Thesis (M.Eng.), Memorial University of Newfoundland, 1998. / Bibliography: leaves 86-97.

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Contamination of soils and groundwater with nitroaromatic compounds such as 2,4,6-trinitrotoluene (TNT) and dinitrotoluenes (DNTs) has drawn considerable attention due to widely distributed contamination sites and substantial efforts for cleanup. Two isomers of DNT, specifically 2,6-dinitrotoluene (2,6-DNT) and 2,4-dinitrotoluene (2,4-DNT), occur as soil and groundwater contaminants at former TNT production sites. The discovery of bacteria that use DNT isomers as electron donors has encouraged bioremediation at contaminated sites. Current work is extending the existing engineered bioremediation to naturally occurring in situ biodegradation and focuses on the application of natural attenuation (NA) as a remediation strategy for residual DNT at contaminated sites. More specifically this research evaluated factors influencing in situ bioremediation of DNTs and TNT in surface soils, vadose zones, and saturated medium. Applications involving surface soils and vadose zones investigated the potential of water infiltration to promote in situ bioremediation. Studies in saturated media were more applicable to NA. Factors that were also considered in studies conduced included: 1) the presence and distribution of degrading microbes in field soils (Barksdale, WI); 2) the dissolution and bioavailability of contaminants in historically contaminated soils; and 3) the effect of mixtures of contaminants (i.e., DNTs and TNT) on biodegradation processes. This research provided information useful for practitioners considering an in situ bioremediation NA as a remedial solution for contaminated sites. Under the condition simulating downflow of surface waters or rainwater, the rapid rate of DNT degradation could be facilitated by the availability of oxygen in the soil gas without concern of toxicity (i.e., nitrite evolution and pH drop) and addition of nutrients. As a result, in situ bioremediation or NA should be strongly considered as a remedial option for Barksdale soils and similar sites where relatively low concentrations of DNT isomers are present as contaminants. At TNT contaminated sites TNT was not mineralized by indigenous microorganisms despite oxidative biotransformation, and mixed culture capable of growth on DNT also could not develop the mineralization of TNT during DNT degradation. This suggests that the mixtures of contamination did not improve the potential for in situ TNT bioremediation.

The rivers, lakes and seas are the major water sources for the animal and plant kingdom in this earth. In recent times, the usage and wastage of water have been increasing due to the uncontrolled population growth. In addition to that, the rapid industrialization over the years has led to the gradual depletion of the natural resources like water, soil and air. Some of these industries discharge contaminants like organic products and inorganic (or) toxic heavy metals without treatment into the environment, leading to its degradation. Zinc is the 24th most abundant element present in the earth crust, amounting 75 ppm (0.0075%). The concentration of zinc present in the soil and seawater is about 64 ppm and 30 ppb respectively (Emsley, 2001). Generally, the zinc is found with the base metals such as copper and lead and it has less affinity with oxides and strong affinity with sulphides. Sphalerite, a zinc sulphide ore, is majorly containing 60-62% of zinc. The other sources of zinc from the minerals are smithsonite, hemimorphite, quartzite, and hydro zincate. The major sources of zinc contamination arise from several industrial activities such as mining, coal, waste combustion and steel and iron processing. Drinking water also contains certain amounts of Zn, which may be higher when it is stored in metal tanks. The acute toxicity arises from the ingestion of excessive amounts of zinc salts, either accidentally or as dietary supplement. Vomiting, nausea and stomach cramps usually occur after the consumption of more than 500 mg of zinc sulfate. In addition to that, the higher amounts of zinc affect gastrointestinal tract, liver, bone and prostate glands. Finally, Zn can interrupt the activity in soils, as it negatively influences the activity of microorganisms and earthworms, thus retarding the breakdown of organic matter. To combat this problem, techniques such as chemical precipitation, ion exchange, reverse osmosis, etc. are adopted, but these processes result in a huge amount of secondary sludge formation, inefficient removal of metals and are not cost effective. In recent times, an innovative, eco-friendly, cost-effective method has been introduced to treat the toxic heavy metals namely bioremediation. ―Bioremediation‖ is a process of removal of organic or inorganic contaminants by using bacteria, fungi, algae and its metabolites In this research work, the potential of four bacterial strains of the Pseudomonas sp. such as P.putida, P.alcaligenes, P.aeruginosa and P.fluorescens and the extracellular proteins secreted by these four species for the bio-sorption of zinc has been investigated through batch experiments. The mechanisms of interaction between the zinc ion and the bacterial biomass as well as with the extracellular proteins have been elucidated. Additionally, a carbon