Biomedical subjects

Craig S Criddle

Publications and source records attributed to Craig S Criddle.

18 recordsLinked to original sources

PubMed2006

Use of reverse osmosis membranes to remove perfluorooctane sulfonate (PFOS) from semiconductor wastewater.

Perfluorooctane sulfonate (PFOS) and related substances are persistent, bioaccumulative, and toxic, and thus of substantial environmental concern. PFOS is an essential photolithographic chemical in the semiconductor industry with no substitutes yet identified. The industry seeks effective treatment technologies. The feasibility of using reverse osmosis (RO) membranes for treating semiconductor wastewater containing PFOS has been investigated. Commercial RO membranes were characterized in terms of permeability, salt rejection, scanning electron microscopy (SEM), transmission electron microscopy (TEM), and membrane surface zeta potential (streaming potential measurements). Filtration tests were performed to determine the membrane flux and PFOS rejection. Over a wide range of feed concentrations (0.5 - 1500 ppm), the RO membranes generally rejected 99% or more of the PFOS. Rejection was better for tighter membranes, but was not affected by membrane zeta potential. Flux decreased with increasing PFOS concentration. While the flux reduction was severe for a loose RO membrane probably due to its higher initial flux, very stable flux was maintained for tighter membranes. At a very high feed concentration (about 500 ppm), all the membranes exhibited an identical stable flux. Isopropyl alcohol, present in some semiconductor wastewaters, had a detrimental effect on membrane flux. Where present it needs to be removed from the wastewater prior to using RO membranes.

Alkanesulfonic Acids↗

PubMed2006

Gene capture and random amplification for quantitative recovery of homologous genes.

The polymerase chain reaction (PCR) is instrumental in molecular analysis of microorganisms, allowing for the selective amplification of nucleic acids directly from clinical and environmental samples. However, the principles that allow for targeted amplification of DNA become a hindrance when attempting to simultaneously discriminate and quantify complex mixtures of homologous genes. Here we present a simple solution to the quantitative problem by separating the enrichment and amplification aspects of a conventional PCR reaction. In this assay, genes are enriched using a DNA oligonucleotide capture probe and subsequently amplified in a two-step random amplification protocol. In order to evaluate the quantitative aspects of the gene capture assay, we used real-time quantitative-PCR to measure initial and final concentrations of homologous genes from constructed mixtures of genomes. Upon sampling for the universal DNA-dependent RNA polymerase gene, rpoC, we were able to demonstrate quantitative recoveries from a mixed DNA sample despite differences in gene copy number ranging up to 4 orders of magnitude. This suggests that minority populations as low as 0.01% of the total community are represented as accurately as populations at higher abundance. These results offer new possibilities for accurately and quantitatively monitoring diverse mixtures of microorganisms.

Base Sequence↗

PubMed2006

Pilot-scale in situ bioremediation of uranium in a highly contaminated aquifer. 1. Conditioning of a treatment zone.

To evaluate the potential for in situ bioremediation of U(VI) to sparingly soluble U(IV), we constructed a pilot test facility at Area 3 of the U.S. Department of Energy Natural and Accelerated Bioremediation Research (NABIR) Field Research Center (FRC) in Oak Ridge, TN. The facility is adjacent to the former S-3 Ponds which received trillions of liters of acidic plating wastes. High levels of uranium are present, with up to 800 mg kg(-1) in the soil and 84-210 microM in the groundwater. Ambient groundwater has a highly buffered pH of approximately 3.4 and high levels of aluminum (12-13 mM), calcium (22-25 mM), and nitrate (80-160 mM). Adjusting the pH of groundwater to approximately 5 within the aquifer would deposit extensive aluminum hydroxide precipitate. Calcium is present in the groundwater at levels that inhibit U(VI) reduction, but its removal by injection of a high pH solution would generate clogging precipitate. Nitrate also inhibits U(VI) reduction and is present at such high concentrations that its removal by in situ denitrification would generate large amounts of N2 gas and biomass. To establish and maintain hydraulic control, we installed a four well recirculation system parallel to geologic strike, with an inner loop nested within an outer loop. For monitoring, we drilled three boreholes perpendicular to strike across the inner loop and installed multilevel sampling tubes within them. A tracer pulse with clean water established travel times and connectivity between wells and enabled the assessment of contaminant release from the soil matrix. Subsequently, a highly conductive region of the subsurface was prepared for biostimulation by removing clogging agents and inhibitors and increasing pH. For 2 months, groundwater was pumped from the hydraulically conductive zone; treated to remove aluminum, calcium, and nitrate, and supplemented with tap water; adjusted to pH 4.3-4.5; then returned to the hydraulically conductive zone. This protocol removed most of the aqueous aluminum and calcium. The pH of the injected treated water was then increased to 6.0-6.3. With additional flushing, the pH of the extracted water gradually increased to 5.5-6.0, and nitrate concentrations fell to 0.5-1.0 mM. These conditions were judged suitable for biostimulation. In a companion paper (Wu et al., Environ. Sci. Technol. 2006, 40, 3978-3987), we describe the effects of ethanol addition on in situ denitrification and U(VI) reduction and immobilization.

