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DOE OSTI2016

Geobacter strains that use alternate organic compounds, methods of making, and methods of use thereof

In preferred embodiments, the present invention provides new isolated strains of a Geobacter species that are capable of using a carbon source that is selected from C.sub.3 to C.sub.12 organic compounds selected from pyruvate or metabolic precursors of pyruvate as an electron donor in metabolism and in subsequent energy production. The wild type strain of the microorganisms has been shown to be unable to use these C.sub.3 to C.sub.12 organic compounds as electron donors. The inventive strains of microorganisms are useful for improving bioremediation applications, including in situ bioremediation (including uranium bioremediation and halogenated solvent bioremediation), microbial fuel cells, power generation from small and large-scale waste facilities (e.g., biomass waste from dairy, agriculture, food processing, brewery, or vintner industries, etc.) using microbial fuel cells, and other applications of microbial fuel cells, including, but not limited to, improved electrical power supplies for environmental sensors, electronic devices, and electric vehicles.

09 BIOMASS FUELS↗

DOE OSTI2011

Direct coupling of a genome-scale microbial in silico model and a groundwater reactive transport model

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, 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.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2012

Use of functional gene arrays for elucidating in situ biodegradation

Microarrays have revolutionized the study of microbiology by providing a high-throughput method for examining thousands of genes with a single test and overcome the limitations of many culture-independent approaches. Functional gene arrays (FGA) probe a wide range of genes involved in a variety of functions of interest to microbial ecology (e.g., carbon degradation, N fixation, metal resistance) from many different microorganisms, cultured and uncultured. The most comprehensive FGA to date is the GeoChip array, which targets tens of thousands of genes involved in the geochemical cycling of carbon, nitrogen, phosphorus, and sulfur, metal resistance and reduction, energy processing, antibiotic resistance and contaminant degradation as well as phylogenetic information (gyrB). Since the development of GeoChips, many studies have been performed using this FGA and have shown it to be a powerful tool for rapid, sensitive, and specific examination of microbial communities in a high-throughput manner. As such, the GeoChip is well-suited for linking geochemical processes with microbial community function and structure. This technology has been used successfully to examine microbial communities before, during, and after in situ bioremediation at a variety of contaminated sites. These studies have expanded our understanding of biodegradation and bioremediation processes and the associated microorganisms and environmental conditions responsible. This review provides an overview of FGA development with a focus on the GeoChip and highlights specific GeoChip studies involving in situ bioremediation.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2016

Influence of triethyl phosphate on phosphatase activity in shooting range soil: Isolation of a zinc-resistant bacterium with an acid phosphatase

Phosphatase-mediated hydrolysis of organic phosphate may be a viable means of stabilizing heavy metals via precipitation as a metal phosphate in bioremediation applications. We investigated the effect of triethyl phosphate (TEP) on soil microbial-phosphatase activity in a heavy-metal contaminated soil. Gaseous TEP has been used at subsurface sites for bioremediation of organic contaminants but not applied in heavy-metal contaminated areas. Little is known about how TEP affects microbial activity in soils and it is postulated that TEP can serve as a phosphate source in nutrient-poor groundwater and soil/sediments. Over a 3-week period, TEP amendment to microcosms containing heavy-metal contaminated soil resulted in increased activity of soil acid-phosphatase and repression of alkaline phosphatase, indicating a stimulatory effect on the microbial population. A soil-free enrichment of microorganisms adapted to heavy-metal and acidic conditions was derived from the TEP-amended soil microcosms using TEP as the sole phosphate source and the selected microbial consortium maintained a high acid-phosphatase activity with repression of alkaline phosphatase. Addition of 5 mM zinc to soil-free microcosms had little effect on acid phosphatase but inhibited alkaline phosphatase. One bacterial member from the consortium, identified as Burkholderia cepacia sp., expressed an acid-phosphatase activity uninhibited by high concentrations of zinc and produced a soluble, indigo pigment under phosphate limitation. The pigment was produced in a phosphate-free medium and was not produced in the presence of TEP or phosphate ion, indicative of purple acid-phosphatase types that are pressed by bioavailable phosphate. Finally, these results demonstrate that TEP amendment was bioavailable and increased overall phosphatase activity in both soil and soil-free microcosms supporting the possibility of positive outcomes in bioremediation applications.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2014