paste electrode has been modified by using Pseudomonas sp. and its metabolites to develop biosensors for zinc and the lower limit of detection of zinc in aqueous solution has been determined. The major objectives of this research work are specified below: • To study the potential of Pseudomonas sp. such as P.putida, P.alcaligenes, P.aeruginosa and P.fluorescens for the bio sorption of zinc, in batch systems. • To determine the speciation of zinc with respect to pH in the growth medium and the maximum inhibitory effect of zinc on the growth of the four chosen Pseudomonas sp. • To isolate and characterize the extracellular proteins from the four Pseudomonas sp. such as P.putida, P.alcaligenes, P.aeruginosa and P.fluorescens. • To study the biosorption of zinc by extracellular proteins secreted by the Pseudomonas sp. • To elucidate the mechanisms involved in the biosorption of zinc at the microbe- metal interface and protein-metal ion interface for all the four systems by different characterization studies such as zeta potential, FTIR analysis and EDAX analysis. • To develop a biomass modified CPE using bacterial cells and extracellular protein to detect the concentration of zinc in aqueous solutions adopting voltammetric techniques. The significant results obtained from this research work are summarized as follows: The initial studies were concentrated on the bio sorption of zinc by using four Pseudomonas species such as P.putida, P.alcaligenes, P.aeruginosa and P.fluorescens. The various factors affecting the bio sorption of zinc by these species were investigated by varying the contact time (10-80 min), pH (2-5±0.2), biomass concentration of the four species in the range of 108- 1011 cells / mL, and the initial zinc concentration from 5 mg/L to 80 mg/L respectively, keeping other parameters such as temperature and agitation speed constant in all the experiments. From the results obtained, the maximum percentage of biosorption achieved by the P.putida, P.alcaligenes, P.aeruginosa and P.fluorescens was found to be 60%, 93%, 70% and 65% respectively for 25 mg/L at pH 5±0.2. The equilibrium time taken by the four species to achieve maximum biosorption was about 10 min and the biosorption kinetics adhered to pseudo-second order reaction and the rate constants were determined for different concentrations of zinc. The biosorption isotherm followed both the Langmuir and Freundlich isotherm models. The Gibbs free energy (ΔG) values determined from the Langmuir isotherm model for all the four systems were found to be -26, -32, -30 and -28 kJ /mole respectively. The Gibbs free energy values indicate that the biosorption of zinc ions onto the bacterial surface is a chemi-sorption process involving co-ordination, complexation or chelation. The characterization studies, namely zeta potential, FTIR analysis and SEM-EDX were also carried out on the bacterial cells before and after interaction with zinc. These studies also provide evidence in support of the complexation of zinc with the functional groups on the bacterial cell surface apart from electrostatic interaction. In the second part of the investigation, the inhibitory effect of zinc on the growth of four Pseudomonas sp. was investigated by varying the concentration of zinc from 50 mg/L to 1000 mg/L and the stability of zinc was analysed with respect to pH (2-12) with different concentrations from 50 - 1700 mg/L. It was found that in the absence of zinc the time taken to reach the exponential phase and the specific growth were almost the same for all the four systems. However, in the presence of zinc ions, the growth of the four Pseudomonas sp. was suppressed beyond 50mg/L of zinc. A control study on the stability of zinc in Luria broth medium showed that zinc was highly stable up to 200 mg/L from pH 2-8. However, the stability of zinc in the growth medium decreased beyond that concentration Additionally, studies on the biosorption of zinc were performed using extracellular proteins isolated from the four Pseudomonas sp. The amount of protein was estimated by the Bradford protein assay method at 594 nm. The biosorption experiments were carried out by varying the protein concentration from 50 to 1000µg/mL and the zinc concentration from 50-1000 mg/L and keeping other parameters fixed, namely such as pH at 5±0.2, reaction time of 20 min, temperature at 30±0.2 and the speed of rotation of 200 rpm. It was found that the maximum percentage of zinc biosorbed by the proteins isolated from P.putida was found to be 91% at 500µg/mL of protein concentration and from the other three species, it was found to be about 60% of biosorption at the same protein concentration. The biosorption isotherms of zinc for extracellular protein adhered to the Giles H1 type for all the four systems. The maximum amount of zinc biosorbed by the protein isolated from P.putida, P.alcaligenes, P.aeruginosa and P.fluorescens was found to be 35.6, 19,18.3 and 10 mg/µg respectively and the Gibbs free energy values were found to be -32, -22,-22 and -23 kJ/mole. The mechanisms involved in protein-zinc interaction were elucidated using FTIR analysis and EDX analysis. The FTIR analysis revealed, that the zinc ions were complexed with carboxylic and amine functional groups. Further, the potential of P.putida, P.alcaligenes, P.aeruginosa and P.fluorescens and their extracellular proteins of P.putida as biosensors for detecting zinc ions in aqueous solutions, using electrochemical methods such as, Cyclic Voltammetry and Differential pulse anodic stripping voltammetry, was assessed. The developed carbon paste electrode coated by the biomass showed an approximately 3-fold increase in the sensing of Zn2+ ion in comparison with the bare electrode. The lower limit of detection of the biosensor for zinc ions by Cyclic voltammetry was found to be 10-6 M, and in case of DPASV the lower limit of detection was about 10-7M. The lower limit of detection of the protein modified biosensor for zinc ions by cyclic voltammetry was found to be 10-7M and in the case of DPASV method the lower limit of detection was found to be 10-9 M.

