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Title: Structure and Function of Metal- and Nitrate-reducing Microbial Communities in the FRC Subsurface

Abstract

The overall goal of this study is to evaluate structure-function relationships of sedimentary microbial communities likely to regulate U(VI) reduction and immobilization in the subsurface of Area 2 at the Field Research Center (FRC), Oak Ridge, TN. Microcosm experiments were conducted under near in situ conditions with FRC subsurface materials cocontaminated with high levels of U(VI) and nitrate. The activity, abundance, and community composition of microorganisms was determined in microcosm samples, stimulated with ethanol or glucose, and compared to those from sediment cores and unamended controls. Activity was assessed by monitoring terminal electron accepting processes (TEAPs; nitrate, sulfate, uranium, and iron reduction) as well as electron donor utilization. Microbial functional groups, nitrate- and iron(III)-reducing bacteria, were enumerated during the nitrate- and metal-reduction phases of the incubation and in sediment core samples using a most probable number (MPN) serial dilution assay. U(VI) and Fe(III) were reduced concurrently in the glucose but not the ethanol treatments. In ethanol-amended microcosms, U(VI) was reduced during a 4-day lag phase between nitrate- and Fe(III)-reduction phases. Biostimulation resulted in 3 to 5 orders of magnitude higher counts of Fe(III)-reducing bacteria, whereas populations of nitrate-reducers were enhanced by 1 to 3 orders of magnitude. One to 2more » orders of magnitude more Fe(III)-reducers were observed in ethanol- as compared to glucose-amended treatments in parallel with enhanced U(VI) removal in ethanol treatments. Cultivatable Fe(III)-reducing bacteria in the ethanol treatments were dominated by Geobacter sp. while those cultured on glucose were dominated by fermentative organisms, i.e., Tolumonas sp. Currently, carbon substrate utilization is being examined through HPLC analysis of microcosm porewaters. In addition, changes in the overall microbial community composition are being assessed using cultivation-independent techniques, including fluorescence in situ hybridization (FISH), terminal restriction fragment length polymorphism analysis (T-RFLP) and cloning/sequencing of structural and functional genes. Our results indicate that the microbially-catalyzed mechanism of U(VI) reduction is electron donor dependent and that more effective U(VI) removal is achieved in parallel with an enrichment of Geobacter sp. upon treatment with ethanol.« less

Authors:
; ; ; ;
Publication Date:
Research Org.:
Department of Oceanography, Florida State University, Tallahassee, FL; Institute of Marine and Coastal Sciences, Rutgers University, New Brunswick, NJ
Sponsoring Org.:
USDOE Office of Science (SC)
OSTI Identifier:
894678
Report Number(s):
CONF-ERSP2006-52
TRN: US200702%%195
Resource Type:
Conference
Resource Relation:
Conference: Annual Environmental Remediation Sciences Program PI Meeting, April 3-5, 2006, Warrenton, VA
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 54 ENVIRONMENTAL SCIENCES; BACTERIA; BINDING ENERGY; ELECTRONS; ETHANOL; FLUORESCENCE; FUNCTIONALS; GENES; GLUCOSE; HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY; INCUBATION; IN-SITU HYBRIDIZATION; IRON; MICROCOSMS; MICROORGANISMS; REMOVAL; SEDIMENTS; SUBSTRATES; URANIUM; VALENCE

