(VI), Co(III), and Mn(IV). The reduction of these metals was dependent upon the presence of cells and hydrogen. The metalloids arsenate and selenate were not reduced. U(VI) was reduced to the insoluble U(IV) mineral uraninite, which was extracellular. Tc(VII) was reduced to insoluble Tc(IV) or Tc(V). Cr(VI) was reduced to the less toxic, less soluble Cr(III). Co(III) was reduced to Co(II). Mn(IV) was reduced to Mn(II) with the formation of manganese carbonate. These results demonstrate that biological reduction may contribute to the speciation of metals in hydrothermal environments and could account for such phenomena as magnetite accumulation and the formation of uranium deposits at ca. 100 degrees C. Reduction of toxic metals with hyperthermophilic microorganisms or their enzymes might be applied to the remediation of metal-contaminated waters or waste streams.}, keywords = {Chromium, Cobalt, Energy Metabolism, Ferric Compounds, Geological Phenomena, Geology, Iron, Manganese, Metals, Heavy, Oxidation-Reduction, Technetium, Thermoproteaceae, Uranium, Water Pollutants, Chemical}, issn = {0099-2240}, author = {Kashefi, K and Lovley, D R} } @article {641, title = {Bioremediation of organic and metal contaminants with dissimilatory metal reduction.}, journal = {J Ind Microbiol}, volume = {14}, year = {1995}, month = {1995 Feb}, pages = {85-93}, abstract = {Dissimilatory metal reduction has the potential to be a helpful mechanism for both intrinsic and engineered bioremediation of contaminated environments. Dissimilatory Fe(III) reduction is an important intrinsic process for removing organic contaminants from aquifers contaminated with petroleum or landfill leachate. Stimulation of microbial Fe(III) reduction can enhance the degradation of organic contaminants in ground water. Dissimilatory reduction of uranium, selenium, chromium, technetium, and possibly other metals, can convert soluble metal species to insoluble forms that can readily be removed from contaminated waters or waste streams. Reduction of mercury can volatilize mercury from waters and soils. Despite its potential, there has as yet been limited applied research into the use of dissimilatory metal reduction as a bioremediation tool.}, keywords = {Bacteria, Biodegradation, Environmental, Chromium, Hydrocarbons, Iron, Mercury, Metals, Oxidation-Reduction, Selenium, Uranium}, issn = {0169-4146}, author = {Lovley, D R} } @article {647, title = {Reduction of uranium by cytochrome c3 of Desulfovibrio vulgaris.}, journal = {Appl Environ Microbiol}, volume = {59}, year = {1993}, month = {1993 Nov}, pages = {3572-6}, abstract = {The mechanism for U(VI) reduction by Desulfovibrio vulgaris (Hildenborough) was investigated. The H2-dependent U(VI) reductase activity in the soluble fraction of the cells was lost when the soluble fraction was passed over a cationic exchange column which extracted cytochrome c3. Addition of cytochrome c3 back to the soluble fraction that had been passed over the cationic exchange column restored the U(VI)-reducing capacity. Reduced cytochrome c3 was oxidized by U(VI), as was a c-type cytochrome(s) in whole-cell suspensions. When cytochrome c3 was combined with hydrogenase, its physiological electron donor, U(VI) was reduced in the presence of H2. Hydrogenase alone could not reduce U(VI). Rapid U(VI) reduction was followed by a subsequent slow precipitation of the U(IV) mineral uraninite. Cytochrome c3 reduced U(VI) in a uranium-contaminated surface water and groundwater. Cytochrome c3 provides the first enzyme model for the reduction and biomineralization of uranium in sedimentary environments. Furthermore, the finding that cytochrome c3 can catalyze the reductive precipitation of uranium may aid in the development of fixed-enzyme reactors and/or organisms with enhanced U(VI)-reducing capacity for the bioremediation of uranium-contaminated waters and waste streams.}, keywords = {Biotransformation, Chemical Precipitation, Cytochrome c Group, Desulfovibrio vulgaris, Oxidation-Reduction, Uranium, Water Pollutants, Radioactive}, issn = {0099-2240}, author = {Lovley, D R and Widman, P K and Woodward, J C and Phillips, E J} } @article {655, title = {Reduction of uranium by Desulfovibrio desulfuricans.}, journal = {Appl Environ Microbiol}, volume = {58}, year = {1992}, month = {1992 Mar}, pages = {850-6}, abstract = {The possibility that sulfate-reducing microorganisms contribute to U(VI) reduction in sedimentary environments was investigated. U(VI) was reduced to U(IV) when washed cells of sulfate-grown Desulfovibrio desulfuricans were suspended in a bicarbonate buffer with lactate or H2 as the electron donor. There was no U(VI) reduction in the absence of an electron donor or when the cells were killed by heat prior to the incubation. The rates of U(VI) reduction were comparable to those in respiratory Fe(III)-reducing microorganisms. Azide or prior exposure of the cells to air did not affect the ability of D. desulfuricans to reduce U(VI). Attempts to grow D. desulfuricans with U(VI) as the electron acceptor were unsuccessful. U(VI) reduction resulted in the extracellular precipitation of the U(IV) mineral uraninite. The presence of sulfate had no effect on the rate of U(VI) reduction. Sulfate and U(VI) were reduced simultaneously. Enzymatic reduction of U(VI) by D. desulfuricans was much faster than nonenzymatic reduction of U(VI) by sulfide, even when cells of D. desulfuricans were added to provide a potential catalytic surface for the nonenzymatic reaction. The results indicate that enzymatic U(VI) reduction by sulfate-reducing microorganisms may be responsible for the accumulation of U(IV) in sulfidogenic environments. Furthermore, since the reduction of U(VI) to U(IV) precipitates uranium from solution, D. desulfuricans might be a useful organism for recovering uranium from contaminated waters and waste streams.}, keywords = {Biodegradation, Environmental, Desulfovibrio, Kinetics, Oxidation-Reduction, Sulfates, Uranium, X-Ray Diffraction}, issn = {0099-2240}, author = {Lovley, D R and Phillips, E J} }