Reductive dechlorination of chlorinated ethenes is dependent upon suitable substrates promoting microbial activity and creating anaerobic conditions. At the periphery of active reductive dechlorinating zones combinations of lesser chlorinated ethenes should exist along with end products of the anaerobic metabolism that is driving reductive dechlorination. Potential end-products of anaerobic metabolism were investigated for their ability to stimulate oxidative cometabolism of dichloro ethenes (DCEs) by the butane-degrading bacterium, Pseudomonas butanovora. Organic acids that supported butane-monooxygenase (BMO) activity were acetate, propionate, lactate, and butyrate. Lactate consistently supported and sustained greater rates of cooxidation than did the other organic acids. When propane replaced butane as the growth substrate, lactate remained the superior electron donor, while the ability of butyrate and acetate to support BMO activity decreased. In contrast, propionate-supported cooxidation was only observed in propane-grown cells. Lactate supported the degradation of 1,2-trans dichloroethylene (1,2-trans DCE), 1,2-cis dichloroethylene (1,2-cis DCE) and 1,1-dichloroethylene (1,1-DCE) in butane-grown P. butanovora. 78 nmoles (25 μM) of 1,2-cis DCE were completely degraded by butane-grown P. butanovora. In contrast, smaller amounts of 1,1-DCE and 1,2-trans DCE were degraded over the twenty minute time course. Decreasing rates of cooxidation over time were observed for of all three DCEs, and 50% of BMO activity was irreversibly lost after 15 min, 6 min, and 0.5 min exposures to 1,2-cis DCE, 1,2 trans-DCE, and 1,1-DCE respectively. Cell viability decreased by over 90, 95, and 99.95% during the transformation of 25 nmoles/mg protein of 1,2-cis DCE, 1,2-trans DCE and 1,1-DCE. These results indicate that cellular viability was more sensitive to cooxidation of 1,2-cis DCE and 1,2-trans DCE than was BMO. 1,2-cis DCE and 1,2-trans DCE induced BMO activity to 25 and 45% of the butane control, respectively. Induction by 1,2-trans DCE was observed at a threshold of about 20 μM and higher concentrations did not increase BMO activity. Fusion of lacZ to the BMO catabolic promoter, with consequent knock out of BMO activity, provided the opportunity to assess substrate induction without the confounding effects of enzyme inactivation and product induction. While BMO substrates, butane, 1,2-cis DCE, and ethylene, were unable to induce lacZ activity the BMO products, 1-butanol, and ethylene oxide, effectively induced lacZ activity. 1,2-trans DCE was unique among the BMO substrates tested in it's ability to induce expression of lacZ, 2-fold above background, in the reporter strain. A wide range of concentrations induced lacZ activities (10 to 100 μM), and low levels of 1,2-trans DCE achieved high levels of induction after 4 hrs. However, lacZ activities were limited to an induction of about four-fold above background and this limit allowed lower concentrations of 1,2-trans DCE to eventually produce equal levels of beta-galactosidease. These data provide proof-of- concept that BMO-dependent cometabolism can occur independently of butane as an inducer and electron donor for BMO gene expression and activity. / Graduation date: 2004

No description available.

The mass transfer and the pressure drop characteristics for countercurrent rotating packed bed (RPB) with a continuous gas phase for the removal of ethylene dichloride (EDC) from water using air stripping have been investigated. The aim of this research was to understand the behaviour of the mass transfer performance and the pressure drop behaviour in a centrifugal environment. The mass transfer results showed that the height of a transfer unit (HTU) in a liquid film limited system can be in the range between 30 to 70mm at moderate centrifugal acceleration between 44 to 280g. Three packings of different packing densities ranging from 870 to 2300 m2/m3 were tested. The HTU values were found to vary with the centrifugal acceleration as HTU a g-0.11 to -0.28. It has been shown that the packing density may not have a notable effect on the separation performance of the bed. The pressure drop results indicated that in RPB the pressure drop is relatively higher than its equivalent packed beds operate at 1g. The usual flooding restrictions were relaxed thus high hydraulic capacities can be achieved per unit size of equipment. At the flooding point, experimental findings indicated that a part of the liquid is not accelerated by the bed. A model to predict the pressure drop was developed. The model was verified against experimental data and good agreement was obtained.

