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.

Tetrachloroethene (PCE) is a major groundwater contaminant that often persists as dense, nonaqueous phase liquids (DNAPLs) in subsurface environments. Dissolved-phase PCE plumes emanate from DNAPL source zones, which act as continuous sources of contamination for decades. Removal of DNAPL source zones is crucial to achieve lasting remedy of contaminated aquifers. This research explored the contributions of the microbial reductive dechlorination process (i.e., anaerobic bioremediation) to PCE-DNAPL source zone remediation, either in isolation or as a polishing step for the removal of residual DNAPL remaining after application of surfactant enhanced aquifer remediation (SEAR), an emerging physical-chemical source zone treatment. Specific objectives of this research were to: (1) evaluate the ability of microorganisms to dechlorinate in the presence of PCE-DNAPL and at high dissolved-phase PCE concentrations expected near/in DNAPL source zones, (2) assess the distribution and activity of key dechlorinating populations during bioenhanced PCE-DNAPL dissolution in continuous-flow column experiments, (3) determine the influence of Tween 80, a biodegradable surfactant commonly used in SEAR, on the microbial reductive dechlorination process, (4) design and optimize quantitative real-time PCR (qPCR) protocols to detect and enumerate key dechlorinating populations (e.g., Geobacter lovleyi, Sulfurospirillum multivorans), and (5) explore the effects of oxygen on Dehalococcoides viability and biomarker quantification. This research demonstrated that microbial dechlorinating activity within DNAPL source zones promotes bioenhanced dissolution although many dechlorinating isolates cannot tolerate saturated PCE concentrations. Application of newly designed qPCR protocols established a direct link between dissolution enhancement and the distribution of relevant dechlorinating populations in the vicinity of PCE-DNAPL. The limited and reversible impact of Tween 80 on key dechlorinators supported the feasibility of a treatment train approach of SEAR followed by microbial reductive dechlorination to remediate PCE-DNAPL source zones. Finally, experiments with oxygen-exposed, Dehalococcoides-containing cultures suggested limitations of using Dehalococcoides DNA and RNA biomarkers for monitoring bioremediation at field sites. These findings advance the scientific understanding of the microbial reductive dechlorination process and are relevant to environmental remediation practitioners. The advantages and current shortcomings of PCE-DNAPL source zone bioremediation, as well as recommendations for future research, are discussed.

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.

Water pollution, especially caused due to the indiscriminate release of heavy metals as a result of anthropogenic activities is a major concern worldwide. Chromium, a heavy metal, regardless of its commercial importance has found to be a potent water pollutant. Chromium generally exist as hexavalent (Cr(VI)) and trivalent (Cr(III)) chromium in the environment. Cr(VI) is ascertained to be more toxic compared to Cr(III) and the former is identified as a carcinogen by the World Health Organisation (WHO). Some of the conventional methods currently available for chromium pollution mitigation are not cost effective and most importantly lead to secondary pollution in the form of sludge. Bioremediation is a promising alternative technique which is also ecofriendly. The bioremediation process utilises biological materials such as microorganisms and agricultural byproducts. Biosorption is a bioremediation process that is a surface related phenomenon involving adsorption of contaminant chromium ions onto the binding sites of the biosorbents. In addition to the efforts made to the remediation of chromium, continuous monitoring of chromium contaminant level in polluted water bodies becomes imperative. The present research study encompasses findings related to bioremediation and detection of chromium ions using bacterial cells. The first part of the dissertation involves studies pertaining to the bioremediation of chromium ions using different bacterial strains as biosorbent. For the study, bacterial strains procured from a microbial culture collection bank as well as those isolated from chromium polluted water samples collected from an industrial site were assessed for their ability to remediate chromium. The next aim of the study was to elucidate the mechanisms involved in the bioremediation of chromium ions by the bacterial cells for which the different characterisation methods such as, Fourier Transform Infrared (FTIR) spectroscopy, Energy Dispersive Spectroscopy (EDS), X-ray Photoelectron Spectroscopy (XPS) and zeta potential measurements which enabled to throw light on the reactions occurring at the bacterial cell surface-chromium solution interface. The later part of the study examines the capability of the bacterial strains used in the bioremediation studies as sensors for the detection of Cr(VI) and Cr(III) ions by adopting electroanalytical