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Bacterial Nitrate Reduction by Paraburkholderia yinzercillus Contributes to Iron Bioremediation in Abandoned Coal Mine Drainage presentation given at the Gordon Research Conference on Renewable Energy: Solar Fuels Conference held February 4-9, 2024, in Ventura, California.
Urban wastewater is a significant by-product of human activities. Conventional urban wastewater treatment plants have limitations in their treatment, mainly concerning the low removal efficiency of conventional and emerging contaminants. Discharged wastewater also contains harmful microorganisms, posing risks to public health, especially by spreading antibiotic-resistant bacteria and genes. Therefore, this study assesses the potential of a native microalgae-bacteria system (MBS) for urban wastewater bioremediation and disinfection, targeting NH4+-N and PO43−-P removal, coliform reduction, and antibiotic resistance gene mitigation. The MBS showed promising results, including a high specific growth rate (0.651 ± 0.155 d−1) and a significant average removal rate of NH4+-N and PO43−-P (9.05 ± 1.24 mg L−1 d−1 and 0.79 ± 0.06 mg L−1 d−1, respectively). Microalgae-induced pH increase rapidly reduces coliforms (r > 0.9), including Escherichia coli, within 3 to 6 days. Notably, the prevalence of intI1 and the antibiotic resistance genes sul1 and blaTEM are significantly diminished, presenting the MBS as a sustainable approach for tertiary wastewater treatment to combat eutrophication and reduce waterborne disease risks and antibiotic resistance spread.
An effective on-line monitoring technique for toxic waste bioremediation using bioluminescent microorganisms has shown great potential for the description and optimization of biological processes. The lux genes of the bacterium Vibrio fischeri are used by this species to produce visible light. The lux genes can be genetically fused to the control region of a catabolic gene, with the result that bioluminescence is produced whenever the catabolic gene is induced. Thus the detection of light from a sample indicates that genetic expression from a specific gene is occurring. This technique was used to monitor biodegradation of specific contaminants from waste sites. For these studies, fusions between the lux genes and the operons for naphthalene and toluene/xylene degradation were constructed. Strains carrying one of these fusions respond sensitively and specifically to target substrates. Bioluminescence from these cultures can be rapidly measured in a nondestructive and noninvasive manner. The potential for this technique in this and other biological systems is discussed.
The present invention provides a method comprising the use of microorganisms for nanotoxicity study and bioremediation. In some embodiment, the microorganisms are bacterial organisms such as Gram negative bacteria, which are used as model organisms to study the nanotoxicity of the fullerene compounds: E. coli W3110, a human related enterobacterium and Shewanella oneidensis MR-1, an environmentally important bacterium with versatile metabolism.
Of central interest in this study were microbial communities attached to sediment particles and in associated groundwater. Are the communities similar? What do the organisms look like and how do they associated with each other? Research was conducted on samples collected from the Department of Energy's (DOE) Rifle Integrated Field Research Challenge (IFRC) site in Rifle, Colorado, USA. This site served first as a test case for in situ bioremediation via biostimulation, and more recently as a location for studying the role of microbial communities in the carbon and other linked biogeochemical cycles. We addressed the question of the nature of planktonic to sediment-attached microbial communities using field and laboratory experimental studies with a range of methods that provide 2- and 3-dimensional topological information coupled to information about chemical speciation, organism type, and activity levels. The research leveraged data from metagenomics and proteomics analyses that obtained through parallel work at the Rifle site in the context of the IFRC. In this project we integrated characterization methods with extensive genomic sequence information and associated environmental data to better understand the processes that occur within groundwater and sediment-attached communities. Notable is the range of characterization methods, including scanning transmission x-ray microscopy (STXM), micro-EXAFS/XANES and microdiffraction and 2D and 3D cryo-electron tomographic analysis, and high-resolutino transmission electron microscope (HRTEM) analysis to characterize these natural microbial communities. A cryo-TEM work was unique because samples for electron microscopic characterization were cryo-plunged directly on site immediately after sampling. This step minimizes post-collection alterations, including cell damages and change of redox state. Among many achievements documented in publications listed at the end of this report, we highlight the following: 1) The development of a platform for routine correlative cryogenic microscopy and spectroscopy with samples prepared on-site. 2) The determination of which organisms dominate planktonic and biofilm communities in the subsurface. 3) Identification of microorganism-mineral associations and discovery of a novel mechanism that sustains activity of iron-reducing bacteria. 4) The detection of bacteria from the OP11-OD1-WWE3 (etc.) radiation and elucidation of their remarkable structural organization by cryog-TEM cryo-electron tomograhpy (cryo-ET). 5) Extensive analysis of biofilms and documentation of the association of cells and Se minerals. 6) The comparison of expressed c-type cytochromes between pure cultures of G. bemidjiensis and related field populations, provided insight into possible molecular mechanisms for U(VI) reduction in the aquifer. At least sixteen publications will result from this project (partial support), which provide both graduate student and post doctoral training.