Protein disulfide isomerase (PDI), a member of the thioredoxin superfamily, contains two domains with significant sequence homology to the active sites in thioredoxin. PDI facilitates the folding of nascent proteins in the endoplasmic reticulum (ER), binds hormones and Ca�����, catalyzes the glutathione dependent reduction of dehydroascorbate, serves as a major chaperone molecule in the ER and serves as a subunit for prolyl-4-hydroxylase and microsomal triglyceride transferase. Because of its abundance in the ER and association with disease and chemically induced toxicity, the goal of this research was to investigate the relative susceptibility of PDI thiols to alkylation. The sensitivity of PDI to 1-chloro-2,4-dinitrobenzene (CDNB), iodoacetamide (IAM) and biotinoylated iodoacetamide (BIAM) was explored. The relative susceptibility of the thiolate anions present in the two active sites of PDI each containing the -CGHC- sequence was investigated with mass spectrometric techniques. PDI was inactivated by CDNB but was not found as sensitive as thioredoxin reductase as shown by Amer and coworkers (1995). IAM and BIAM were used as model alkylating agents to explore the two active sites of PDI and determine the residues most susceptible to alkylation. Alkylation by IAM and BIAM was first detected at the N-terminal cysteine in each active site (-C*GHC-) followed by alkylation at the second cysteine residue (-C*GHC*-) as shown by tandem mass spectrometry. Mass spectroscopy showed that the episulfonium ion derived from the glutathione conjugate of 1,2-dichloroethane, S-(2-chloroethyl)glutathione (CEG), decreased activity and protein thiols of PDI. CEG produced two protein adducts at very low excesses of CEG over PDI; however, higher concentrations resulted in several protein adducts. Only one modification in each active site at the N-terminal cysteine residue can be identified, indicating that while these thiolate anions of PDI are susceptible, it would appear that the episulfonium ion may present itself to other sites as well. This may have important toxicologic significance regarding the mechanism of 1,2-dichloroethane toxicity and the role of PDI in the redox status of the cell. / Graduation date: 2003

1,2-Dichloroethane (1,2-DCA), a highly toxic and recalcitrant compound, is produced anthropogenically in larger quantities than any other chlorinated compound. It is regarded as a mutagen and carcinogen, thus making it a priority target molecule for biological degradation. In addition, the intermediates of 1,2-DCA degradation are highly reactive and toxic, due to the electrophilic nature of the carbonyl groups in these compounds. Aerobic biodegradation of 1,2-DCA, resulting in complete mineralization, has previously been reported in Xanthobacter autotrophicus GJ10 and some Ancylobacter aquaticus strains. X. autotrophicus GJ10 has been found to possess chloroacetaldehyde (CAA) dehydrogenase and haloacid (HA) dehalogenase enzymes, both of which play a crucial role in 1,2-DCA degradation. Five strains of Ancylobacter aquaticus capable of utilizing 1,2-DCA as a sole carbon and energy source have recently been isolated in our laboratory. The degradation potential and specific dehalogenase activities of these bacterial isolates against 1,2-DCA and other halogenated compounds as a carbon source were investigated and compared to previously characterized organisms, viz., X. autotrophicus GJ10 and Ancylobacter aquaticus strains AD25 and AD27. Furthermore, this study proposed to detect the presence of the CAA dehydrogenase (aldB) and HA dehalogenase (dhlB) encoding genes in these isolates. Growth of all strains in the presence of 1,2-DCA as a carbon source was monitored over an 84 h period, in minimal medium supplemented with either vitamins or yeast extract. Dehalogenase activities were measured colorimetrically by monitoring halide release by crude cell extracts of the isolates. In order to detect the presence of dhlB and aldB genes, genomic DNA of the isolates was digested with individual restriction endonucleases, viz., EcoRI, PstI, HindIII and BamHI, and then subjected to Southern hybridization experiments. All isolates demonstrated significant growth rates in both vitamin and yeast extract supplemented media, with the former having a greater overall growth effect. Ancylobacter aquaticus DH5 demonstrated the highest growth rate of 0.147.h-1 in the presence of vitamins while Ancylobacter aquaticus DH12 displayed the highest growth rate of 0.118.h-1 with yeast extract. Optimum haloalkane dehalogenase activities of these bacterial isolates were confirmed at pH 8, similar to the activity in X. autotrophicus GJ10, while haloaciddehalogenase activity had a broader pH range. Hydrolytic dehalogenase activity of the bacterial isolates using a range of halogenated aliphatic compounds was also determined. Results demonstrated a wide substrate range with activity being observed on 1,3- dibromopropane, 1,2-dibromoethane and 1,3-dichoropropene, for all isolates. Southern Hybridization experiments confirmed the presence of both aldB and dhlB genes in X. autotrophicus GJ10. The dhlB probe produced a positive signal for an EcoRI fragment in Ancylobacter aquaticus DH12 while the aldB probe hybridized and produced a single positive signal on similar sized PstI fragments for all organisms except A. aquaticus AD25 which produced two positive signals. The results in this study demonstrate the potential application of the newly isolated strains of Ancylobacter aquaticus. in future bioremediation strategies. The detection of the genes involved in 1,2-DCA degradation further support the use of these isolates and/or their enzymes for the degradation of 1,2- DCA as well as other halogenated compounds. Future work need to determine sequence similarity of these genes detected in A. aquaticus strains to the genes in Xanthobacter autotrophicus GJ10 and other previously reported genes. It may also be important to investigate the activity of the enzymes under various environmental conditions and to determine enzyme structure and the catalytic sites, so as to gain knowledge of their degradation potential on site. Characterization of enzymes at both the molecular and protein levels may be necessary and beneficial for implementation in strategies involving bioremediation for the biological degradation of a wide range of halogenated aliphatic hydrocarbons. / Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2011.

