Browsing by Author "Hughes, Joseph B."
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Item 2,4,6-trinitrotoluene reduction by hydrogenase in Clostridium acetobutylicum(2003) Watrous, Mary Margaret; Hughes, Joseph B.Unique genetic modifications to C. acetobutylicum altered the level of hydrogenase expression, allowing study of the resulting effects on the 2,4,6-trinitrotoluene (TNT) reduction rates to be possible. A strain designed to over-express the hydrogenase gene resulted in maintained TNT reduction during late growth phases when it is not typically observed. Strains exhibiting under-expression of hydrogenase produced slower TNT rates of reduction correlating to the expected inhibition of each strain type. Hydrogenase activity, measured by hydrogen production, in Clostridium acetobutylicum correlates strongly (R2 = 0.89) to TNT reduction rates. Indications suggested that hydrogenase potentially played an integral role in catalysis of TNT transformation by reducing its nitro substituents to the corresponding hydroxylamines. A mechanistic pathway is proposed by which this transformation takes place and may enhance the understanding of commonly found hydrogenases in other microorganisms and their ability to transform nitroaromatic compounds.Item Anaerobic transformation of 2,4,6-trinitrotoluene by Clostridium acetobutylicum(1996) Khan, Tariq A.; Hughes, Joseph B.In this work, rapid TNT ($>$150 mg/L) transformation by Clostridium acetobutylicum via a reductive pathway to monoaminonitrotoluenes, diaminonitrotoluenes, and on to undetectable end products was observed. The maximum velocities were greatest for TNT reduction, followed by 2A46DNT, 4A26DNT, and 24DA6NT. Initial reduction was preferred at the para position. The measured TNT transformation rates were considerably higher than those observed by others. The transformation of hydroxylamino intermediates was rate limiting. Rates and extents of transformation of TNT and its metabolites were affected by the organism's growth phase. When TNT was added in the early acidogenic phase, rapid transformation to undetectable compounds occurred. When added at the end of exponential growth, TNT and its intermediates were recalcitrant. The decline in transformation was putatively due to ferrodoxin and electron transfer inactivity. The results show potential for this organism to remediate TNT contamination, as in an ex situ bioslurry reactor amended with soluble glucose (sugar).Item Bioavailability of polycyclic aromatic hydrocarbons as sediment-associated, desorption-resistant contaminants(2000) Beckles, Denise Maria; Hughes, Joseph B.This study investigated the bioavailability of sediment-sorbed polycyclic aromatic hydrocarbons (PAHs), specifically naphthalene, phenanthrene and fluoranthene to a mixed microbial culture after extensive step desorption. The culture was tested for its ability to produce extracellular biomolecules that could affect PAR partitioning. Also, the relative importance of mass transfer and biodegradation rates to PAR bioavailability was analyzed. The study was designed to overcome certain problems associated with previous bioavailability studies. The use of a unified approach (same sediments, contaminants and inocula) made the effects of each parameter on bioavailability more readily identifiable. Also, because the effect of extracellular biomolecules on PAH partitioning was identified separately, an analysis of abiotic desorption and biodegradation was possible. The PAHs were all bioavailable, even at low aqueous concentrations. Naphthalene and phenanthrene mineralization could be enhanced by adding excess naphthalene. The excess naphthalene supported higher biomass concentrations in the phenanthrene systems. Fluoranthene mineralization was enhanced for only a short time, then was statistically indistinguishable from systems without excess naphthalene. After biodegradation, the final sediment concentration of all PAHs became independent of the initial concentration. The desorption of all the sediment-sorbed PAHs was not affected by either biologically produced molecules or the presence of excess naphthalene. The PAHs all exhibited biphasic abiotic desorption, and the extent of desorption varied inversely with the Kow of the contaminant. The amount of phenanthrene and fluoranthene biodegraded was less than that available through abiotic desorption. Naphthalene biodegradation differed. Without excess naphthalene, biodegradation closely followed abiotic desorption. With excess naphthalene, biodegradation exceeded predicted abiotic desorption. In conclusion, PAHs are bioavailable, even at low initial aqueous concentrations. The mixed