Citation Formats

Akob, Denise M., Mills, Heath J., Kerkhof, Lee, Gihring, Thomas M., and Kostk, Joel E. Structure and Function of Metal- and Nitrate-reducing Microbial Communities in the FRC Subsurface. United States: N. p., 2006. Web.
Akob, Denise M., Mills, Heath J., Kerkhof, Lee, Gihring, Thomas M., & Kostk, Joel E. Structure and Function of Metal- and Nitrate-reducing Microbial Communities in the FRC Subsurface. United States.
Akob, Denise M., Mills, Heath J., Kerkhof, Lee, Gihring, Thomas M., and Kostk, Joel E. Wed . "Structure and Function of Metal- and Nitrate-reducing Microbial Communities in the FRC Subsurface". United States. doi:. https://www.osti.gov/servlets/purl/894678.
@article{osti_894678,
title = {Structure and Function of Metal- and Nitrate-reducing Microbial Communities in the FRC Subsurface},
author = {Akob, Denise M. and Mills, Heath J. and Kerkhof, Lee and Gihring, Thomas M. and Kostk, Joel E.},
abstractNote = {The overall goal of this study is to evaluate structure-function relationships of sedimentary microbial communities likely to regulate U(VI) reduction and immobilization in the subsurface of Area 2 at the Field Research Center (FRC), Oak Ridge, TN. Microcosm experiments were conducted under near in situ conditions with FRC subsurface materials cocontaminated with high levels of U(VI) and nitrate. The activity, abundance, and community composition of microorganisms was determined in microcosm samples, stimulated with ethanol or glucose, and compared to those from sediment cores and unamended controls. Activity was assessed by monitoring terminal electron accepting processes (TEAPs; nitrate, sulfate, uranium, and iron reduction) as well as electron donor utilization. Microbial functional groups, nitrate- and iron(III)-reducing bacteria, were enumerated during the nitrate- and metal-reduction phases of the incubation and in sediment core samples using a most probable number (MPN) serial dilution assay. U(VI) and Fe(III) were reduced concurrently in the glucose but not the ethanol treatments. In ethanol-amended microcosms, U(VI) was reduced during a 4-day lag phase between nitrate- and Fe(III)-reduction phases. Biostimulation resulted in 3 to 5 orders of magnitude higher counts of Fe(III)-reducing bacteria, whereas populations of nitrate-reducers were enhanced by 1 to 3 orders of magnitude. One to 2 orders of magnitude more Fe(III)-reducers were observed in ethanol- as compared to glucose-amended treatments in parallel with enhanced U(VI) removal in ethanol treatments. Cultivatable Fe(III)-reducing bacteria in the ethanol treatments were dominated by Geobacter sp. while those cultured on glucose were dominated by fermentative organisms, i.e., Tolumonas sp. Currently, carbon substrate utilization is being examined through HPLC analysis of microcosm porewaters. In addition, changes in the overall microbial community composition are being assessed using cultivation-independent techniques, including fluorescence in situ hybridization (FISH), terminal restriction fragment length polymorphism analysis (T-RFLP) and cloning/sequencing of structural and functional genes. Our results indicate that the microbially-catalyzed mechanism of U(VI) reduction is electron donor dependent and that more effective U(VI) removal is achieved in parallel with an enrichment of Geobacter sp. upon treatment with ethanol.},
doi = {},
journal = {},
number = ,
volume = ,
place = {United States},
year = {Wed Apr 05 00:00:00 EDT 2006},
month = {Wed Apr 05 00:00:00 EDT 2006}
}