Bacteria↗

PubMed2006

Pilot-scale in situ bioremedation of uranium in a highly contaminated aquifer. 2. Reduction of u(VI) and geochemical control of u(VI) bioavailability.

In situ microbial reduction of soluble U(VI) to sparingly soluble U(IV) was evaluated at the site of the former S-3 Ponds in Area 3 of the U.S. Department of Energy Natural and Accelerated Bioremediation Research Field Research Center, Oak Ridge, TN. After establishing conditions favorable for bioremediation (Wu, et al. Environ. Sci. Technol. 2006, 40, 3988-3995), intermittent additions of ethanol were initiated within the conditioned inner loop of a nested well recirculation system. These additions initially stimulated denitrification of matrix-entrapped nitrate, but after 2 months, aqueous U levels fell from 5 to approximately 1 microM and sulfate reduction ensued. Continued additions sustained U(VI) reduction over 13 months. X-ray near-edge absorption spectroscopy (XANES) confirmed U(VI) reduction to U(IV) within the inner loop wells, with up to 51%, 35%, and 28% solid-phase U(IV) in sediment samples from the injection well, a monitoring well, and the extraction well, respectively. Microbial analyses confirmed the presence of denitrifying, sulfate-reducing, and iron-reducing bacteria in groundwater and sediments. System pH was generally maintained at less than 6.2 with low bicarbonate level (0.75-1.5 mM) and residual sulfate to suppress methanogenesis and minimize uranium mobilization. The bioavailability of sorbed U(VI) was manipulated by addition of low-level carbonate (< 5 mM) followed by ethanol (1-1.5 mM). Addition of low levels of carbonate increased the concentration of aqueous U, indicating an increased rate of U desorption due to formation of uranyl carbonate complexes. Upon ethanol addition, aqueous U(VI) levels fell, indicating that the rate of microbial reduction exceeded the rate of desorption. Sulfate levels simultaneously decreased, with a corresponding increase in sulfide. When ethanol addition ended but carbonate addition continued, soluble U levels increased, indicating faster desorption than reduction. When bicarbonate addition stopped, aqueous U levels decreased, indicating adsorption to sediments. Changes in the sequence of carbonate and ethanol addition confirmed that carbonate-controlled desorption increased bioavailability of U(VI) for reduction.

Bacteria↗

PubMed2006

Thermodynamic constraints on the oxidation of biogenic UO2 by Fe(III) (Hydr)oxides.

Uranium mobility in the environment is partially controlled by its oxidation state, where it exists as either U(VI) or U(IV). In aerobic environments, uranium is generally found in the hexavalent form, is quite soluble, and readily forms complexes with carbonate and calcium. Under anaerobic conditions, common metal respiring bacteria can reduce soluble U(VI) species to sparingly soluble UO2 (uraninite); stimulation of these bacteria, in fact, is being explored as an in situ uranium remediation technique. However, the stability of biologically precipitated uraninite within soils and sediments is not well characterized. Here we demonstrate that uraninite oxidation by Fe(III) (hydr)oxides is thermodynamically favorable under limited geochemical conditions. Our analysis reveals that goethite and hematite have a limited capacity to oxidize UO2(biogenic) while ferrihydrite can lead to UO2(biogenic) oxidation. The extent of UO2(biogenic) oxidation by ferrihydrite increases with increasing bicarbonate and calcium concentration, but decreases with elevated Fe(II)(aq) and U(VI)(aq) concentrations. Thus, our results demonstrate that the oxidation of UO2(biogenic) by Fe(III) (hydr)oxides may transpire under mildly reducing conditions when ferrihydrite is present.