culture produced consistent, low final sediment concentrations for all three PAHs. While additional naphthalene can increase PAH bioavailability, the effect is PAH specific, and probably due to higher biomass concentrations. Because the partitioning of the sorbed PAHs is the same in abiotic and biotic systems, a direct comparison of mass transfer and biodegradation is valid. This analysis determined that the biodegradation of the higher molecular weight PAHs is controlled by biological activity, not mass transfer rates.Item Biodegradation of trichloroethene by ammonia-oxidizing bacteria: Kinetics, effects of mixtures, and toxicity(1995) Ochoa, Martin Humberto; Hughes, Joseph B.Trichloroethene is degraded by ammonia-oxidizing bacteria. In the absence of ammonia, TCE transformation proceeds at moderate rates. Under the experimental conditions tested, the presence of PCE and cis-DCE, competitive inhibitors of TCE, slowed the rate and extent of TCE transformation. The capacity of these microorganisms to degrade TCE in the absence or presence of mixtures is diminished by the inactivation of the cells as a consequence of the transformation of the chlorinated compounds. The potential application of ammonia oxidizers as a stand-alone in situ treatment process for TCE degradation is diminished by the inactivation of the cells and the inhibitory effects that mixtures of TCE and other chlorinated compounds pose to the microorganisms.Item Characterization of tetrachloroethene-dechlorinating bacteria and investigation into their ability to enhance removal rates of tetrachloroethene-containing nonaqueous phase liquids(2000) Carr, Cynthia Schmidt; Hughes, Joseph B.The influence of electron donors on the ability to sustain tetrachloroethene (PCE) dechlorination was investigated in recycle columns containing a PCE-dechlorinating mixed culture. Over a period exceeding one year, it was demonstrated that equivalent amounts of lactate, methanol, and hydrogen could sustain rate and extent of dechlorination equally. Dechlorination was not impacted by competition for electron donor at high hydrogen partial pressures, despite the presence of an actively methanogenic community. Results from these experiments imply that cost and method of delivery may dictate electron donor selection for stimulation of anaerobic in situ dechlorination. Efforts to isolate PCE-dechlorinating organisms from the mixed culture resulted in the development of a highly purified co-culture that dechlorinated PCE to cis-dichloroethene and was dominated by curved, motile rods and cocci. Preliminary molecular biology techniques were employed to characterize the ecology of the purified culture. It was discovered that multiple microorganisms, one of which shared 98% 16S rDNA sequence identity with the fermentative coccus, Lactosphaera pasteurii, were present. Experiments characterizing the nutritional requirements of the purified coculture determined dechlorination sustainment required the addition of filter-sterilized cell extract prepared from the parent mixed culture. It was determined that hydrogen, pyruvate, glucose, ethanol, and yeast extract could sustain dechlorination, while formate, acetate, acetaldehyde, lactate, propionate, butyrate, and methanol could not. Experiments to determine the effect of dechlorination on the longevity and composition of PCE-containing NAPLs were conducted in continuous-flow stirred-tank reactors (CFSTRs). Comparisons between biotic and abiotic CFSTRs demonstrated that dechlorination resulted in a factor of 14 increase in PCE removal rates from the NAPL. Dechlorination daughter products partitioned between the aqueous and NAPL phases, resulting in temporal changes in NAPL composition. The combined effects of dissolution and dechlorination on the removal of chlorinated ethenes from the NAPL were described using a mathematical model that approximated dechlorination as a pseudo first-order process. It was determined that total chlorinated ethenes removal from the NAPL would be achieved in 13 days in the biotic CFSTRs, as compared to 77 days in the abiotic CFSTRs---corresponding to an 83% reduction in the longevity of the chlorinated ethenes component of the NAPL.Item Demonstration-scale analysis of anaerobic bioremediation of tetrachloroethene DNAPL source zone using bioaugmentation and electron donor delivery(2002) McDade, James Michael; Hughes, Joseph B.An experiment was designed to test the hypothesis that bioremediation within DNAPL source zones is feasible provided all limiting factors are delivered in adequate amounts. A demonstration-scale aboveground aquifer simulation system was bioaugmented with a mixed anaerobic