A reactive transport benchmark problem set has been developed based on in situ uranium bio-immobilization experiments that have been performed at a former uranium mill tailings site in Rifle, Colorado, USA. Acetate-amended groundwater stimulates indigenous microorganisms to catalyze the reduction of U(VI) to a sparingly soluble U(IV) mineral. The interplay between the flow, acetate loading periods and rates, microbially-mediated and geochemical reactions leads to dynamic behavior in metal- and sulfate-reducing bacteria, pH, alkalinity, and reactive mineral surfaces. The benchmark is based on an 8.5 m long one-dimensional model domain with constant saturated flow and uniform porosity. The 159-day simulation introduces acetate and bromide through the upgradient boundary in 14-day and 85-day pulses separated by a 10 day interruption. Acetate loading is tripled during the second pulse, which is followed by a 50 day recovery period. Terminal electron accepting processes for goethite, phyllosilicate Fe(III), U(VI), and sulfate are modeled using Monod-type rate laws. Major ion geochemistry modeled includes mineral reactions, as well as aqueous and surface complexation reactions for UO2++, Fe++, and H+. In addition to the dynamics imparted by the transport of the acetate pulses, U(VI) behavior involves the interplay between bioreduction, which is dependent on acetate availability, and speciation-controlled surface complexation, which is dependent on pH, alkalinity and available surface complexation sites. The general difficulty of this benchmark is the large number of reactions (74), multiple rate law formulations, a multisite uranium surface complexation model, and the strong interdependency and sensitivity of the reaction processes. Results are presented for three simulators: HYDROGEOCHEM, PHT3D, and PHREEQC.
Chemicals involved in plutonium uranium reduction extraction (PUREX) can be released from nuclear reprocessing facilities and accumulate in the environment. We exposed chemically diverse soils to a range of concentrations of key chemicals used in the PUREX process. The responses of soil microbial communities are dependent on soil type, and tributyl phosphate exposure generates the most reproducible changes in microbial communities. We reconstructed the genomes of key bacteria and find several phosphotriesterase genes found only in Rhizobiaceae. The abundance of phosphotriesterase genes is significantly higher in samples exposed to tributyl phosphate. In conclusion, these phosphotriesterase genes may be involved in breakdown of tributyl phosphate, and a means of accessing phosphate for these bacteria.
