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Groundwater parameter analysis, microcosm experiments, and microcosms modeling were undertaken to assess the potential of Monitored Natural Attenuation as a remediation strategy at Site 12 at the Naval Amphibious Base (NAB) Little Creek. Site 12 was contaminated with PCE waste disposed by a former dry cleaning facility. In the groundwater analysis, contaminant characteristics and redox indicators were evaluated to assess the reductive dechlorination potential of Site 12. The results of the groundwater analysis indicated that Site 12 exhibited sulfate-reducing and methanogenic conditions which provide the required environment for reductive dechlorination. However, Site 12 only demonstrated partial reductive dechlorination to cis-1,2-DCE and possible anaerobic oxidation of cis-1,2-DCE and VC to CO₂. Microcosms were designed to further evaluate the extent of microbial degradation of the chlorinated ethenes at Site 12 and to provide concentration versus time data for the estimation of chlorinated ethenes' biodegradation rates. The extent of degradation in the microcosms was consistent with the groundwater data. However, ethene production was not observed and the quantity of TCE measured for two of the microcosms differed substantially when compared to the groundwater data. The microcosm model used SEAM3D to simulate the results of the microcosm experiments (concentration versus time data) to estimate the biodegradation rates of PCE and its daughter products. The SEAM3D reductive dechlorination package, based on Monod kinetics, predicted for the MLS12-Shallow microcosm maximum specific utilization rates for PCE, TCE, cis-1,2-DCE and VC at 0.4, 0.42, 0.05, and 0.25 day⁻¹, respectively and half saturation coefficients for PCE, TCE, cis-1,2-DCE and VC at 0.41, 0.01, 0.07, and 0.02 mg/L, respectively. The results of this study suggest that while the groundwater environment provides the necessary conditions for reductive dechlorination, Site 12 is not an efficient system for reductive dechlorination. This lack of efficiency may stem from sparse microbial populations capable of reducing cis-1,2-DCE or the system may contain levels of PCE which inhibit the further reduction of cis-1,2-DCE. Based on the observed inhibitory relationship between PCE and cis-1,2-DCE and VC production, source removal would reduce the PCE levels and encourage further reductive dechlorination at Site 12. Therefore, the recommended first step for a monitered natural attenuation-based remediation strategy at Site 12 should be source removal. / Master of Science

This feasibility study was performed at Virginia Polytechnic Institute and State University (Blacksburg, VA) in collaboration with BioSystems Technology, Inc. (Blacksburg, VA). In this study, degradability of PCBs (Aroclor 1242) from an aged surface soil was evaluated using serum bottle microcosms containing aceticlastic methanogenic consortium, enriched from a municipal anaerobic digester. Two different experiments, "intermediate feed" and "starve and feed" were conducted by manipulating the methanogenic consortium with different amounts of acetate feeding, during 30 days of incubation. Disappearance of Aroclor 1242 in the microcosms was quantified using gas chromatography (GC). Significant differences in Aroclor 1242 removal between inoculated and uninoculated (control) microcosms were observed suggesting that the methanogenic consortium was responsible for Aroclor 1242 disappearance. However, GC-mass spectrometry (GC-MS) results could not confirm that disappearance of Aroclor 1242 was due to anaerobic dehalogenation. From another experiment, it was confirmed that removal of Aroclor 1242 was not due to evaporation losses during sample extraction. Toxicity of an aged Aroclor 1242 contaminated surface soil was evaluated on an aceticlastic methanogenic consortium, enriched from a municipal anaerobic digester. Microcosms were set up using different amounts of soil and inoculum. Total gas production in the microcosms was monitored during 30 days of incubation, using a glass syringe. Total methane production in the microcosms was quantitated using GC. Toxicity of the soil on the methanogenic inoculum was determined based on the decreased rate of methane production in the microcosms relative to non- soil containing controls. Compared to the control, there was reduction in total methane production in soil containing microcosms. Between 3-27% reduction in total methane production was noticed in microcosms containing different amounts of soil and consortium. Reduction in methane production seemed to increase with increasing amount of soil. Whether this decrease in methane production was due to toxicity of Aroclor 1242 on the methanogenic consortium or due possibly to the toxicity of trapped oxygen in the soil could not be determined. The rate of gas production in the soil microcosm was linear. / Master of Science