Bacteria↗

PubMed2006

Occurrence of ammonia-oxidizing archaea in wastewater treatment plant bioreactors.

We report molecular evidence that ammonia-oxidizing archaea (AOA) occur in activated sludge bioreactors used to remove ammonia from wastewater. Using PCR primers targeting archaeal ammonia monooxygenase subunit A (amoA) genes, we retrieved and compared 75 sequences from five wastewater treatment plants operating with low dissolved oxygen levels and long retention times. All of these sequences showed similarity to sequences previously found in soil and sediments, and they were distributed primarily in four major phylogenetic clusters. One of these clusters contained virtually identical amoA sequences obtained from all five activated sludge samples (from Oregon, Wisconsin, Pennsylvania, and New Jersey) and accounted for 67% of all the sequences, suggesting that this AOA phylotype may be widespread in nitrifying bioreactors.

Ammonia↗

PubMed2005

A parametric transfer function methodology for analyzing reactive transport in nonuniform flow.

We analyze reactive transport during in-situ bioremediation in a nonuniform flow field, involving multiple extraction and injection wells, by the method of transfer functions. Gamma distributions are used as parametric models of the transfer functions. Apparent parameters of classical transport models may be estimated from those of the gamma distributions by matching temporal moments. We demonstrate the method by application to measured data taken at a field experiment on bioremediation conducted in a multiple-well system in Oak Ridge, TN. Breakthrough curves (BTCs) of a conservative tracer (bromide) and a reactive compound (ethanol) are measured at multi-level sampling (MLS) wells and in extraction wells. The BTCs of both compounds are jointly analyzed to estimate the first-order degradation rate of ethanol. To quantify the tracer loss, we compare the approaches of using a scaling factor and a first-order decay term. Results show that by including a scaling factor both gamma distributions and inverse-Gaussian distributions (transfer functions according to the advection-dispersion equation) are suitable to approximate the transfer functions and estimate the reactive rate coefficients for both MLS and extraction wells. However, using a first-order decay term for tracer loss fails to describe the BTCs at the extraction well, which is affected by the nonuniform distribution of travel paths.

Biodegradation, Environmental↗

PubMed2005

Mass-transfer limitations for nitrate removal in a uranium-contaminated aquifer.

A field test on in situ subsurface bioremediation of uranium(VI) is underway at the Y-12 National Security Complex in the Oak Ridge Reservation, Oak Ridge, TN. Nitrate has a high concentration at the site, which prevents U(VI) reduction, and thus must be removed. An acidic-flush strategy for nitrate removal was proposed to create a treatment zone with low levels of accessible nitrate. The subsurface at the site contains highly interconnected fractures surrounded by matrix blocks of low permeability and high porosity and is therefore subject to preferential flow and matrix diffusion. To identify the heterogeneous mass transfer properties, we performed a novel forced-gradient tracer test, which involved the addition of bromide, the displacement of nitrate, and the rebound of nitrate after completion of pumping. The simplest conceptualization consistent with the data is that the pore-space consists of a single mobile domain, as well as a fast and a slowly reacting immobile domain. The slowly reacting immobile domain (shale matrix) constitutes over 80% of the pore volume and acts as a long-term reservoir of nitrate. According to simulations, the nitrate stored in the slowly interacting immobile domain in the fast flow layer, at depths of about 12.2-13.7 m, will be reduced by an order of magnitude over a period of about a year. By contrast, the mobile domain rapidly responds to flushing, and a low average nitrate concentration can be maintained if the nitrate is removed as soon as it enters the mobile domain. A field-scale experiment in which the aquifer was flushed with acidic solution confirmed our understanding of the system. For the ongoing experiments on microbial U(VI) reduction, nitrate concentrations must be low in the mobile domain to ensure U(VI) reducing conditions. We therefore conclude that the nitrate leaching out of the immobile pore space must continuously be removed by in situ denitrification to maintain favorable conditions.