techniques, such as, Cyclic Voltammetry (CV) and Cathodic Stripping Voltammetry (CSV), wherein a microbe-modified Carbon Paste Electrode (CPE) was used as the working electrode in a typical three electrode electrochemical cell with Saturated Calomel Electrode (SCE) and platinum wire used as the reference and auxiliary electrodes respectively. The key objectives of the present study are as follows: (i) To study the bioremediation of Cr(VI) and Cr(III) ions present in aqueous solutions using two bacterial strains procured from a microbial culture collection bank as biosorbents. The bacterial strains used were Corynebacterium paurometabolum (Cp), a Gram positive bacterium and Citrobacter freundii (Cf), a Gram negative bacterium. The various factors affecting the biosorption process are to be investigated. (ii) To isolate and identify bacterial strains from water samples collected from chromium contaminated mining site in Sukinda, Odisha, India, by adopting appropriate microbiological and molecular biological procedures. (iii) To study the various factors affecting bioremediation of Cr(VI) using the mine isolates (Chromobacterium sp. (Cb) and Sphingopyxis sp. (Sp)) both Gram negative, as biosorbents. (iv) To elucidate the mechanisms adopted by the chosen bacterial cells in the bioremediation of chromium. (v) To develop an electrochemical-microbial sensor by modifying the Carbon Paste Electrode (CPE) using the bacterial strains for the detection of Cr(VI) and Cr(III) ions present in aqueous solutions. (vi) To determine the capability of the developed sensor in the detection of Cr ions in mine water samples collected from Sukinda chromite mine in Odisha, India. (vii) To elucidate the mechanisms occurring at the bio-modified electrode–solution interface. A compendious description of the findings from the present work is given below: The capability of two bacterial strains procured from a microbial culture collection bank (MTCC), Corynebacterium paurometabolum (Gram positive bacterium) and Citrobacter freundii (Gram negative bacterium) as biosorbents for Cr (VI) and Cr(III) ions was assessed. Further, it became of interest to translate the studies related to bioremediation to an industrial situation. For this, bacterial strains were isolated from chromium contaminated water samples collected from surface water of Sukinda chromite mine in Odisha, India. Based on detailed microbiological and molecular biological protocols, two strains of bacteria were identified and characterised as Chromobacterium sp. and Sphingopyxis sp. The bioremediation efficiency of the strains was evaluated taking into consideration the various factors such as effect of contact time of bacterial cells with the chromium ions, pH of the chromium ion solution, biomass loading and initial chromium ion concentration. The Cr(VI) biosorption efficiency obtained for C. freundii was found to be about 59 %, followed by Sphingopyxis sp. and C. paurometabolum ≈ Chromobacterium sp. in the range of 50 % to 55 %. Subsequent to interaction of the bacterial cells with the Cr(VI) solution, the residual chromium was found to be in the form of Cr(III) ions. Hence, complete bioremediation of Cr(VI) could be achieved in terms of both biosorption and bioreduction processes using all the bacterial strains. It was found that the bioreduction process occurring in conjunction with the biosorption process resulted in nil concentration of Cr(VI) ions in the bulk solution. Similarly, studies related to bioremediation of Cr(III) using C. paurometabolum and C. freundii bacterial strains were also performed with higher biosorption efficiency achieved for the former, 50 % compared to 30 % obtained for C. freundii bacterial cells. The bioremediation of Cr(III) ions by the bacterial cells is achieved by the biosorption process. Biosorption of Cr ions by all the bacterial strains were found to follow a typical Langmuirian behaviour. The bioremediation process by the bacterial strains was also evaluated using suitable kinetic models and the results indicated that the bioremediation of Cr(VI) and Cr(III) by C. paurometabolum and C. freundii respectively followed pseudo first order kinetics, while the bioremediation of Cr(VI) by C. freundii, Chromobacterium sp. and Sphingopyxis sp. followed pseudo second order kinetics. It becomes of importance to ascertain the mechanisms of bioremediation of chromium ions by the bacterial cells and for this, different characterisation methods were adopted that helped in deducing the reactions occurring at the bacterial cell surface-chromium solution interface. The involvement of chemical forces in the bioremediation process was corroborated by the achievement of only partial desorption of chromium ions from the biosorbed bacterial cells. This was further confirmed by the Gibbs free energy (∆G) values, which were found to be in the range of -25 to -30 kJ/mol. FTIR spectral studies provided evidence in support of the key functional groups present on the bacterial cell surface such as, –OH, -COOH and –NH, which facilitated the binding with chromium. The EDS data for chromium