dechlorinating culture after the addition of neat PCE below the water table. Hydrogen Releasing Compound RTM was introduced to the tank to provide a long-term and steady supply of reducing equivalents for dechlorination. The system effluent was monitored for chlorinated ethene concentrations, and results demonstrated the dechlorination of PCE to trichloroethene (TCE) and cis-dichloroethene (cis-DCE). Measurements at the end of the study indicated the following mole fractions in the effluent: cis-DCE (66%), TCE (24%), and PCE (10%).Item Effects of surfactants on a PCE dechlorinating mixed culture(2002) McGuire, Travis Martin; Hughes, Joseph B.The influence of surfactants on a perchloroethene dechlorinating mixed culture was investigated in laboratory experiments. Seven surfactants, representing each of the surfactant classes, were screened for their effects on the rate and extent of perchloroethene dechlorination. Based on screening results, a nonionic surfactant, Tween 80, and an anionic surfactant, Steol CS-330, were selected for use in further studies. Dechlorination of perchloroethene to dichloroethene, vinyl chloride, and ethene occurred in all Tween 80 amended microcosms with a depressed rate of ethene production as the only adverse effect. Steol CS-330, however, inhibited dechlorination beyond dichloroethene at all surfactant concentrations exceeding 25 mg/L. Attempts to acclimate a culture to the surfactant were unsuccessful. Inhibition of vinyl chloride and ethene production was reversible upon dilution of the surfactant to 10 mg/L or below indicating that surfactant interactions with the enzyme system responsible for reductive dechlorination of dichloroethene was the most likely cause of inhibition.Item Impacts of unsaturated zone reaeration on the bioattenuation of organic contaminants in groundwater systems(2000) Neale, Charles Nelson; Hughes, Joseph B.; Ward, C. H.Reaeration, or the diffusion of O2 through the unsaturated zone and into an aquifer system, is a mechanism that influences the extent of hydrocarbon contaminant bioattenuation in ground water. A series of laboratory and numerical modeling experiments were completed to determine the amount of O2 supplied to an anaerobic aquifer by reaeration under various unsaturated zone conditions and to quantify the impacts of reaeration on the migration of hydrocarbon plumes in ground water. Results from laboratory experiments indicated that reaeration flux rates exhibited a bimodal distribution and either approached 12,000 Mg/M2-day or were less than 2,000 Mg/M2-day. Soil water content significantly affected the reaeration flux rate while soil type, soil O2 utilization rate, and unsaturated zone thickness did not influence the reaeration rate. In some cases, the capillary rise impeded O2 transport into the bulk ground water due to high water content near the base of the capillary region. An unsaturated zone transport model was developed to predict reaeration flux based on the effective diffusion coefficient of soil (Ds), liquid-side mass transfer coefficient (KL), Soil O2 utilization rate (Rsoil), and unsaturated zone thickness (z). Good agreement was generally found between the predicted and experimental flux results. Results from numerical modeling experiments indicated that reaeration reduced the steady-state length of hydrocarbon plumes in ground water for aquifer hydraulic conductivities of K = 10--2 cm/s and K = 10--4cm/s. Important groundwater reaeration parameters having an influence on the steady-state hydrocarbon plume length included the concentration of O2 in the overlying soil gas (C O2(g)), the ground water mass transfer coefficient (KL,GW), the vertical dispersivity in the saturated zone (alphav), and the zone of reaeration or the thickness of the top model layer (ZR). The relative importance of each of these parameters was on the order of alphav > > KL,GW > CO2(g) > ZR.Item Interpretation and modeling of slurry reactor performance to provide monitoring strategies for the aerobic bioremediation of dinitrotoluene-contaminated soils(2001) Daprato, Rebecca C.; Hughes, Joseph B.In a previous study, pilot-scale bioslurry reactors were used to treat soils highly contaminated with 2,4-dinitrotoluene (2,4-DNT) and 2,6-dinitrotoluene (2,6-DNT). The treatment scheme involved a soil washing process followed by two sequential aerobic slurry reactors augmented with DNT mineralizing bacteria. This previous work found that constant monitoring was necessary to avoid long lag periods upon refeeding. In this study, it was determined that the heterogeneous distribution of soil in the reactor deleteriously impacted direct monitoring of DNT