Bromides↗

PubMed2005

Impacts on microbial communities and cultivable isolates from groundwater contaminated with high levels of nitric acid-uranium waste.

Microbial communities were characterized at contaminated sites that had elevated levels of nitrate, nickel, aluminum, and uranium (up to 690 mM, 310 microM, 42 mM, and 30 microM, respectively). The bacterial community structure based upon clonal libraries of the SSU rRNA genes (screened clones = 876) was diverse at the background site, but the three acidic samples had decreased diversity and the majority of clones were closely related to Azoarcus and Pseudomonas species. Arthrobacter and Novosphingobium sequences were recovered from the background samples but not the acidic sites, and similar pseudomonad populations were present at the background and acidic sites albeit at different relative abundances. Heterologous sequence coverage analyses indicated the microbial communities at the contaminated sites were very similar (p = 0.001) but different from the background site. Bacterial isolates (n = 67) classified as beta-or gamma-Proteobacteria, high G+C Gram-positive or low G+C Gram-positive were obtained from the background and one contaminated sample, and some of the isolates had less than 95% sequence identity with previously observed microorganisms. Despite variations in nitrate and heavy metal levels and different proximities to the source ponds, the three acidic samples had similar microbial populations. However, the least contaminated site (lowest nitrate and aluminum) had increased diversity compared to the other acidic samples. The results suggested that the combined contamination has decreased the microbial diversity, and Azoarcus populations were observed at a drastically increased frequency compared to the background site that had a more even distribution of multiple taxa.

Aluminum↗

PubMed2005

Bioreduction of uranium in a contaminated soil column.

The bioreduction of soluble uranium [U(VI)] to sparingly soluble U(IV) species is an attractive remedial technology for contaminated soil and groundwater due to the potential for immobilizing uranium and impeding its migration in subsurface environments. This manuscript describes a column study designed to simulate a three-step strategy proposed for the remediation of a heavily contaminated site at the U.S. Department of Energy's NABIR Field Research Center in Oak Ridge, TN. The soil is contaminated with high concentrations of uranium, aluminum, and nitrate and has a low, highly buffered pH (approximately 3.5). Steps proposed for remediation are (i) flushing to remove nitrate and aluminum, (ii) neutralization to establish pH conditions favorable for biostimulation, and (iii) biostimulation for U(VI) reduction. We simulated this sequence using a packed soil column containing undisturbed aggregates of U(VI)-contaminated saprolite that was flushed with an acidified salt solution (pH 4.0), neutralized with bicarbonate (60 mM), and then biostimulated by adding ethanol. The column was operated anaerobically in a closed-loop recirculation setup. However, during the initial month of biostimulation, ethanol was not utilized, and U(VI) was not reduced. A bacterial culture enriched from the site groundwaterwas subsequently added, and the consumption of ethanol coupled with sulfate reduction immediately ensued. The aqueous concentration of U(VI) initially increased, evidently because of the biological production of carbonate, a ligand known to solubilize uranyl. After approximately 50 days, aqueous U(VI) concentrations rapidly decreased from approximately 17 to <1 mg/L. At the conclusion of the experiment,the presence of reduced solid phase U(IV) was confirmed using X-ray absorption near edge structure spectroscopy. The results indicate that bioreduction to immobilize uranium is potentially feasible at this site; however, the stability of the reduced U(IV) and its potential reoxidation will require further investigation, as do the effects of groundwater chemistry and competitive microbial processes, such as methanogenesis.

Bacteria, Anaerobic↗

PubMed2005

Quantitative determination of perfluorochemicals in sediments and domestic sludge.