Uranium Immobilization through Fe(II) bio-oxidation: A Column study

Current research on the bioremediation of heavy metals and radionuclides is focused on the ability of reducing organisms to use these metals as alternative electron acceptors in the absence of oxygen and thus precipitate them out of solution. However, many aspects of this proposed scheme need to be resolved, not the least of which is the time frame of the treatment process. Once treatment is complete and the electron donor addition is halted, the system will ultimately revert back to an oxic state and potentially result in the abiotic reoxidation and remobilization of the immobilized metals. In addition, the possibility exists that the presence of more electropositive electron acceptors such as nitrate or oxygen will also stimulate the biological oxidation and remobilization of these contaminants. The selective nitrate-dependent biooxidation of added Fe(II) may offer an effective means of “capping off” and completing the attenuation of these contaminants in a reducing environment making the contaminants less accessible to abiotic and biotic reactions and allowing the system to naturally revert to an oxic state. Our previous DOE-NABIR funded studies demonstrated that radionuclides such as uranium and cobalt are rapidly removed from solution during the biogenic formation of Fe(III)-oxides. In the case of uranium, X-ray spectroscopy analysis indicated that the uranium was in the hexavalent form (normally soluble) and was bound to the precipitated Fe(III)-oxides thus demonstrating the bioremediative potential of this process. We also demonstrated that nitrate-dependent Fe(II)-oxidizing bacteria are prevalent in the sediment and groundwater samples collected from sites 1 and 2 and the background site of the NABIR FRC in Oakridge, TN. However, all of these studies were performed in batch experiments in the laboratory with pure cultures and although a significant amount was learned about the microbiology of nitrate-dependent bio-oxidation of Fe(II), the effects of complex processes (such as advective flow) present in the natural environment are unknown. The objective of the current studies was to address some of these short-comings in an attempt to develop this bioremediative strategy into a robust, field applicable technology. This objective was approached by both pure culture studies investigating the mechanism of Fe(II) oxidation by nitrate reducing bacteria and examining the flow dynamics and microbial processes in advective flow columns amended with Fe(II) and nitrate over an extended period.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2015

Metaproteomics Identifies the Protein Machinery Involved in Metal and Radionuclide Reduction in Subsurface Microbiomes and Elucidates Mechanisms and U(VI) Reduction Immobilization