A groundwater monitoring study was conducted as part of a comprehensive program to remediate a former wood-preserving site that was contaminated with creosote. Twenty-five multi-level samplers (MLSs) were installed on-site and groundwater samples were collected and tested regularly between March 1998 and July 2000. Nearly one-thousand hybrid poplar trees were planted on-site in 1997 to help contain the groundwater plume and enhance phytoremediation. Ten polycyclic aromatic hydrocarbons (PAHs) were monitored along with several terminal electron acceptors (TEAs) and their reduced end products. The focus of the study was to determine the extent of natural biodegradation in the subsurface and assess the role of the poplar trees in site remediation. Since monitoring began, considerable progress has been made remediating the site and the contaminant plume has been shrinking consistently. PAH levels in the groundwater and soil have been reduced and individual MLSs show consistently decreasing contamination. At this point in the study it cannot be conclusively determined what impact the poplar trees are having on the progressing site remediation. However, there is a wealth of evidence indicating that natural biodegradation is playing a major role in site cleanup. Monitoring of TEAs indicates suggests that there are aerobic zones in the site aquifer, but that reduced conditions exist as well. Dissolved oxygen (DO) was found in many MLS ports, but other ports were devoid of both DO and nitrate and contained large quantities of aqueous Fe(II). Oxygen, nitrate and Fe(III) are being reduced on-site and data suggests that they are being used in the biological oxidation of PAHs. Although laboratory studies document the oxidation of PAHs under sulfate-reducing conditions, high aqueous sulfate values were recorded throughout the site, regardless of the level of contamination. Several possible mechanisms are proposed to explain the coexistence of high sulfate and PAHs in the site aquifer. The system may be redox-buffered by excess solid Fe(III) and Mn(III, IV) oxides. Also, dissimilatory sulfate-reducers are strict anaerobes and oxygen-rich rainwater may be toxic to them. The presence of a layer of coal below land surface creates pyrite oxidation conditions similar to those encountered in conjunction with acid mine drainage. The MLSs most affected by the coal layer have less PAHs and DO, lower pH, and higher sulfate and Fe(II) levels than other wells. The oxidation-reduction status of each MLS, based on oxygen, nitrate and Fe(II) measurements, appears to be closely related to the level of PAH contamination, suggesting that PAHs are the primary substrate being biologically oxidized in the site aquifer. These findings tend to support the general belief that the major limitation to natural biodegradation in subsurface environments is the delivery of adequate supplies of suitable TEAs to contaminated zones. / Master of Science

Remediation of chlorinated ethene contaminated sites presents a problem for the environmental industry. Many innovative technologies exist to remove these chemicals from the subsurface; however, most of these technologies require extensive time and incur significant cost. A technology called bioremediation utilizes microorganisms to break down contaminants such as perchloroethene (PCE), trichloroethene (TCE), dichloroethene (DCE), and vinyl chloride (VC) to non-toxic compounds in a process called reductive dechlorination. Microorganisms that are capable of dechlorination usually require reducing conditions as well as bioavailable hydrogen and carbon sources. Emulsified vegetable oil has emerged as a cost-effective source of degradable organic matter to facilitate reductive dechlorination in the subsurface. Through Æ Ã -oxidation, microorganisms can break down the long chain fatty acids in vegetable oil into smaller fatty acids such as acetate, propionate, and butyrate. The fermentation of the oil provides reduced conditions as well as a slow release of hydrogen and carbon into the subsurface. This study consisted of an evaluation the effectiveness of emulsified vegetable oil in stimulating reductive dechlorination using sixteen laboratory microcosms constructed from soil and groundwater from an aquifer contaminated with TCE located at the Naval Weapons Station in Charleston, South Carolina. Each microcosm was monitored for chloroethenes, volatile fatty acids, long chain fatty acids, and total carbon on a weekly basis. Results show successful fermentation of fatty acids and reduced conditions favorable for dechlorination. / Master of Science