There is a growing need to assess long-term impacts of active remediation strategies on treated aquifers. A variety of biogeochemical alterations can result from interactions of the amendment with the aquifer, conceivably leading to a geophysical footprint of the long-term alteration of an aquifer. This concept of post-remediation geophysical assessment was investigated in a shallow, chlorinated solvent-contaminated aquifer six to eight years after amendment delivery. Surface resistivity imaging and cross-borehole resistivity and induced polarization (IP) imaging were performed on a transect that spanned treated and untreated zones of the aquifer. Established relationships between IP parameters and surface electrical conductivity were used to predict vertical profiles of electrolytic conductivity and surface conductivity from the inverted cross-borehole images. Aqueous geochemistry data, along with natural gamma and magnetic susceptibility logs, were used to constrain the interpretation. The electrical conductivity structure determined from surface and borehole imaging was foremost controlled by the electrolytic conductivity of the interconnected pore space, being linearly related to fluid specific conductance. The electrolytic conductivity (and thus the conductivity images alone) did not discriminate between treated and untreated zones of the aquifer. In contrast, inverted phase angles and surface conductivities did discriminate between treated and untreated zones of the aquifer, with the treated zone being up to an order of magnitude more polarizable in places. Supporting aqueous chemistry and borehole logging datasets indicate that this geophysical footprint of the long-term impact of the remediation on the aquifer is most likely associated with the formation of polarizable, dispersed iron sulfide minerals. The study opens the door to the possibility of employing time-lapse electrical geophysical measurements to assist with long-term environmental stewardship of legacy sites undergoing active remediation.
No abstract prepared.
Field biostimulation experiments at the U.S. Department of Energy's Integrated Field Research Challenge (IFRC) site in Rifle, Colorado, have demonstrated that uranium concentrations in groundwater can be decreased to levels below the U.S. Environmental Protection Agency's (EPA) drinking water standard (0.126 {micro}M). During successive summer experiments - referred to as 'Winchester' (2007) and 'Big Rusty' (2008) - acetate was added to the aquifer to stimulate the activity of indigenous dissimilatory metal-reducing bacteria capable of reductively immobilizing uranium. The two experiments differed in the length of injection (31 vs. 110 days), the maximum concentration of acetate (5 vs. 30 mM), and the extent to which iron reduction ('Winchester') or sulfate reduction ('Big Rusty') was the predominant metabolic process. In both cases, rapid removal of U(VI) from groundwater occurred at calcium concentrations (6 mM) and carbonate alkalinities (8 meq/L) where Ca-UO2-CO3 ternary complexes constitute >90% of uranyl species in groundwater. Complete consumption of acetate and increased alkalinity (>30 meq/L) accompanying the onset of sulfate reduction corresponded to temporary increases in U(VI); however, by increasing acetate concentrations in excess of available sulfate (10 mM), low U(VI) concentrations (0.1-0.05 {micro}M) were achieved for extended periods of time (>140 days). Uniform delivery of acetate during 'Big Rusty' was impeded due to decreases in injection well permeability, likely resulting from biomass accumulation and carbonate and sulfide mineral precipitation. Such decreases were not observed during the short-duration 'Winchester' experiment. Terminal restriction fragment length polymorphism (TRFLP) analysis of 16S rRNA genes demonstrated that Geobacter sp. and Geobacter-like strains dominated the groundwater community profile during iron reduction, with 13C stable isotope probing (SIP) results confirming these strains were actively utilizing acetate to replicate their genome during the period of optimal U(VI) removal. Gene transcript levels during 'Big Rusty' were quantified for Geobacter-specific citrate synthase (gltA), with ongoing transcription during sulfate reduction indicating that members of the Geobacteraceae were still active and likely contributing to U(VI) removal. The persistence of reducible Fe(III) in sediments recovered from an area of prolonged (110 day) sulfate reduction is consistent with this conclusion. These results indicate that acetate availability and its ability to sustain the activity of iron- and uranyl-respiring Geobacter strains during sulfate reduction exerts a primary control on optimized U(VI) removal from groundwater at the Rifle IFRC site over extended timescales (>50 days).