biosorbed bacterial cells showed peaks corresponding to chromium, thereby confirming the binding of chromium by the bacterial cells. The redox state of chromium bound on the bacterial cell surface was determined with the help of XPS analysis. In the Cr2p XPS spectra obtained for the bacterial cells interacted with Cr(VI), it was interesting to observe a peak corresponding to Cr(III) in addition to Cr(VI), unequivocally indicating that the Cr(III) formed via bioreduction was not only released into the bulk solution but also got biosorbed on the bacterial cell surface. Apparent shifts in the binding energy values for the bacterial cells interacted with chromium were observed in the spectra recorded corresponding to C1s, O1s, N1s, P2p and S2p as compared to the spectra obtained for the bacterial cells alone. This attests to the fact that the functional groups corresponding to the elements mentioned are involved in chemical interaction with the chromium ions or are involved in the donation of electrons to bring about reduction of Cr(VI) to the less toxic Cr(III). The variation in the charge of the bacterial cell surface before and after interaction with chromium ions was monitored by performing zeta potential measurements as a function of pH. The surface charge of the bacterial cells alone was found to be negative over a wide range of pH. Subsequent to interaction of the bacterial cells with the negatively charged oxyanions of Cr(VI) ions, the surface charge was observed to be less electronegative, which further confirmed the binding of the positively charged Cr(III) ions formed via bioreduction on the bacterial cell surface. Similar results were also observed in the case when cells were allowed to interact with Cr(III) ions. The shifts in the iso-electric point for bacterial cells interacted with chromium ions further testified to the involvement of chemical binding forces in the bioremediation process. The findings obtained from the different characterisation methods enabled in understanding the reactions that are occurring at the bacterial cell surface-Cr solution interface. Initially, biosorption via electrostatic interaction of negatively charged oxyanions of Cr(VI) with the positively charged amino groups present on the bacterial cell surface takes place. Subsequent to the biosorption of Cr(VI) ions, the adjacent electron donating functional groups containing ligands present on the bacterial cell surface reduce Cr(VI) to Cr(III) via the reactions shown below: Bioreduction involving –OH group Bioreduction involving –SH group It can be seen that, the reactions involving bioreduction of Cr(VI) in the form of chromate oxyanion to Cr(III) involving hydroxyl and thiol group present on the bacterial cell surface result in the formation of intermediates, chromate-oxy and chromate-thio ester respectively. These intermediates facilitate the transfer of electrons from oxygen/sulphur donor centers to Cr(VI) acceptor molecule, thereby resulting in the reduction of Cr(VI) to Cr(III). The Cr(III) ions thus formed are then either released into the bulk solution or get complexed with the binding groups present on the bacterial cell surface. The next objective was to explore the potential of bacterial strains as sensors for the detection of Cr(VI) and Cr(III) ions. The chromium ions were detected using CV and CSV, both of which are electroanalytical techniques. For this, CPE was coated with the bacterial strains, C. paurometabolum, C. freundii, Chromobacterium sp. and Sphingopyxis sp., and the modified electrode was used as the working electrode in a typical three electrode electrochemical cell. These biosensors developed using each of the aforementioned strains resulted in a ~ 2 to 2.5 fold improved performance compared to the bare CPE for the detection of Cr(VI) ions, due to the binding ability of the various functional groups present on the bacterial cell surface. The lower limit of detection (LLOD) obtained for Cr(VI) and Cr(III) ions using CV technique was found to be 1x10-4 M and 5x10-4 M respectively. The LLOD was further improved to 1x10-9 M and 1x10-7 M for Cr(VI) and Cr(III) respectively using CSV. From the voltammograms obtained, it was postulated that the different functional groups present on the bacterial cell surface facilitate the detection of the chromium ions. Additionally, the developed microbial sensors were also found to be capable of detecting Cr(VI) ions in mine water samples collected from Sukinda chromite mine Odisha, India. In summary, the mechanisms of bioremediation of toxic Cr(VI) ions have been delineated as comprising of both biosorption and bioreduction processes. The residual Cr(VI) concentration subsequent to the treatment of the Cr(VI) aqueous solution with the bacterial cells was found to be nil, which meets the regulatory limit of 0.05 mg L-1 put forward by the US-Environmental Protection Agency (EPA) for a safe effluent discharge. Moreover, it has also been demonstrated that the chosen bacterial strains could be used as sensors for the detection of upto nanomolar concentration of Cr(VI) ions, under optimum conditions.