Perfluorochemicals (PFCs) are the subject of increasingly intense environmental research. Despite their detection both in biota and in aqueous systems, little attention has been paid to the possible presence of this class of compounds in solid environmental matrixes. The limited available data indicate that some PFCs such as perfluorooctane sulfonate (PFOS) may strongly sorb to solids, and sewage sludge is widely suspected as a major sink of PFCs entering municipal waste streams. A quantitative analytical method was developed that consists of liquid solvent extraction of the analytes from sediments and sludge, cleanup via solid-phase extraction, and injection of the extracts with internal standards into a high-performance liquid chromatography (HPLC) system coupled to a tandem mass spectrometer (LC/MS/MS). The limits of detections of the method were analyte and matrix dependent, but ranged from 0.7 to 2.2 ng/g and 0.041 to 0.246 ng/g (dry weight) for sludge and sediment, respectively. A demonstration of the method was performed by conducting a limited survey of domestic sludge and sediments. The concentration of PFCs in domestic sludge ranged from 5 to 152 ng/g for total perfluorocarboxylates and 55 to 3370 ng/g for total perfluoroalkyl sulfonyl-based chemicals. Data from a survey of San Francisco Bay Area sediments suggest widespread occurrence of PFCs in sediments at the low ng/g to sub-ng/g level. Furthermore, substances that may be transformed to PFOS, such as 2-(N-ethylperfluorooctanesulfonamido) acetic acid (N-EtFOSAA) and 2-(N-methylperfluorooctanesulfonamido) acetic acid (N-MeFOSAA), are present in both sediments and sludge at levels often exceeding PFOS.

Alkanesulfonic Acids↗

PubMed2005

Global transcriptional profiling of Shewanella oneidensis MR-1 during Cr(VI) and U(VI) reduction.

Whole-genome DNA microarrays were used to examine the gene expression profile of Shewanella oneidensis MR-1 during U(VI) and Cr(VI) reduction. The same control, cells pregrown with nitrate and incubated with no electron acceptor, was used for the two time points considered and for both metals. U(VI)-reducing conditions resulted in the upregulation (> or = 3-fold) of 121 genes, while 83 genes were upregulated under Cr(VI)-reducing conditions. A large fraction of the genes upregulated [34% for U(VI) and 29% for Cr(VI)] encode hypothetical proteins of unknown function. Genes encoding proteins known to reduce alternative electron acceptors [fumarate, dimethyl sulfoxide, Mn(IV), or soluble Fe(III)] were upregulated under both U(VI)- and Cr(VI)-reducing conditions. The involvement of these upregulated genes in the reduction of U(VI) and Cr(VI) was tested using mutants lacking one or several of the gene products. Mutant testing confirmed the involvement of several genes in the reduction of both metals: mtrA, mtrB, mtrC, and menC, all of which are involved in Fe(III) citrate reduction by MR-1. Genes encoding efflux pumps were upregulated under Cr(VI)- but not under U(VI)-reducing conditions. Genes encoding proteins associated with general (e.g., groL and dnaJ) and membrane (e.g., pspBC) stress were also upregulated, particularly under U(VI)-reducing conditions, pointing to membrane damage by the solid-phase reduced U(IV) and Cr(III) and/or the direct effect of the oxidized forms of the metals. This study sheds light on the multifaceted response of MR-1 to U(VI) and Cr(VI) under anaerobic conditions and suggests that the same electron transport pathway can be used for more than one electron acceptor.

Bacterial Proteins↗

PubMed2004

Correspondence between community structure and function during succession in phenol- and phenol-plus-trichloroethene-fed sequencing batch reactors.

The effects of more than 2 years of trichloroethene (TCE) application on community succession and function were studied in two aerobic sequencing batch reactors. One reactor was fed phenol, and the second reactor was fed both phenol and TCE in sequence twice per day. After initiation of TCE loading in the second reactor, the TCE transformation rates initially decreased, but they stabilized with an average second-order rate coefficient of 0.044 liter mg(-1) day(-1) for 2 years. In contrast, the phenol-fed reactor showed higher and unstable TCE transformation rates, with an average rate coefficient of 0.093 liter mg(-1) day(-1). Community analysis by terminal restriction fragment length polymorphism (T-RFLP) analysis of the 16S rRNA genes showed that the phenol-plus-TCE-fed reactor had marked changes in community structure during the first 100 days and remained relatively stable afterwards, corresponding to the period of stable function. In contrast, the community structure of the phenol-fed reactor changed periodically, and the changes coincided with the periodicity observed in the TCE transformation rates. Correspondence analysis of each reactor community showed that different community structures corresponded with function (TCE degradation rate). Furthermore, the phenol hydroxylase genotypes, as determined by restriction fragment length polymorphism analysis, corresponded to community structure patterns identified by T-RFLP analysis and to periods when the TCE transformation rates were high. Long-term TCE stress appeared to select for a different and stable community structure, with lower but stable TCE degradation rates. In contrast, the community under no stress exhibited a dynamic structure and dynamic function.