The overall goal for this funded project was to develop and exploit environmental metaproteomics tools to identify biomarkers for monitoring microbial activity affecting U speciation at U-contaminated sites, correlate metaproteomics profiles with geochemical parameters and U(VI) reduction activity (or lack thereof), elucidate mechanisms contributing to U(VI) reduction, and provide remediation project managers with additional information to make science-based site management decisions for achieving cleanup goals more efficiently. Although significant progress has been made in elucidating the microbiology contribution to metal and radionuclide reduction, the cellular components, pathway(s), and mechanisms involved in U trans-formation remain poorly understood. Recent advances in (meta)proteomics technology enable detailed studies of complex samples, including environmental samples, which differ between sites and even show considerable variability within the same site (e.g., the Oak Ridge IFRC site). Additionally, site-specific geochemical conditions affect microbial activity and function, suggesting generalized assessment and interpretations may not suffice. This research effort integrated current understanding of the microbiology and biochemistry of U(VI) reduction and capitalize on advances in proteomics technology made over the past few years. Field-related analyses used Oak Ridge IFRC field ground water samples from locations where slow-release substrate biostimulation has been implemented to accelerate in situ U(VI) reduction rates. Our overarching hypothesis was that the metabolic signature in environmental samples, as deciphered by the metaproteome measurements, would show a relationship with U(VI) reduction activity. Since metaproteomic and metagenomic characterizations were computationally challenging and time-consuming, we used a tiered approach that combines database mining, controlled laboratory studies, U(VI) reduction activity measurements, phylogenetic analyses, and gene expression studies to support the metaproteomics characterizations. Growth experiments of target microorganisms (Anaeromyxobacter, Shewanella, Geobacter) revealed tremendous respiratory versatility, as evidenced by the ability to utilize a range of electron donors (e.g. acetate, hydrogen, pyruvate, lactate, succinate, formate) and electron acceptors (e.g. nitrate, fumarate, halogenated phenols, ferric iron, nitrous oxide, etc.). In particular, the dissimilatory metabolic reduction of metals, including radionuclides, by target microorganisms spurred interest for in situ bioremediation of contaminated soils and sediments. Distinct c-type cytochrome expression patterns were observed in target microorganisms grown with the different electron acceptors. For each target microorganism, the core proteome covered almost all metabolic pathways represented by their corresponding pan-proteomes. Unique proteins were detected for each target microorganism, and their expression and possible functionalities were linked to specific growth conditions through proteomics measurements. Optimization of the proteomic tools included in-depth comprehensive metagenomic and metaproteomic analyses on a limited number of samples. The optimized metaproteomic analyses were then applied to Oak Ridge IFRC field samples from the slow-release substrate biostimulation. Metaproteomic analysis and pathway mapping results demonstrated the distinct effects of metal and non-metal growth conditions on the proteome expression. With these metaproteomic tools, we identified which previously hypothetical metabolic pathways were active during the analyzed time points of the slow release substrate biostimulation. Thus, we demonstrated the utility of these tools for site assessment, efficient implementation of bioremediation and long-term monitoring. This research of detailed protein analysis linked with metal reduction activity did (1) show that c-type cytochrome isoforms, previously associated with radionuclide reduction activity, are suitable biomarkers, (2) identify new biomarker targets for site assessment and bioremediation monitoring, and (3) provide new information about specific proteins and mechanisms involved in U(VI) reduction and immobilization. This expanded metagenomic and metaproteomic toolbox contributed to implementing science-driven site management with broad benefits to the DOE mission.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2019

Detection of Organohalide-Respiring Enzyme Biomarkers at a Bioaugmented TCE-Contaminated Field Site

RNA-based biomarkers have been successfully detected at field sites undergoing in situ bioremediation, but the detection of expressed enzymes is a more direct way to prove activity for a particular biocatalytic process of interest since they provide evidence of potential in situ activity rather than simply confirming presence and abundance of genes in a given population by measurement of DNA copies using qPCR. Here we successfully applied shotgun proteomics to field samples from a trichloroethene (TCE)-contaminated industrial site in southern Ontario, Canada that had been bioaugmented with the commercially available KB-1 TM microbial culture. The KB-1 TM culture contains multiple strains of Dehalococcoides mccartyi (D. mccartyi) as well as an organohalide respiring Geobacter species. The relative abundances of specific enzymatic proteins were subsequently compared to corresponding qPCR-derived levels of DNA and RNA biomarkers in the same samples. Samples were obtained from two wells with high hydraulic connectivity to the KB-1 TM -bioaugemented enhanced in situ bioremediation system, and two control wells that showed evidence of low levels of native organohalide respiring bacteria (OHRB), Dehalococcoides and Geobacter . Enzymes involved in organohalide respiration were detected in the metaproteomes of all four field samples, as were chaperonins of D. mccartyi , chemotaxis proteins, and ATPases. The most highly expressed RDase in the bioaugmentation culture (VcrA) was the most highly detected enzyme overall in the bioaugmented groundwater samples. In one background groundwater well, we found high expression of the Geobacter pceA RDase. The DNA and RNA biomarkers detected using qPCR-based assays were a set of orthologs of Dehalococcoides reductive dehalogenases (VcrA, TceA, BvcA, dehalogenase “DET1545”), and the Ni-Fe uptake hydrogenase, HupL. Within a sample, RNA levels for key enzymes correlated with relative protein abundance. These results indicate that laboratory observations of TCE-bioremediation biomarker protein expression are recapitulated in field environmental systems and that both RNA and protein biomarker monitoring hold promise for activity monitoring of in situ populations of OHRB.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS2004