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  • The reduction of structural Fe in smectite may be mediated either abiotically by reaction with chemical reducing agents or biotically by reaction with various bacterial species. The effects of abiotic reduction on clay surface chemistry are much better known than the effects of biotic reduction, and differences between them are still in need of investigation. The purpose of the present study was to compare the effects of dithionite (abiotic) and bacteria (biotic) reduction of structural Fe in nontronite on the clay structure as observed by variable-temperature Mössbauer spectroscopy. Biotic reduction was accomplished by incubating Na-saturated Garfield nontronite (sample API 33a)more » with« less
  • The objectives of this project were to: (1) isolate and characterize novel anaerobic prokaryotes from subsurface environments exposed to high levels of mixed contaminants (U(VI), nitrate, sulfate), (2) elucidate the diversity and distribution of metabolically active metal- and nitrate-reducing prokaryotes in subsurface sediments, and (3) determine the biotic and abiotic mechanisms linking electron transport processes (nitrate, Fe(III), and sulfate reduction) to radionuclide reduction and immobilization. Mechanisms of electron transport and U(VI) transformation were examined under near in situ conditions in sediment microcosms and in field investigations at the Oak Ridge Field Research Center (ORFRC), in Oak Ridge, Tennessee, where themore » subsurface is exposed to mixed contamination predominated by uranium and nitrate. A total of 20 publications (16 published or 'in press' and 4 in review), 10 invited talks, and 43 contributed seminars/ meeting presentations were completed during the past four years of the project. PI Kostka served on one proposal review panel each year for the U.S. DOE Office of Science during the four year project period. The PI leveraged funds from the state of Florida to purchase new instrumentation that aided the project. Support was also leveraged by the PI from the Joint Genome Institute in the form of two successful proposals for genome sequencing. Draft genomes are now available for two novel species isolated during our studies and 5 more genomes are in the pipeline. We effectively addressed each of the three project objectives and research highlights are provided. Task I - Isolation and characterization of novel anaerobes: (1) A wide range of pure cultures of metal-reducing bacteria, sulfate-reducing bacteria, and denitrifying bacteria (32 strains) were isolated from subsurface sediments of the Oak Ridge Field Research Center (ORFRC), where the subsurface is exposed to mixed contamination of uranium and nitrate. These isolates which are new to science all show high sequence identity to sequences retrieved from ORFRC subsurface. (2) Based on physiological and phylogenetic characterization, two new species of subsurface bacteria were described: the metal-reducer Geobacter daltonii, and the denitrifier Rhodanobacter denitrificans. (3) Strains isolated from the ORFRC show that Rhodanobacter species are well adapted to the contaminated subsurface. Strains 2APBS1 and 116-2 grow at high salt (3% NaCl), low pH (3.5) and tolerate high concentrations of nitrate (400mM) and nitrite (100mM). Strain 2APBS1 was demonstrated to grow at in situ acidic pHs down to 2.5. (4) R. denitrificans strain 2APBS1 is the first described Rhodanobacter species shown to denitrify. Nitrate is almost entirely converted to N2O, which may account for the large accumulation of N2O in the ORFRC subsurface. (5) G. daltonii, isolated from uranium- and hydrocarbon-contaminated subsurface sediments of the ORFRC, is the first organism from the subsurface clade of the genus Geobacter that is capable of growth on aromatic hydrocarbons. (6) High quality draft genome sequences and a complete eco-physiological description are completed for R. denitrificans strain 2APBS1 and G. daltonii strain FRC-32. (7) Given their demonstrated relevance to DOE remediation efforts and the availability of detailed genotypic/phenotypic characterization, Rhodanobacter denitrificans strain 2APBS1 and Geobacter daltonii strain FRC-32 represent ideal model organisms to provide a predictive understanding of subsurface microbial activity through metabolic modeling. Tasks II and III-Diversity and distribution of active anaerobes and Mechanisms linking electron transport and the fate of radionuclides: (1) Our study showed that members of genus Rhodanobacter and Geobacter are abundant and active in the uranium and nitrate contaminated subsurface. In the contaminant source zone of the Oak Ridge site, Rhodanobacter spp. are the predominant, active organisms detected (comprising 50% to 100% of rRNA detected). (2) We demonstrated for the first time that the function of microbial communities can be quantified in subsurface sediments using messenger RNA assays (molecular proxies) under in situ conditions. (3) Active Geobacteraceae were identified and phylogenetically characterized from the cDNA of messenger RNA extracted from ORFRC subsurface sediment cores. Multiple clone sequences were retrieved from G. uraniireducens, G. daltonii, and G. metallireducens. (4) Results show that Geobacter strain FRC-32 is capable of growth on benzoate, toluene and benzene as the electron donor, thereby providing evidence that this strain is physiologically distinct from other described members of the subsurface Geobacter clade. (5) Fe(III)-reducing bacteria transform structural Fe in clay minerals from their layer edges rather than from their basal surfaces.« less
  • The purpose of this study was to provide comparative information regarding the changes in clay structure that occur due to biotic or abiotic reduction, as probed by variable-temperature Mössbauer spectroscopy.
  • The goal of this research project was to employ a multi-disciplinary team to investigate the DOE-ERSP Field Research Center at Oak Ridge, TN (ORFRC), which contains well-defined subsurface contaminant plumes with contrasting pH and redox conditions. Part of the team would pursue cultivation-independent characterization of the microbial groups catalyzing relevant biogeochemical reactions to gain an understanding of the physiological mechanisms controlling radionuclide immobilization. Other team members would focus on cultivation and physiological characterization of model microorganisms from the site using single cell sorting methods. In order to understand and predict the in situ function of microbial communities, the PIs hopemore » to develop new strategies for cultivation and to couple phylogenetic structure with microbial community function. Specific objectives by the Rutgers group was to discern the active bacteria at the Oak Ridge Research Field Challenge Site: 1. by applying stable isotope probing techniques to enrichment cultures developed from Florida State University; 2. by fingerprinting intact rRNA from groundwater samples obtained along the various flow pathways at ORFRC; and 3. by identifying functional genes for N and S cycling along the flowpaths to aid in detection of active bacteria.« less