Bacteria↗

PubMed2003

Cometabolism of Cr(VI) by Shewanella oneidensis MR-1 produces cell-associated reduced chromium and inhibits growth.

Microbial reduction is a promising strategy for chromium remediation, but the effects of competing electron acceptors are still poorly understood. We investigated chromate (Cr(VI)) reduction in batch cultures of Shewanella oneidensis MR-1 under aerobic and denitrifying conditions and in the absence of an additional electron acceptor. Growth and Cr(VI) removal patterns suggested a cometabolic reduction; in the absence of nitrate or oxygen, MR-1 reduced Cr(VI), but without any increase in viable cell counts and rates gradually decreased when cells were respiked. Only a small fraction (1.6%) of the electrons from lactate were transferred to Cr(VI). The 48-h transformation capacity (Tc) was 0.78 mg (15 micromoles) Cr(VI) reduced. [mg protein](-1) for high levels of Cr(VI) added as a single spike. For low levels of Cr(VI) added sequentially, Tc increased to 3.33 mg (64 micromoles) Cr(VI) reduced. [mg protein](-1), indicating that it is limited by toxicity at higher concentrations. During denitrification and aerobic growth, MR-1 reduced Cr(VI), with much faster rates under denitrifying conditions. Cr(VI) had no effect on nitrate reduction at 6 microM, was strongly inhibitory at 45 microM, and stopped nitrate reduction above 200 microM. Cr(VI) had no effect on aerobic growth at 60 microM, but severely inhibited growth above 150 microM. A factor that likely plays a role in Cr(VI) toxicity is intracellular reduced chromium. Transmission electron microscopy (TEM) and electron energy loss spectroscopy (EELS) of denitrifying cells exposed to Cr(VI) showed reduced chromium precipitates both extracellularly on the cell surface and, for the first time, as electron-dense round globules inside cells.

Biodegradation, Environmental↗

PubMed2003

Understanding bias in microbial community analysis techniques due to rrn operon copy number heterogeneity.

Molecular tools based on rRNA (rrn) genes are valuable techniques for the study of microbial communities. However, the presence of operon copy number heterogeneity represents a source of systematic error in community analysis. To understand the types and magnitude of such bias, four commonly used rrn-based techniques were used to perform an in silico analysis of a hypothetical community comprised organisms from the Comprehensive Microbial Resource database. Community profiles were generated, and diversity indices were calculated for length heterogeneity PCR, automated ribosomal integenic spacer analysis, denaturing gradient gel electrophoresis, and terminal RFLP (using RsaI, MspI, and HhaI). The results demonstrate that all techniques present a quantitative bias toward organisms with higher copy numbers. In addition, techniques may underestimate diversity by grouping similar ribotypes or overestimate diversity by allowing multiple signals for one organism. The results of this study suggest that caution should be used when interpreting rrn-based community analysis techniques.

DNA, Bacterial↗

PubMed2002

Development, operation, and long-term performance of a full-scale biocurtain utilizing bioaugmentation.