Heavy metals are released into the environment either by natural processes or by anthropogenic activities. Industries such as leather tanning, textiles, metallurgical, electroplating and mining activities discharge the chromium along with other heavy metals, which causes water pollution and environmental degradation. There are many conventional methods to overcome this problem such as chemical precipitation, ion exchange, reverse osmosis, etc but, these methods have certain drawbacks like generation of secondary sludge, inefficient removal of metal ions of low concentration, high cost etc. To overcome these limitations by conventional methods, an environmental friendly method, namely bioremediation has been adopted. Bioremediation uses microorganisms, biodegradable industrial wastes, or plants to mitigate this problem. In this investigation, bacterial strains have been isolated from the soil and water samples collected from a chromite mine in Karnataka. The capability of these bacterial strains have been assessed to remediate Cr (VI) in batch experiments in order to achieve the prescribed standards of regulatory agencies, and to elucidate the mechanisms of bioremediation of Cr (VI). Additionally, using these bacterial strains, biosensors have been developed to detect Cr (VI) ions in the solution by electroanalytical techniques. The major objectives of this research investigation are: a) Isolation, characterization and identification of bacterial strains from water and soil samples obtained from a chromite mine in Karnataka. b) To study the ability of three isolated bacterial strains namely Arthrobacter sp, Exiguobacterium sp. and Micrococcus sp. to remediate Cr (VI) during growth in media, amended with different concentrations of Cr (VI) c) Delineation of the probable mechanisms of bioremediation of Cr (VI) by three bacterial strains with the aid of proteomic and metabolomic studies d) Optimization of factors influencing the bioremoval of Cr (VI) using the isolated bacterial strains as biosorbents in batch experiments. c) Elucidation of mechanisms of bioremoval of Cr (VI) at the microbe – metal interface for all three bacterial strains, adopting characterization techniques like FTIR, XPS, SEM – EDS and zeta potential measurements. d) Micrococcus sp. was chosen for the fabrication of biomodified carbon paste electrode (CPE) to sense the Cr (VI) ions using voltammetric techniques, namely cyclic voltammetry (CV) and differential pulse cathodic stripping voltammetry (DPCSV). The salient findings of this research work are highlighted as follows: Firstly, bioremediation experiments were carried out using the bacterial strains isolated from soil and water samples collected from the chromite mines of Mysore Minerals Limited, Hassan district, Karnataka, India. Initially, the characterisation of the isolated bacterial strains were carried out with respect to their biochemical aspects, antibiotic susceptibility, morphology using scanning electron microscopy and cell wall nature by Gram’s staining. The identification of the three isolated bacterial strains were accomplished by 16S rRNA method and the three bacterial strains have been identified as Arthrobacter sp., Exiguobacterium sp. and Micrococcus sp.. The experiments were conducted to assess the potential of the isolated bacterial strains namely, Arthrobacter sp., Exiguobacterium sp. and Micrococcus sp., for the remediation at two different concentrations of 10 mg/L and 30 mg/L of Cr (VI) ions, during cell growth i.e. using metabolically active cells of bacteria. It was found that the three bacterial strains could bioreduce toxic Cr (VI) to the less toxic Cr (III) form, by 95% to 99%, within a time span of 12 h to 120 h. In the experiment with sulphate as the competitive ion in the growing mode of the bacterial strains Arthrobacter sp., Exiguobacterium sp. and Micrococcus sp., the percentage bioreduction of Cr (VI) to Cr (III) was not hampered. Scanning electron microscopic studies on the bacterial cells of Arthrobacter sp., Exiguobacterium sp. and Micrococcus sp., before and after interaction