Chlorinated solvents such as trichloroethene (TCE) and perchloroethene (PCE) are commonly found in industrialized areas and can have major impact on human health and groundwater quality. The techniques for removing these substances from the subsurface environment is constantly being tuned and revised, and as such, the need for monitoring at such remediation sites is crucial. To find important correlations and hidden patterns between variables principle component analyses (PCA) and correlations matrixes were used on sets of field data from an existing remediation site in southern Sweden. Four important components were extracted in the following order; End products of dechlorination (EPD), second wave of dechlorination (SWD), first wave of dechlorination (FWD) and indicators of dechlorination (ID). The underlying pattern found in the data set was most likely derived from thermodynamic preference, explaining important correlations such as the correlation between iron and sulfate, the correlation between redox and degree of dechlorination. The law of thermodynamic preference means that we can (roughly) estimate the level of difficulty and/or the time it will take to remediate a polluted site.  These findings show that similar results shown in theory and laboratory environments also applies in the field and also that PCA is a potent tool for evaluating large data sets in this field of science. However, it is of great importance that the correlations are examined thoroughly, as correlation it not equal to causation.

Soil and groundwater at many existing and former industrial areas and disposal sites is contaminated by halogenated organic compounds that were released into the environment. Halogenated organic compounds are heavier than water. When they are released into the subsurface, they tend to adsorb onto the soils and cause the appearance of DNAPL (dense-non-aqueous phase liquid) pool. Among those halogenated organic compounds, trichloroethylene (TCE), a human carcinogen, is one of the commonly observed contaminants in groundwater. Thus, TCE was used as the target compound in this study. The objective of this study was to develop the emulsified carbon-releasing substrate and apply it as the filling material in the permeable reactive barrier to remediate TCE-contaminated groundwater. In this study, the developed emulsified carbon-releasing substrate contained soybean oil, lactate, biodegradable surfactant (Simple GreenTM and lecithin), and nutrients. Results of emulsion test show that up to 90% of the emulsified carbon-releasing substrate was distributed effectively in the soil pores. The emulsified carbon-releasing substrate was able to provide carbon for the enhancement of in situ anaerobic biodegradation for a long period of time. A pilot-scale study was operated at a TCE-contaminated site located in southern Taiwan. Emulsified carbon-releasing substrate emulsion was pressure-injected into the remediation wells. A total of 120 L of emulsified carbon-releasing substrate was injected into the test site. Based on the groundwater analytical results, dissolved oxygen, oxidation-reduction potential, and sulfate concentrations decreased after injection. However, the anaerobic degradation byproduct, acetic acid, increased after injection. Results also show that the total viable bacteria increased in the upgradient injection (remediation) well. Decrease in TCE concentration (dropped to below 0.01 mg/L) was also observed after substrate injection, and TCE degradation byproducts, cis-1,2-dichloroethene (cDCE) and vinyl chloride (VC) were also observed. Result of microbial analyses show that various TCE-degrading bacteria exist in the groundwater samples including Ralstonia sp., Clostridium sp., Uncultured Burkholderiales bacterium, Hydrogenophaga sp., Acidovorax sp., Zoogloea sp., Hydrocarboniphaga sp., Uncultured Curvibacter sp., Pseudomonas sp., Comamonas sp., Aquabacterium sp., and Variovorax strains. This reveals that the anaerobic dechlorination of TCE is a feasible technology at this site. Slug test result show that only a slight variation in soil permeability of the injection well was observed. This indicates that the substrate injection would not cause clogging of the soil pores. Results from the cost analysis show that the total cost for the test site remediation was approximately USD13,442 per year. This indicates that the developed system has the potential to be developed into an environmentally, economically, and naturally acceptable remedial technology. Knowledge obtained from this study will aid in designing a carbon-released substrate biobarrier system for site remediation.