A pilot-scale system was established to examine the feasibility of in situ U(VI) immobilization at a highly contaminated aquifer (U.S. DOE Integrated Field Research Challenge site, Oak Ridge, TN). Ethanol was injected intermittently as an electron donor to stimulate microbial U(VI) reduction, and U(VI) concentrations fell to below the Environmental Protection Agency drinking water standard (0.03 mg liter 1). Microbial communities from three monitoring wells were examined during active U(VI) reduction and maintenance phases with GeoChip, a high-density, comprehensive functional gene array. The overall microbial community structure exhibited a considerable shift over the remediation phases examined. GeoChip-based analysis revealed that Fe(III)-reducing bacterial (FeRB), nitrate-reducing bacterial (NRB), and sulfate-reducing bacterial (SRB) functional populations reached their highest levels during the active U(VI) reduction phase (days 137 to 370), in which denitrification and Fe(III) and sulfate reduction occurred sequentially. A gradual decrease in these functional populations occurred when reduction reactions stabilized, suggesting that these functional populations could play an important role in both active U(VI) reduction and maintenance of the stability of reduced U(IV). These results suggest that addition of electron donors stimulated the microbial community to create biogeochemical conditions favorable to U(VI) reduction and prevent the reduced U(IV) from reoxidation and that functional FeRB, SRB, and NRB populations within this system played key roles in this process.
A pilot-scale system was established to examine the feasibility of in situ U(VI) immobilization at a highly contaminated aquifer (U.S. DOE Integrated Field Research Challenge site, Oak Ridge, TN). Ethanol was injected intermittently as an electron donor to stimulate microbial U(VI) reduction, and U(VI) concentrations fell to below the Environmental Protection Agency drinking water standard (0.03 mg liter{sup -1}). Microbial communities from three monitoring wells were examined during active U(VI) reduction and maintenance phases with GeoChip, a high-density, comprehensive functional gene array. The overall microbial community structure exhibited a considerable shift over the remediation phases examined. GeoChip-based analysis revealed that Fe(III)-reducing bacterial (FeRB), nitrate-reducing bacterial (NRB), and sulfate-reducing bacterial (SRB) functional populations reached their highest levels during the active U(VI) reduction phase (days 137 to 370), in which denitrification and Fe(III) and sulfate reduction occurred sequentially. A gradual decrease in these functional populations occurred when reduction reactions stabilized, suggesting that these functional populations could play an important role in both active U(VI) reduction and maintenance of the stability of reduced U(IV). These results suggest that addition of electron donors stimulated the microbial community to create biogeochemical conditions favorable to U(VI) reduction and prevent the reduced U(IV) from reoxidation and that functional FeRB, SRB, and NRB populations within this system played key roles in this process.
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Chromium contaminated soil and ground water has become a serious problem caused by the rapid expansion of industries, leading to direct or indirect discharge of Cr(VI). The conventional methods that include pump and treat result into toxic byproducts and residual. Cr(VI) reducing bacteria in the contrary have been envisioned as an economically and ecologically friendly method to treat contaminated sites. Biological Cr(VI) reduction processes include biotransformation, biosorption which involve detoxification/ removal of hazardous substances and bioaccumulation. A continuous flow column system for Cr(VI) reduction was investigated in three different treatments and the control, using dried sludge and soil from target site. Cultures identified using effluent samples in the 16S rRNA sequencing showed a prominent of Exiguobacterium aestuarii species as it was identified from all column systems. Other cultures that showed 100% query cover included; Enterobacter aerogenes, Enterobacter kobei and Pseudomonas aeruginosa. More than 90% of Cr(VI) reduction was observed in all columns over a 60 day period at 40 mgL{sup -1}. Cr(VI) was reduced from 38.12 mgL{sup -1} to 0.39 mgL{sup -1}. A more unstable profile was observed at 60 mgL{sup -1} over 60 days. Subsequently, there was near complete reduction from System 1 and 3 of 1.152 mgL{sup -1} and 1.215 mgL{sup -1} Cr(VI) concentration, respectively. A Cr(VI) reduction profile along the column proved to be uniform at assumed quasi-steady state from the influent to the effluent for both 40 and 60 mgL{sup -1} Cr(VI) concentration. Biological remediation strategies for Cr(VI) contaminated soil and groundwater is an emerging field that has been proposed to successfully treat Cr(VI) contaminated sites using Cr(VI) reduction species of bacteria. (authors)
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