with Cr (VI) showed the morphological changes after interaction with Cr (VI), as an adaptive strategy to counter the toxic effect of Cr (VI). Further, to elucidate the mechanisms of bioreduction of Cr (VI) to Cr (III) by the three bacterial strains, the proteins and metabolites were isolated from the pristine bacterial cells and Cr (VI) interacted bacterial cells. The proteins were isolated from different parts of the cells and assessed for the differential expression of proteins under Cr (VI) stress. It was found that, seven differentially expressed protein bands were observed on SDS PAGE profile of Arthrobacter sp. interacted with Cr (VI), from the soluble protein isolated from the crude extract, devoid of cell membrane. A single band of differentially expressed protein was observed in the extracellular secretion in Exiguobacterium sp. and in the case of Micrococcus sp. four differentially expressed proteins were observed in the membrane fraction of proteins. The mass spectrometry data of the differentially expressed proteins were used to identify the probable protein candidates using MASCOT search in NCBIr database. It was found that some of these proteins were a class of transport proteins and a few belong to the reactive oxygen species scavengers. These findings suggested that the bioreduction of Cr (VI) to Cr (III) involved the efflux mechanism and ROS scavenger production, to resist the toxicity of Cr (VI). The metabolite concentration profile was studied for the all three bacterial cells in the absence and presence of Cr (VI) using NMR spectroscopy. The results of this study showed an increase and decrease in the concentration of various metabolite components after interaction with Cr (VI), and this was observed in all the three bacterial strains. Some of the metabolites identified using Chenomx 8.1 metabolite library, were found to be osmoprotectants like betaine, proline etc, which combat the stress of Cr (VI). Therefore, the overall bioremediation of Cr (VI) by metabolically active bacterial cells is through bioreduction of toxic Cr (VI) to the less toxic Cr (III) form and the resistance mechanisms to overcome the toxic effect of Cr (VI) is by the efflux mechanism, production of osmoprotectants and expression of ROS scavengers. In the third part of investigation, the bioremoval of Cr (VI) ions in batch experiments using metabolically inactive cells as biosorbents, for all the three bacterial strains, were studied. The bioremediation efficiency of each bacterial strain was evaluated, considering the various parameters like effect of contact time of bacterial cells with the Cr (VI) ions, pH of Cr ion solution, biomass loading and initial concentration of Cr (VI) ion. The Cr (VI) biosorption efficiency obtained for the bacterial strains Arthrobacter sp., Exiguobacterium sp. and Micrococcus sp. was found to be 93 %, 85 % and 100 % respectively. Apart from the biosorption of Cr (VI) by bacterial cells, the residual Cr was found to be in the form of Cr (III) ions. Therefore, complete bioremoval of Cr (VI) ions could be achieved as a combined process of biosorption and bioreduction, for all three bacterial strains, meeting the acceptable limits prescribed for Cr (VI) ion for drinking water, by regulatory agencies i.e. 0.05 mg/L of Cr (VI) ions. The biosorption of Cr ions by all the three bacterial strains were found to follow a typical Langmurian behaviour. The bioremediation process by the bacterial strains was also evaluated using suitable kinetic models and the results indicated that the bioremoval of Cr (VI) by Arthrobacter sp., Exiguobacterium sp. and Micrococcus sp. followed pseudo second order kinetics. The next aim was to ascertain the mechanism of bioremoval of Cr (VI) ions by the metabolically inactive cells. For this, different characterisation techniques were adopted that aided in the elucidation of reactions occurring at the interface of bacterial cell surface and Cr solution. The nature of interacting forces in bioremoval process was found out by desorption studies, and it was observed that only partial desorption of Cr ions