Uranium contaminations of the subsurface in the vicinity of nuclear materials processing sites pose a health risk as the uranyl ion in its oxidized state, U(VI), is highly mobile in aquifers. Current remediation strategies such as pump and treat or excavation are invasive and expensive to implement on a large scale. In situ bioremediation represents an alternative strategy that uses the ability of local microbial communities to immobilize contaminants and is actively studied for uranium remediation. The immobilization of U(VI) in groundwater is achieved either by bioreduction to solid uraninite (U(IV)), adsorption to the soil matrix, or non-reductive precipitation of uranium phosphate minerals through the activity of bacterial phosphatases. Bioreduction has been widely studied for remediation of the saturated zone, as anaerobic conditions typically prevail in these environments. This process is only efficient at circumneutral pH, however, and the end product uraninite is unstable under aerobic conditions or in the presence of manganese oxides, nitrite, or even freshly formed iron oxides. Although non-reductive biomineralization of uranium catalyzed by bacterial phosphatase activity successfully removes uranium from the vadose zone, further studies are needed to assess the ability of microbial communities to hydrolyze organophosphate compounds in the saturated zone where oxygen is often depleted and uranium bioreduction may be significant. To investigate this process under anaerobic conditions, low pH soil samples from a uranium contaminated site at the Oak Ridge Field Research Center were incubated anaerobically in flow through reactors in the presence of exogenic organophosphate compounds to stimulate the natural microbial communities in the original soil matrix. Aqueous uranium was injected continuously in the reactors to determine the fraction of uranium removed during these incubations. The reactors amended with organophosphate produced inorganic phosphate in the effluent, suggesting that bacterial phosphatase activity can be stimulated even in anaerobic environments at low pH. Removal of U(VI) in a control amended with organophosphate over a short time period was similar compared to reactors amended with organophosphate for long times suggesting that adsorption may also play a role in U(VI) immobilization. A sequential extraction technique was optimized to differentiate the fraction of uranium loosely adsorbed and the fraction of uranium precipitated as phosphate minerals and batch adsorption experiments were performed to obtain thermodynamic parameters that could be used to predict the fraction of U(VI) adsorbed onto the soil matrix. Results indicated that 100% uranium adsorption was favorable from pH 5 to 10 (without the presence of phosphate), and that most of the solid phase uranium was extracted in the step defined for the strongly adsorbed/uranium phosphate mineral in both long and short-term amended reactors. Overall, these results demonstrate that the biomineralization of uranium phosphate minerals is a viable bioremediation strategy in both the vadose and saturated zones of aquifers at both low and high pH, provided an organophosphate source is available.