Using Iron to Treat Chlorohydrocarbon-Contaminated Soil

A method of in situ remediation of soil contaminated with chlorinated hydrocarbon solvents involves injection of nanometer-size iron particles. The present method exploits a combination of prompt chemical remediation followed by longer-term enhanced bioremediation and, optionally, is practiced in conjunction with the method of bioremediation described earlier. Newly injected iron particles chemically reduce chlorinated hydrocarbons upon contact. Thereafter, in the presence of groundwater, the particles slowly corrode via chemical reactions that effect sustained release of dissolved hydrogen. The hydrogen serves as an electron donor, increasing the metabolic activity of the anaerobic bacteria and thereby sustaining bioremediation at a rate higher than the natural rate.

Hitchens, G. Duncan↗

DOE OSTI2011

Microbial reductive dehalogenation of vinyl chloride

Compositions and methods are provided that relate to the bioremediation of chlorinated ethenes, particularly the bioremediation of vinyl chloride by Dehalococcoides-like organisms. An isolated strain of bacteria, Dehalococcoides sp. strain VS, that metabolizes vinyl chloride is provided; the genetic sequence of the enzyme responsible for vinyl chloride dehalogenation; methods of assessing the capability of endogenous organisms at an environmental site to metabolize vinyl chloride; and a method of using the strains of the invention for bioremediation.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2014

Microbial reductive dehalogenation of vinyl chloride

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2013

Hiereachical Bayesian Model for Combining Geochemical and Geophysical Data for Environmental Applications Software

Development of a hierarchical Bayesian model to estimate the spatiotemporal distribution of aqueous geochemical parameters associated with in-situ bioremediation using surface spectral induced polarization (SIP) data and borehole geochemical measurements collected during a bioremediation experiment at a uranium-contaminated site near Rifle, Colorado. The SIP data are first inverted for Cole-Cole parameters including chargeability, time constant, resistivity at the DC frequency and dependence factor, at each pixel of two-dimensional grids using a previously developed stochastic method. Correlations between the inverted Cole-Cole parameters and the wellbore-based groundwater chemistry measurements indicative of key metabolic processes within the aquifer (e.g. ferrous iron, sulfate, uranium) were established and used as a basis for petrophysical model development. The developed Bayesian model consists of three levels of statistical sub-models: 1) data model, providing links between geochemical and geophysical attributes, 2) process model, describing the spatial and temporal variability of geochemical properties in the subsurface system, and 3) parameter model, describing prior distributions of various parameters and initial conditions. The unknown parameters are estimated using Markov chain Monte Carlo methods. By combining the temporally distributed geochemical data with the spatially distributed geophysical data, we obtain the spatio-temporal distribution of ferrous iron, sulfate and sulfide, and their associated uncertainity information. The obtained results can be used to assess the efficacy of the bioremediation treatment over space and time and to constrain reactive transport models.

Chen, Jinsong↗

DOE OSTI2011

Evidence for multiple modes of uranium immobilization by an anaerobic bacterium

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

12 MANAGEMENT OF RADIOACTIVE AND NON-RADIOACTIVE W↗

DOE OSTI2024

Bioelectrochemical reactor to manage anthropogenic sulfate pollution for freshwater ecosystems: Mathematical modeling and experimental validation