was achieved from the biosorbed bacterial cells. This was further confirmed, by calculation of Gibbs free energy and the values were found to be in the range of – 25 to -32 kJ/mol, thus indicating that the process of bioremoval of Cr (VI) ions by the bacterial cells, is by chemisorption process. The variation in the charge of the bacterial cell surface, before and after interaction with chromium ions, was studied by performing zeta potential measurements as a function of pH. The surface charge of the bacterial cells alone was found to be negatively charged over a wide range of pH. Subsequent to interaction of the bacterial cells with the negatively charged oxyanions of Cr (VI) ions, the surface charge was observed to be less electronegative, which further confirmed the binding of the positively charged Cr (III) ions, formed via bioreduction on the bacterial cell surface. FTIR spectral studies revealed the functional groups involved, in bioremoval of Cr ions, present on bacterial cell surface. The functional group facilitating the bioremoval of Cr ions are –NH, -COOH and phosphate. EDS studies confirmed the Cr peak for the bacterial cells interacted with Cr ions. The oxidation state of Cr ion bound to the bacterial cell surface was determined with the help of XPS analysis. It was interesting to observe the Cr (III) peaks along with their Cr (VI) peaks. These studies provided evidence in support of the bioreduction of Cr (VI) to Cr (III) and biosorption of bioreduced Cr (III) ions onto the surface of bacterial cells, apart from the fraction present in bulk solution. The next objective was to assess the potential of Micrococcus sp. as sensor for the detection of Cr (VI) ions, using electroanalytical techniques such as, cyclic voltammetery (CV) and differential pulse cathodic stripping voltammetry (DPCSV). For this, Carbon Paste Electrode (CPE) was coated with the bacterial strain namely, Micrococcus sp and the modified electrode was used as the working electrode in a three electrode system. The developed biomodified electrode showed an approximately 3-fold increase in the sensing of Cr (VI) ion in comparison with the unmodified electrode CPE, which is attributed to the binding of Cr (VI) ions to functional groups present on the bacterial cell surface. The lower limit of detection obtained for Cr (VI) ions using CV was found to be 1 x10-4 M. The lower limit of detection was improved to 1 x 10-9 M of Cr (VI) using DPCSV.

Thesis (M. Tech.: Biotechnology)-Dept. of Biotechnology and Food Technology, Durban University of Technology, 2008. xiv, 103 leaves : ill. / Phytoremediation is an emerging technology where specially selected and engineered metal-accumulating plants are used for bioremediation. Amaranthus dubius (marog or wild spinach) is a popular nutritious leafy vegetable crop which is widespread especially in the continents of Africa, Asia and South America. Their rapid growth and great biomass makes them some of the highest yielding leafy crops which may be beneficial for phytoremediation. This study was undertaken to evaluate the potential of A. dubius for the phytoremediation of Chromium (Cr), Mercury (Hg), Arsenic (As), Lead (Pb), Copper (Cu) and Nickel (Ni). Locally gathered soil and plants of A. dubius were investigated for the metals from a regularly cultivated area, a landfill site and a sewage site. Metals were extracted from the samples using microwave-digestion and analyzed using Inductively Coupled Plasma – Mass Spectroscopy (ICP-MS). Further experiments were conducted with plants from locally collected seeds of A. dubius, in a tunnel house under controlled conditions. The mode of phytoremediation, the effect of the metals on the plants, the ability of the plant to extract metals from soil (Bioconcentration Factor - BCF), and the ability of the plants to move the metals to the aerial parts of the plants (Translocation Factor - TF) were evaluated for the different metals. Finally, A. dubius was micro-propagated in a tissue culture system with and without exposure to the metal, and the effect was studied by electron microscopy.