This study describes a laboratory scale development of an in-situ bioremediation method, which uses a water table management system to supply nutrients to subsoil microorganisms, for biostimulation and subsequent biodegradation of pollutants such as fertilizer-nitrate and hydrocarbons (e.g., diesel oils), in the unsaturated zone of the soil. The study, which was divided into two parts: first nitrate bioremediation and secondly diesel biodegradation, was carried out on packed soil columns. / For the nitrate study, different levels of glucose were introduced into packed soil columns, 1,000 mm long x 200 mm, diameter, via subirrigation in order to supplement the organic carbon levels in the soil. Two sandy soils were used, with 1.6% and 3.4% organic matter content, respectively; and the water table in the soil columns was maintained at a depth of 350 mm below the surface. Fertilizer-nitrate was applied to the soil surface at a rate of 180 kg/ha nitrate-N. Simulated rainfall was used to leach nitrates to lower depths. The efficacy of using the subirrigation system, as a method for nutrient delivery in the bioremediation of leached nitrate, was monitored with time and with reference to the nitrate residue, redox potential of the soil solution, and solubilized Fe and Mn. / Leached nitrate was denitrified to less than 10 mg/L nitrate-N, which is the limit permitted in drinking water. The ideal organic carbon range was considered to be the glucose level (20 mg/L glucose-C) that reduced mom nitrate and gave redox potential and soluble Fe and Mn levels, similar to the control soil solution, when subjected to 96 days of subirrigation. Successful delivery of nutrient for the bioremediation of nitrate, within the farm boundaries, will be considered a "break through" toward nitrate residue control if this novel approach to nitrate control is demonstrated in the field. The delivery method will offer a technical solution to on-farm nitrate pollution. It is inexpensive, easy to adopt, and does not require major changes in the current farm practices. / In the second part of the study, a diesel contaminated sandy soil was packed in columns, 2,000 nun long x 200 nun diameter. The subirrigation method was used to supply two different combinations of treatments to the microorganisms in the soil for the biodegradation of the diesel namely: air, water and nutrients (N, P etc.), and air and water. The success of using subirrigation, to deliver nutrients to the soil in the columns, was monitored by measuring the trend in the reduction of soil diesel-TPH (diesel-total petroleum hydrocarbon) residue with time. Results obtained from the treated columns were compared with each other, and with the control columns undergoing passive biodegradation. / The study showed that subirrigation can be used as a method of nutrient delivery in the -bioremediaton of diesel contaminated soil. The TPH in the contaminated soil decreased, from an initial 670 mg diesel TPH/kg soil to an acceptable level of 40 mg diesel TPH/kg soil, in 82 days in the columns subjected to a combination of nutrient, air and water treatments. If this method of delivering biostimulants to the subsoil microbial population is demonstrated in the field, it will be invaluable to in-situ bioremediation of contaminated soils.

Uranium has been released into the environment due to improper practices associated with mining and refinement for energy and weapons production. Soluble U(VI) species such as uranyl carbonate can be reduced to form the insoluble U(IV) mineral uraninite (UO2) via microbial respiratory processes. Formation of UO2 diminishes uranium mobility and prevents uranium-laden groundwater from being discharged into surface water; however, oxygen and other oxidants re-solubilize UO2. Many organisms have been shown to reduce uranium, but variations in microbial physiology change the dynamics of microbial uranium reduction in situ and affect uraninite stability. Anaeromyxobacter dehalogenans is a metal-reducing delta-Proteobacterium in the myxobacteria family that displays remarkable respiratory versatility and efficiently reduces U(VI). The approach of this research was to enhance characterization of A. dehalogenans by identifying unique genetic traits, describing variability within the species, and examining the environmental distribution of A. dehalogenans strains. Genome analysis revealed that A. dehalogenans shares many traits with the myxobacteria including type IV pilus-based motility and an aerobic-like electron transport chain. In addition, the genome revealed genes that share sequence similarity with strict anaerobes and other metal-reducing organisms. Physiological examination of microaerophilism in A. dehalogenans strain 2CP-C revealed growth at sub-atmospheric oxygen partial pressure. Physiological characterization of novel isolates demonstrated that strain-level variation in the 16S rRNA gene coincides with metabolic changes that can be linked to the loss of specific gene homologs. Anaeromyxobacter spp. were present at the Oak Ridge Integrated Field-scale Subsurface Research Challenge (IFC) site and multiplex qPCR tools designed using a minor-groove binding probe gave insights into strain and species differences in the community. Finally, 16S rRNA gene sequences were identified which suggest a novel Anaeromyxobacter species that is responsible for uranium reduction at the Oak Ridge IFC site. This research contributes new knowledge of the ecophysiology of a widely distributed, metal-reducing bacterial group capable of uranium immobilization. The characterization of Anaeromyxobacter spp. helps to elucidate the dynamics of biological cycling of metals at oxic-anoxic interfaces, like those at the Oak Ridge IFC, and contributes to the broader study of microbial ecology in groundwater and sediment environments.

Thesis (Ph.D.)--Civil and Environmental Engineering, Georgia Institute of Technology, 2008. / Committee Chair: Dr. Joseph B. Hughes; Committee Member: Dr. Jian Luo; Committee Member: Dr. Jim C. Spain; Committee Member: Dr. Patricia Sobecky; Committee Member: Dr. Spyros G. Pavlostathis