concentrations. Instead, the use of nitrite production or NaOH consumption as surrogate monitoring parameters proved to be more accurate predictors of reactor performance. A model was developed to predict the distribution and biodegradation of DNT in the reactors. Analysis of model results showed that the maximum substrate utilization rate controlled DNT degradation rates in the reactor, a population shift occurred after approximately 26 days, and phosphorous was limiting at high solids loading rates.Item Laboratory investigations into biologically enhanced removal of PCE from NAPL source zones(2001) Cope, Nathan; Hughes, Joseph B.The influence of dechlorinating microorganisms on PCE and its reduction products in a residual non-aqueous phase liquid (NAPL) source zone was investigated. Experiments were conducted in upflow anaerobic columns containing glass beads contaminated with a residual NAPL consisting of tridecane and PCE. Columns were inoculated with a mixed PCE-dechlorinating culture, and fed a nutrient medium that included electron donor (pyruvate and/or lactate) at a wide range of concentrations. The systems that were monitored for effluent organic acids (pyruvate-fed) demonstrated evidence of active fermentation. Comparisons between biotic and abiotic columns demonstrated that dechlorination resulted in an increase in total PCE removal, up to a factor of 16 over dissolution. PCE was sequentially reduced as far as vinyl chloride. Total chlorinated ethenes removal was enhanced up to 6.5 times over dissolution removal. The system fed the highest electron donor level, interestingly, lost dechlorinating activity early in the experiment.Item Population dynamics of tetrachloroethene dechlorinating consortia for surfactant and bioaugmentation remediation applications(2007) Daprato, Rebecca C.; Hughes, Joseph B.Three anaerobic, dechlorinating consortia were enriched from different origins using methanol and tetrachloroethene (PCE) and maintained for approximately three years. Characterization of the consortia with terminal restriction fragment length polymorphism (TRFLP) and qualitative and quantitative PCR (qPCR) demonstrated that all three dechlorinating communities were dominated by Dehalococcoides and Dehalobacter spp. Monitoring methane production combined with qPCR for archaea demonstrated that complete PCE dechlorination occurred in the presence and absence of methanogenesis. Combining results for denaturing gradient gel electrophoresis (DGGE) and qPCR for reductive dehalogenase genes suggested that one consortium contained a strain 195-type organism with the ability to respire vinyl chloride (VC). The effect of the anionic surfactant SteolRTM CS-330 on PCE dechlorination was evaluated using pure and mixed dechlorinating cultures. Sulfurospirillum multivorans was the only pure culture able to dechlorinate in the presence of SteolRTM CS-330. S. multivorans was present in consortia OW and CH, and these consortia were capable of partial dechlorination to cis-dichloroethene ( cis-DCE) in the presence of SteolRTM CS-330. Monitoring Dehalobacter spp., Sulfurospirillum spp. and Dehalococcoides cell numbers in consortium OW during exposure to SteolRTM CS-330 with qPCR demonstrated that the cell numbers were reduced by 79%, 88% and 99%, respectively. Dechlorination past cis-DCE was never recovered after resuspension into surfactant free media. Two experimental controlled release systems (ECRS) were employed to examine PCE source zone bioremediation and the efficacy of bioaugmentation. Results obtained demonstrated that bioaugmentation enhanced PCE removal by a factor of 1.6 over biostimulation alone, but minimal ethene production was observed in both systems. Interestingly, both systems contained Dehalococcoides capable of growth on VC; but VC dechlorination was not observed. It was also demonstrated that the bioaugmented populations became dominant, and that the dechlorinating organisms were not washed out of either system. Analysis of energy flow demonstrated that the dechlorinating populations consumed more energy than the methanogens until chloroethenes became limiting. A comparison of cell numbers between archaea and dechlorinating organisms showed that cell numbers did not correlate to activity, since methanogens had higher cell numbers throughout the experiment.Item The behavior of TNT in model chemical redox systems and the fate of TNT reduction products in aerobic microbial systems(1996) Pucik, Lara Ellen; Hughes, Joseph B.When exposed to the Fenton oxidation process, approximately 25% of the initial TNT was mineralized and approximately 63% was converted to unknown soluble products. When exposed to