published_or_final_version / Environmental Management / Master / Master of Science in Environmental Management

published_or_final_version / abstract / Biological Sciences / Doctoral / Doctor of Philosophy

Uranium contamination of groundwater from mining and milling operations is an environmental concern. Reductive precipitation of soluble and mobile hexavalent uranium (U(VI)) contamination to insoluble and immobile tetravalent uranium (U(IV)) constitutes the most promising remediation approach for uranium in groundwater. Previous research has shown that many microorganisms are able to catalyze this reaction in the presence of suitable electron-donors. The purpose of this work is to explore lowcost, effective alternatives for biologically catalyzed reductive precipitation of U(VI). Methanogenic granular sludge from anaerobic reactors treating industrial wastewaters was tested for its ability to support U(VI)-reduction. Due to their high microbial diversity, methanogenic granules displayed intrinsic activity towards U(VI)-reduction. Endogenous substrates from the slow decomposition of sludge biomass provided electron-equivalents to support efficient U(VI)-reduction without external electrondonors. Continuous columns with methanogenic granules also demonstrated sustained reduction for one year at high uranium loading rates. One column fed with ethanol, only enabled a short-term enhancement in the uranium removal efficiency, and no enhancement over the long term compared to the endogenous column. Nitrate, a common co-contaminant of uranium, remobilized previously deposited biogenic U(IV). U(VI) also caused inhibition to denitrification. An enrichment culture (EC) was developed from a zero-valent iron (Fe⁰)/sand packed-bed bioreactor. During 28 months, the EC enhanced U(VI)-reduction rates by Fe⁰ compared with abiotic Fe⁰ controls. Additional experiments indicated that the EC prevented the passivation of Fe⁰ surfaces through the use of cathodic H₂ for the reduction of Fe(III) in passivating corrosion mineral phases (e.g. magnetite) to Fe²⁺. This contributed to the formation of secondary minerals more enriched with Fe(II), which are known to be chemically reactive with U(VI). To determine the toxicity of U(VI) to different populations present in uranium contaminated sites, including methanogens, denitrifiers and uranium-reducers, experiments were carried out with anaerobic mixed cultures at increasing U(VI) concentrations. Significant inhibition to the presence of U(VI) was observed for methanogens and denitrifiers. On the other hand uranium-reducing microorganisms were tolerant to high U(VI) concentrations. The results of this dissertation indicate that direct microbial reduction of U(VI) and microbially enhanced reduction of U(VI) by Fe⁰ are promising approaches for uranium bioremediation.

The purpose of this research was to investigate the microorganisms, genetics and biochemistry of anaerobic dechlorination of chlorinated ethanes, which are common groundwater contaminants. Specifically, this project used mixed microbial cultures to study the dechlorination of 1,2-dichloroethane (1,2-DCA), 1,1,2-trichloroethane (1,1,2-TCA) and 1,1,1-trichloroethane (1,1,1-TCA). A mixed microbial culture enriched from a contaminated multilayered aquifer in West Louisiana dechlorinated 1,2-DCA, 1,1,2-TCA, tetrachloroethene, trichloroethene, cis-dichloroethene and vinyl chloride (VC) to non-toxic ethene when amended with ethanol as the electron donor. 16S rRNA gene sequence analysis revealed the presence of the putative dechlorinating organisms Dehalobacter and Dehalococcoides spp. Denaturing gradient gel electrophoresis analysis and quantitative PCR (qPCR) with species-specific primers demonstrated that both organisms grew during the dichloroelimination of 1,2-DCA to ethene. Conversely, during the dichloroelimination of 1,1,2-TCA to VC only Dehalobacter grew, while during the reductive dechlorination of VC to ethene only Dehalococcoides grew. Further enrichment with 1,2-DCA, H2 and acetate yielded a co-culture of Dehalobacter and Acetobacterium spp. that did not dechlorinate other chlorinated ethanes or ethenes. Dehalobacter grew in the presence but not in the absence of 1,2-DCA, while Acetobacterium growth was not affected by 1,2-DCA. A novel putative Dehalobacter-associated 1,2-DCA reductive dehalogenase gene was identified and was shown to be transcribed only in the presence of 1,2-DCA. An enrichment microbial culture derived from a 1,1,1-TCA-contaminated site in the northeastern United States was also studied. This culture, referred to as MS, reductively dechlorinated 1,1,1-TCA to 1,1-dichloroethane (1,1-DCA) and then to monochloroethane (CA) when amended with methanol, ethanol, acetate and lactate. 16S rRNA gene sequence analysis revealed the presence of the putative dechlorinating organism Dehalobacter sp., whose growth during 1,1,1-TCA and 1,1-DCA dechlorination was confirmed by qPCR. In the presence of chlorinated ethenes, dechlorination 1,1,1-TCA by the culture MS was slowed, while dechlorination of 1,1-DCA was completely inhibited. Experiments with cell-free extracts and whole cell suspensions of culture MS suggested that chlorinated ethenes have direct inhibitory effects on 1,1,1-TCA reductive dehalogenase(s), while the inhibition of 1,1-DCA dechlorination may be due to effects on non-dehalogenase components of Dehalobacter sp. cells. Additionally, two novel reductive dehalogenase genes associated with 1,1,1-TCA reductive dechlorination were identified.