Currently, the application of bioremediation requires extensive and costly monitoring due to limited understanding of the terminal electron accepting processes (TEAPs) that control biodegradation, which impairs the accurate quantification of contaminant mass loss. The measurement of redox conditions and evaluation of TEAPs are critical in assessing the capacity for bioremediation at any site. A series of batch microcosm experiments, using sediment collected from a gasoline-contaminated aquifer at Fort McCoy, Wisconsin, was designed to: 1) evaluate the role of Fe(III) in the development of TEAPs during biodegradation of benzene, toluene, ethylbenzene, and the xylenes (BTEX); 2) examine the biodegradation potential in different portions of the plume; and 3) compare methods of TEAP characterization. In general, the presence of Fe-oxides in microcosms inhibited methanogenesis. Although Fe-reducers did not actively degrade BTEX, Fe-reduction did occur, and most probable number (MPN) counts showed that added Fe(III) increased numbers of Fe-reducers in the microcosms. Methane production in microcosms constructed from sediment near the source area was ~5 times lower than levels produced by the mid-plume sediment. No Fe-reduction occurred in microcosms containing sediment from the source area. These results suggest that the source area has much lower biological activity than the mid-plume. TEAP characterization was conducted using a variety of methods, including geochemical indicators, redox dyes, MPN, and hydrogen concentrations. Monitoring of CH4 concentration yielded useful information in delineation of redox processes; Fe(II) monitoring was unreliable as a geochemical indicator. Redox dyes supplied basic information on reducing environments. MPN counts estimated microbial populations in lieu of faulty geochemical indicators, i.e., Fe(II). The measurement of H2 proved to be one of the more simple and reliable methods for TEAP identification. Results of this study indicate that TEAP characterization should include use of multiple methods; relying on geochemical indicators alone is not sufficient. / Master of Science

A contaminant fate and transport computer model, Bioplume II, which allows simulation of bioremediation in ground water systems, was used to compare the effects of 11 remediation scenarios on a benzene plume. The plume was created in three different hydrogeologic settings from the simulation of an underground gasoline storage tank leak.

Les hydrocarbures aromatiques polycycliques (HAPs) sont des polluants organiques persistants (POP) émis essentiellement par les activités humaines suite à la combustion incomplète de la matière organique (industrie, chauffage, trafic routier...). En raison de leur faible taux de dégradation, de leur toxicité et de leur bioaccumulation, les HAPs font l'objet de plusieurs études d'écotoxicologie. La présente thèse, entreprise dans ce contexte par le biais d'une étude microcosmique se propose d'évaluer l'impact des HAPs sur la microflore et la méiofaune et d'appliquer différentes techniques de bioremédiation (biostimulation, bioaugmentation et combinaison des deux techniques) dans le but de dégrader les HAPs. Nos résultats ont montré que les HAPs sont toxiques aussi bien pour la microflore que pour la méiofaune de la lagune de Bizerte. Sur la microflore, l'effet toxique de ces contaminants s'est manifesté par l'inhibition de l'activité bactérienne à l'interface eau–sédiment et par la modification profonde de la structure des communautés bactériennes. En ce qui concerne la méiofaune, les HAPs ont entrainé une altération de la structure des communautés nématologiques. En effet, nous avons pu caractériser des espèces indicatrices de pollution par les HAPs. Ainsi, l'espèce Spirinia parasitifera dont la densité s'accroît dans tous les microcosmes contaminés par les HAPs paraît être une espèce “opportuniste” à la pollution par les HAPs. Cependant, Oncholaimus campylocercoïdes, fortement dominante dans tous les microcosmes témoins, a diminué de densité dans tous les microcosmes contaminés et a été considérée comme HAP-sensible. En analysant la structure des communautés microbiennes et méiofaunistiques, nous avons observé que la méiofaune joue un rôle structurant dans le maintien d'une communauté microbienne peu sensible aux effets des HAPs. Ce rôle structurant a été moins prononcé dans le cas d'ajout des sels nutritifs par la technique de biostimulation ; technique qui a présenté des effets non significatifs vis-à-vis des communautés nématologiques et a semblé plus efficace dans la minéralisation des HAPs et par conséquent à la réduction de leur effet toxique sur les organismes benthiques. Ces résultats suggèrent que la bioremédiation serait une alternative prometteuse à la dégradation des HAPs. / Polycyclic aromatic hydrocarbons (PAHs) are persistent organic pollutants (POP) emitted mainly by human activities due to the incomplete combustion of organic matter (industry, heating, traffic ...). Because of their low rate of degradation, their toxicity and their bioaccumulation, PAHs are main of concern in ecological studies. In this context, the objectives of this thesis were to assess the impact of PAHs on the benthic microflora and meiofauna and apply different techniques of bioremediation (biostimulation, bioaugmentation and combination of both treatments) in order to degrade PAHs, using experimental approach with microcosms.Our results showed that PAHs are toxic for microflora but also for meiofauna of Bizerta lagoon. The toxic effects of these contaminants were demonstrated by the inhibition of bacterial activity in the sediment/water interface and by profound changes in the structure of bacterial communities. PAHs provoked significant changes on meiofaunal community with the selection of nematode species that could be proposed as bioindicators of PAH pollution. Thus, Spirinia parasitifera which significantly (p<0.05) increased in PAH contaminated microcosms, suggesting that it is an "opportunistic» species to PAH pollution. In contrast, Oncholaimus campylocercoïdes, strongly dominant in control microcosms, decreased in PAH contaminated microcosms and seemed to be a ‘‘PAH-sensitive'' species.By analyzing the structure of microbial and meiofaunal community, we observed that the structural role of meiofauna on bacteria community structure was still evident even under PAH contamination despite the toxic effects on meiofauna. However, this structural role of meiofauna disappeared when nutrients were added to the sediment resulting in an almost complete removal of PAHs. Biostimulation seemed to be the most effective bioremediation strategy in the reduction of PAH toxic effects on benthic organisms. Overall, these results suggest that bioremediation using nutrient addition is a promising alternative technique for the degradation of PAHs in coastal polluted environments.

Microbial metal reduction contributes to biogeochemical cycling, and reductive precipitation provides the basis for bioremediation strategies designed to immobilize radionuclide contaminants present in the subsurface. Facultatively anaerobic ×-proteobacteria of the genus Shewanella are present in many aquatic and terrestrial environments and are capable of respiration on a wide range of compounds as terminal electron acceptor including transition metals, uranium and transuranics. S. putrefaciens is readily cultivated in the laboratory and a genetic system was recently developed to study U(VI) reduction in this organism. U(VI) reduction-deficient S. putrefaciens point mutant Urr14 (hereafter referred to as CCMB1) was found to retain the ability to respire several alternate electron acceptors. In the present study, CCMB1 was tested on a suite of electron acceptors and found to retain growth on electron acceptors with high reduction potential (E¡¬0) [O2, Fe(III)-citrate, Mn(IV), Mn(III)-pyrophosphate, NO3-] but was impaired for anaerobic growth on electron acceptors with low E¡¬0 [NO2-, U(VI), dimethyl sulfoxide, trimethylamine N-oxide, fumarate, ×-FeOOH, SO32-, S2O32-]. Genetic complementation and sequencing analysis revealed that CCMB1 contained a point mutation (H108Y) in a CcmB homolog, an ABC transporter permease subunit required for c-type cytochrome maturation in E. coli. The periplasmic space of CCMB1 contained low levels of cytochrome c and elevated