Anthropogenic sulfate loading into otherwise low-sulfate freshwater systems can cause significant ecological consequences as a biogeochemical stressor. To address this challenge, in situ bioremediation technologies have been developed to leverage naturally occurring microorganisms that transform sulfate into sulfide rather than implementing resource-intensive physio-chemical processes. However, bioremediation technologies often require the supply of electron donors to facilitate biological sulfate reduction. Bioelectrochemical systems (BES) can be an alternative approach for supplying molecular hydrogen as an electron donor for sulfate-reducing bacteria through water electrolysis. Although the fundamental mechanisms behind BESs have been studied, limited research has evaluated the design and operational parameters of treatment systems when developing BESs on a scale relevant to environmental systems. Further, this study aimed to develop an application-based mathematical model to evaluate the performance of BESs across a range of reactor configurations and operational modes. The model was based on sulfate transformation by hydrogenotrophic sulfate-reducing bacteria coupled with the recovery of solid iron sulfide species formed by the oxidative dissolution of dissolved ferrous iron from a stainless steel anode. Sulfate removal closely corresponded to the rate of electrolytic hydrogen production and hydraulic residence time but was less sensitive to specific microbial rate constants. The mathematical model results were compared to experimental data from a pilot-scale BES tested with nonacidic mine drainage as a case study. The close agreement between the mathematical model and the pilot-scale BES experiment highlights the efficacy of using a mathematical model as a tool to develop a conceptual design of a scaled-up treatment system.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2015

Bicarbonate impact on U(VI) bioreduction in a shallow alluvial aquifer

Field-scale biostimulation and desorption tracer experiments conducted in a uranium (U) contaminated, shallow alluvial aquifer have provided insight into the coupling of microbiology, biogeochemistry, and hydrogeology that control U mobility in the subsurface. Initial experiments successfully tested the concept that Fe-reducing bacteria such as Geobacter sp. could enzymatically reduce soluble U(VI) to insoluble U(IV) during in situ electron donor amendment (Anderson et al., 2003; Williams et al., 2011). In parallel, in situ desorption tracer tests using bicarbonate amendment demonstrated rate-limited U(VI) desorption (Fox et al., 2012). These results and prior laboratory studies underscored the importance of enzymatic U(VI)-reduction and suggested the ability to combine desorption and bioreduction of U(VI). Here in this paper we report the results of a new field experiment in which bicarbonate-promoted uranium desorption and acetate amendment were combined and compared to an acetate amendment-only experiment in the same experimental plot. Results confirm that bicarbonate amendment to alluvial aquifer sediments desorbs U(VI) and increases the abundance of Ca-uranyl-carbonato complexes. At the same time, the rate of acetate-promoted enzymatic U(VI) reduction was greater in the presence of added bicarbonate in spite of the increased dominance of Ca-uranyl-carbonato aqueous complexes. A model-simulated peak rate of U(VI) reduction was ~3.8 times higher during acetate-bicarbonate treatment than under acetate-only conditions. Lack of consistent differences in microbial community structure between acetate-bicarbonate and acetate-only treatments suggest that a significantly higher rate of U(VI) reduction in the bicarbonate-impacted sediment may be due to a higher intrinsic rate of microbial reduction induced by elevated concentrations of the bicarbonate oxyanion. The findings indicate that bicarbonate amendment may be useful in improving the engineered bioremediation of uranium in aquifers.

58 GEOSCIENCES↗

DOE OSTI2018

Carbon-dependent chromate toxicity mechanism in an environmental Arthrobacter isolate

Arthrobacter spp. are widespread in soil systems and well-known for their Cr(VI) reduction capabilities making them attractive candidates for in situ bioremediation efforts. Cellulose drives carbon flow in soil systems; yet, most laboratory studies evaluate Arthrobacter-Cr(VI) interactions solely with nutrient-rich media or glucose. This work aims to determine how various cellulose degradation products and biostimulation substrates influence Cr(VI) toxicity, reduction, and microbial growth of an environmental Arthrobacter sp. isolate. Laboratory culture-based studies suggest there is a carbon-dependent Cr(VI) toxicity mechanism that affects subsequent Cr(VI) reduction by strain LLW01. Strain LLW01 could only grow in the presence of, and reduce, 50 μM Cr(VI) when glucose or lactate were provided. Compared to lactate, Cr(VI) was at least 30-fold and 10-fold more toxic when ethanol or butyrate was the sole carbon source, respectively. The addition of sulfate mitigated toxicity somewhat, but had no effect on the extent of Cr(VI) reduction. Cell viability studies indicated that a small fraction of cells were viable after 8 days suggesting cell growth and subsequent Cr(VI) reduction may resume. These results suggest when designing bioremediation strategies with Arthrobacter spp. such as strain LLW01, carbon sources such as glucose and lactate should be considered over ethanol and butyrate.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2011