Fe(0) under acidic conditions, TNT was completely reduced to unknown products, approximately 20% of which were not soluble. Reduction of TNT by Fe(0) did not occur above pH 3.0. When exposed to sulfide, TNT was reduced to a mixture of 4ADNT, 2ADNT, DA6NT and unknown soluble products. TNT and its reduction products from chemical (iron and sulfide), aquatic plant, and Clostridia systems were not mineralized by either an activated sludge culture or a mixed aerobic culture grown on anthranilic acid. Biological transformation of TNT and sulfide-reduced TNT products, however, did appear to occur in the aerobic microbial culture grown on anthranilic acid.Item The biodegradation patterns of mixtures of the polycyclic aromatic hydrocarbons naphthalene, acenaphthene and fluoranthene in sediments(1997) Beckles, Denise Maria; Hughes, Joseph B.; Ward, C. H.The biodegradation patterns of polycyclic aromatic hydrocarbon (PAH) mixtures were studied in systems with and without sediments. Naphthalene showed no change in its behaviour in the presence of any other PAH. In sediment-free systems, acenaphthene showed a diauxic pattern in the presence of naphthalene, but was unaffected with fluoranthene. Diauxy was not observed in the same mixture in sediment-containing systems. Sediment-free experiments with fluoranthene indicated it was the only PAH not degraded when present alone, and it was only degraded with naphthalene. In sediment-containing systems, fluoranthene degradation only occurred above a critical naphthalene concentration. Experiments using all three PAHs in both sediment-free and -containing systems showed results similar to those obtained using pairs. This work indicated that mixtures of PAHs do have different biodegradation patterns to single compounds. These patterns may be additive. Sediments act as a sink for PAHs, highlighting the concentration dependence of the patterns observed.Item The reactivity of partially reduced metabolities of 2,4,6-trinitrotoluene in natural systems(2001) Ahmad, Farrukh; Hughes, Joseph B.The reactivity of partially reduced metabolites of 2,4,6-trinitrotoluene (TNT), namely arylhydroxylamines and nitrosoarenes, was evaluated with a simple biological system and with components of soil natural organic matter (NOM). This study was carried out to address the long-standing problem of irreversible binding to soil NOM and biomass, commonly observed during the reductive transformation of polynitroaromatic contamination. The study focused on partially reduced metabolites rather than the completely reduced arylamine metabolites that have already been extensively investigated for their role in binding to soil NOM. In the simple bioreduction system of Clostridium acetobutylicum cell-free extract/molecular hydrogen (electron donor), 10% of the initial 14C was found bound to solid proteinaceous material following sequential anaerobic/aerobic treatment. A review of the nitroso and hydroxylamino functional group chemistry revealed that the nitroso-thiol reaction was most likely responsible for the reaction with proteins. The introduction of a model thiol, 1-thioglycerol, into an anaerobic mixture of 4-hydroxylamino-2,6-dinitrotoluene (4HADNT) and 2,4-dihydroxylamino-6-nitrotoluene (DHA6NT) resulted in the formation of a new product, only when the reaction mixture was exposed to air. The results from the model reaction confirmed that thiols could act as competing nucleophiles for nitroso compounds, which are readily formed from hydroxylamino compounds upon exposure to air. The reactivity of arylhydroxylamines and nitrosoarenes with standard humic acids was investigated using 4HADNT and nitrosobenzene as model compounds, respectively. Contrary to results reported by others, 4HADNT was found to be nonreactive towards humic acid at humic acid concentrations in excess of dissolved organic matter concentrations found in nature. Conversely, nitrosobenzene reacted rapidly with humic acids, with the extent of reaction being highest for humic acids that had a high protein content. Humic acids that were pretreated with a thiol derivatizing agent showed diminished capacity for reaction with nitrosobenzene. Since nitroso intermediates from TNT reduction are difficult to synthesize and are rarely observed in nature due to their high instability, their electrophilic characteristics were evaluated using a molecular modeling approach. Molecular models of potential TNT nitroso intermediates were compared with those of the strongly electrophilic nitrosobenzene. The comparison revealed that 2-nitroso-4-hydroxylamino-6-nitrotoluene was more likely to react similarly to nitrosobenzene than 4-nitroso-2,6-dinitrotoluene.