A full-scale field evaluation of bioaugmentation was conducted in a carbon tetrachloride (CT)- and nitrate-impacted aquifer at Schoolcraft, MI. The added organism was Pseudomonas stutzeri KC (strain KC), a denitrifying bacterium that cometabolically degrades CT without producing chloroform (CF). To introduce and maintain strain KC in the aquifer, a row of closely spaced (1-m) injection/extraction wells were installed normal to the direction of groundwater flow near the leading edge of the CT plume. The resulting system of wells was used to establish and maintain a "biocurtain" for CT degradation through the intermittent addition of base to create favorable pH conditions; inoculation; and weekly addition of acetate (electron donor), alkali, and phosphorus. Although half of the test zone was inoculated twice, the long-term performance of both sections was indistinguishable: both had high CT removal efficiencies (median of 98-99.9%) and similar levels of strain KC colonization (>10(5) strain KC/g). Sustained and efficient (98%) removal of CT has now been observed over 4 yr. Transient low levels of CF (<20 ppb) and H2S (<2 ppm) were observed, but both disappeared when the concentration of acetate in the weekly feed was reduced. Nitrate removal efficiencies ranged from 60% at low acetate concentrations to nearly 100% at high acetate concentrations. We conclude that closely spaced wells and intermittent substrate addition are effective means of delivering organisms and substrates to subsurface environments. At the Schoolcraft site, we achieved uniform removal efficiencies over a significant vertical depth (15 m), despite significant variability in hydraulic conductivity. This was accomplished by pumping 65% (v/v) of the natural gradient flow passing through the biocurtain during a given week in a single 6-h pumping event. Approximately 18,600 m3 of contaminated groundwater was treated during the project.

Biodegradation, Environmental↗

PubMed2002

Analysis of regulatory elements and genes required for carbon tetrachloride degradation in Pseudomonas stutzeri strain KC.

Previously, we described the generation and initial characterization of four Tn5 mutants of Pseudomonas stutzeri strain KC with impaired ability to degrade carbon tetrachloride (Sepúlveda-Torres et al., 1999). In this study, we show cloning and sequencing of an 8.3 kbp region in which all four transposons were located. This fragment encodes eight potential genes and is located in the central part of the 25 kbp fragment recently identified by Lewis et al. (2000) and shown by them to be sufficient to confer carbon tetrachloride transformation capability upon other pseudomonads. The four transposon insertion mutants mapped in ORF's F and I designated by Lewis et al. (2000). This is consistent with the results by Lewis et al. (2000) that orfFis required for carbon tetrachloride degradation. We further established that orfl is required for CCl4 degradation since the three mutants in this ORF were unable to degrade carbon tetrachloride. We present our analysis of the gene and protein sequences from the 8.3 kbp region and propose a tentative model for the role of different genes in the synthesis and activity of pyridine-2,6-bis(thiocarboxylate) (PDTC), the secreted factor responsible for carbon tetrachloride dechlorination. We also found a putative promoter that overlaps with a Fur-box-like sequence in the region upstream of mutated genes. To test this putative promoter region and Fur-box, we generated and ligated DNA fragments containing wild-type and mutant Fur-boxes to a lacZ reporter. The wild-type fragment showed promoter activity that is regulated by the concentration of iron in the medium. Finally, we screened a selection of Pseudomonas strains, including P. putida DSMZ 3601--a strain known to produce PDTC--for the presence of the genes characterized in this study. None of the strains tested positive, suggesting that Pseudomonas stutzeri strain KC may possess a distinct biosynthetic pathway for PDTC production.

Bacterial Proteins↗

PubMedDate not supplied

A nested-cell approach for in situ remediation.

We characterize the hydraulics of an extraction-injection well pair in arbitrarily oriented regional flow by the recirculation ratio, area, and average residence time in the recirculation zone. Erratic regional flow conditions may compromise the performance of the reactor between a single well pair. We propose an alternative four-well system: two downgradient extraction and two upgradient injection wells creating an inner cell nested within an outer cell. The outer cell protects the inner cell from the influence of regional flow. Compared to a two-well system, the proposed four-well system has several advantages: (1) the recirculation ratio within the nested inner cell is less sensitive to the regional flow direction; (2) a transitional recirculation zone between the inner and outer cells can capture flow leakage from the inner cell, minimizing the release of untreated contaminants; and (3) the size of the recirculation zone and residence times can be better controlled within the inner cell by changing the pumping rates. The system is applied at the Field Research Center in Oak Ridge, Tennessee, where experiments on microbial in situ reduction of uranium (VI) are under way.

Bacteria↗