Use of immunomagnetic separation for the detection of Desulfovibrio vulgaris from environmental samples

Immunomagnetic separation (IMS) has proved highly efficient for recovering microorganisms from heterogeneous samples. Current investigation targeted the separation of viable cells of the sulfate-reducing bacterium, Desulfovibrio vulgaris. Streptavidin-coupled paramagnetic beads and biotin labeled antibodies raised against surface antigens of this microorganism were used to capture D. vulgaris cells in both bioreactor grown laboratory samples and from extremely low-biomass environmental soil and subsurface drilling samples. Initial studies on detection, recovery efficiency and viability for IMS were performed with laboratory grown D. vulgaris cells using various cell densities. Efficiency of cell isolation and recovery (i.e., release of the microbial cells from the beads following separation) was followed by microscopic imaging and acridine orange direct counts (AODC). Excellent recovery efficiency encouraged the use of IMS to capture Desulfovibrio spp. cells from low-biomass environmental samples. The environmental samples were obtained from a radionuclide-contaminated site in Germany and the chromium (VI)-contaminated Hanford site, an ongoing bioremediation project of the U.S. Department of Energy. Field deployable IMS technology may greatly facilitate environmental sampling and bioremediation process monitoring and enable transcriptomics and proteomics/metabolomics-based studies directly on cells collected from the field.

09 BIOMASS FUELS↗

DOE OSTI2020

Burkholderia : An Untapped but Promising Bacterial Genus for the Conversion of Aromatic Compounds

Burkholderia , a bacterial genus containing greater than 120 species, is typically reported to inhabit soil and water environments. These Gram-negative bacteria are known to harbor a variety of aromatic catabolic pathways and are thus potential organisms for the bioremediation of sites contaminated with aromatic pollutants. However, there are still substantial knowledge gaps in these catabolic processes that must be elucidated before harnessing these pathways and organisms for biotechnological applications. This review presents recent discoveries on the catabolism of monoaromatic and polycyclic aromatic hydrocarbons as well as heterocyclic compounds by a diversity of Burkholderia strains. We emphasize the advantageous features of Burkholderia for bioremediation processes. Furthermore, future directions for its utilization in bioconversion and upgrading of aromatic compounds are also presented.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2020

Metagenome-Guided Proteomic Quantification of Reductive Dehalogenases in the Dehalococcoides mccartyi -Containing Consortium SDC-9

At groundwater sites contaminated with chlorinated ethenes, fermentable substrates are often added to promote reductive dehalogenation by indigenous or augmented microorganisms. Contemporary bioremediation performance monitoring relies on nucleic acid biomarkers of key organohalide-respiring bacteria, such as Dehalococcoides mccartyi (Dhc). In this work, metagenome sequencing of the commercial, Dhc-containing consortium, SDC-9, identified 12 reductive dehalogenase (RDase) genes, including pceA (two copies), vcrA, and tceA, and allowed for specific detection and quantification of RDase peptides using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Shotgun (i.e., untargeted) proteomics applied to the SDC-9 consortium grown with tetrachloroethene (PCE) and lactate identified 143 RDase peptides, and 36 distinct peptides that covered greater than 99% of the protein-coding sequences of the PceA, TceA, and VcrA RDases. Quantification of RDase peptides using multiple reaction monitoring (MRM) assays with 13 C-/ 15 N-labeled peptides determined 1.8 × 10 3 TceA and 1.2 × 10 2 VcrA RDase molecules per Dhc cell. Ultimately, the MRM mass spectrometry approach allowed for sensitive detection and accurate quantification of relevant Dhc RDases and has potential utility in bioremediation monitoring regimes.

59 BASIC BIOLOGICAL SCIENCES↗