levels of free thiol equivalents (-SH), an indication that redox homeostasis was disrupted. Anaerobic growth ability, but not cytochrome c maturation activity, was restored to CCMB1 by adding exogenous disulfide bond-containing compounds (e.g., cystine) to the growth medium. To test the possibility that CcmB transports heme from the cytoplasm to the periplasm in S. putrefaciens, H108 was replaced with alanine, leucine, methionine and lysine residues via site-directed mutagenesis. Anaerobic growth, cytochrome c biosynthesis or redox homeostasis was disrupted in each of the site-directed mutants except H108M. The results of this study demonstrate, for the first time, that S. putrefaciens requires CcmB to produce c-type cytochromes under U(VI)-reducing conditions and maintain redox homeostasis during growth on electron acceptors with low E¡¬0. The present study is the first to examine CcmB activity during growth on electron acceptors with widely-ranging E¡¬0, and the results suggest that cytochrome c or free heme maintains periplasmic redox poise during growth on electron acceptors with E¡¬0 < 0.36V such as in the subsurface engineered for rapid U(VI) reduction or anoxic environments dominated by sulfate-reducing bacteria. A mechanism for CcmB heme translocation across the S. putrefaciens cytoplasmic membrane via heme coordination by H108 is proposed.

Nitroglycerin (NG) is a toxic explosive commonly found in soil and contaminated groundwater at old manufacturing plants and military ranges. When NG enters an aquifer, it behaves as a dense non-aqueous phase liquid (DNAPL). Nitroglycerin is an impact sensitive explosive and therefore excavating the area to remove or treat the contaminant can be dangerous. In situ bioremediation and natural attenuation of NG have been proposed as remediation alternatives and it is therefore necessary to understand the degradation mechanisms of NG in contaminated soil and groundwater and investigate the potential for using bioremediation at contaminated sites. Many bacteria have been isolated for the ability to transform NG as a source of nitrogen, but no isolates have used NG as a sole source of carbon, nitrogen, and energy. We isolated Arthrobacter JBH1 from NG contaminated soil by selective enrichment with NG as the sole growth substrate. The degradation pathway involves a sequential denitration to 1,2-dinitroglycerin (DNG) and 1-mononitroglycerin (MNG) with simultaneous release of nitrite. Flavoproteins of the Old Yellow Enzyme (OYE) family capable of removing the first and second nitro groups from NG have been studied in the past and we identified an OYE homolog in JBH1 capable of selectively producing the 1 MNG intermediate. To our knowledge, there is no previous report on enzymes capable transforming MNG. Here we show evidence that a glycerol kinase homolog in JBH1 is capable of transforming 1 MNG into 1-nitro-3-phosphoglycerol, which could be later introduced into a widespread pathway, where the last nitro group is removed. Overall, NG is converted to CO2 and biomass and some of the nitrite released during denitration is incorporated into biomass as well. As a result, NG can be now considered a growth substrate, which changes the potential to bioremediate NG contaminated sites. The magnitude of the effect of biodegradation processes in the fate of NG in porous systems was unknown, and we have been able to quantify these effects, determine degradation rates, and have evidence that bioaugmentation with Arthrobacter sp. strain JBH1 could result in complete mineralization in contaminated soil and sediments contaminated with NG, without the addition of other carbon sources. Site specific conditions have the potential to affect NG degradation rates in situ. Experiments were conducted to investigate NG degradation at various pH values and NG concentrations, and the effects of common co-contaminants on NG degradation rates. Arthrobacter JBH1 was capable of growing on NG at pH values as low as 5.1 and NG concentrations as high as 1.2 mM. The presence of explosive co-contaminants at the site such as trinitrotoluene and 2,4-dinitrotoluene lowered NG degradation rates, and could potentially result in NG recalcitrance. Collectively, these results provide the basis for NG bioremediation and natural attenuation at sites contaminated with NG without the addition of other sources of carbon. Nonetheless, careful attention should be paid to site-specific conditions that can affect degradation rates.