1,2-Dichloroethane (1,2-DCA), a short chain chlorinated aliphatic compound, is one of the most hazardous toxic pollutant of soil and groundwater, with an annual production in excess of 5.44 × 109 kg. The major concern over soil contamination with 1,2-DCA stems largely from health risks. Owing to their toxicity, persistence and potential for bioaccumulation, there is a growing interest in technologies for their removal. Many sites are, however, co-contaminated with a complex mixture of 1,2-DCA and heavy metal contaminants. Co-contaminated environments are considered difficult to remediate because of the mixed nature of the contaminants and the fact that the two components often must be treated differently. Therefore, the objective of this study was to evaluate the aerobic biodegradation of 1,2-DCA by autochthonous microorganisms in soil co-contaminated with 1,2-DCA and heavy metals, namely; arsenic (As3+), cadmium (Cd2+), mercury (Hg2+) and lead (Pb2+), via a direct and quantitative measurement of the inhibitory effects of heavy metals in a microcosm setting. Effects of various metal concentrations and their combinations were evaluated based on the following: (i) degradation rate constants; (ii) estimated minimal inhibitory concentrations (MICs) of metals; (iii) concentrations of heavy metals that caused biodegradation half-life doublings (HLDs); and (iv) heavy metal concentrations that caused a significant effect on biodegradation (> 10% increase in t½ of 1,2-DCA). The effects of biostimulation, bioaugmentation and the addition of treatment additives on the biodegradation process were evaluated. The presence of heavy metals was observed to have a negative impact on the biodegradation of 1,2-DCA in both clay and loam soil samples, with the toxic effect being more pronounced in loam soil for all heavy metal concentrations except for Hg2+, after 15 days. Heavy metal concentrations of 75 mg/kg As3+, 840 mg/kg Hg2+, and 420 mg/kg Pb2+, resulted in 34.24%, 40.64%, and 45.94% increases in the t½ of 1,2-DCA, respectively, in loam soil compared to clay soil. Moreover, the combination of four heavy metals in loam soil resulted in 6.26% less degradation of 1,2-DCA compared to clay soil, after 15 days. Generally, more than 127.5 mg/kg Cd2+, 840 mg/kg Hg2+ and 420 mg/kg of Pb2+ was able to cause a > 10% increase in the t½ of 1,2-DCA in clay soil, while less than 75 mg/kg was required for As3+. An increased reduction in 1,2-DCA degradation was observed with increasing concentration of the heavy metals. In clay soil, a dose-dependant relationship between k1 and metal ion concentrations in which k1 decreased with higher initial metal concentrations was observed for all the heavy metals tested except Hg2+. Ammonium nitrate-extractable fractions of bioavailable As3+ and Cd2+ concentrations varied greatly, with approximately < 2.73% and < 0.62% of the total metal added to the system being bioavailable, respectively. Although bioavailable heavy metal fractions were lower than the total metal concentration added to the system, indigenous microorganisms were sensitive to the heavy metals. Biostimulation, bioaugmentation and amendment with treatment additives were all effective in enhancing the biodegradation of 1,2-DCA in the co-contaminated soil. In particular, biostimulation with fertilizer, dual-bioaugmentation and amendment with CaCO3 were most efficient in enhancing 1,2-DCA degradation resulting in 41.93%, 59.95% and 51.32% increases in the degradation rate constant of 1,2-DCA in the As3+ co-contaminated soil, respectively, after 20 days. Among all the treatments, dualbioaugmentation produced the highest 1,2-DCA degrading population of up to 453.33 × 107 cfu/ml in the Cd2+ co-contaminated soil. On comparison of the As3+ and Cd2+ co-contaminated soil undergoing either biostimulation or dual-bioaugmentation, similarity in the denaturing gradient gel electrophoresis (DGGE) banding patterns was observed. However, the banding patterns for the different bioremediation options demonstrated a difference in bacterial diversity between the fertilized and dual-bioaugmented samples. DGGE profiles also indicate that while numerous bands were common in the fertilized co-contaminated soils, there were also changes in the presence and intensity of bands due to treatment and temporal effects. Dehydrogenase and urease activities provided a more accurate assessment of the negative impact of heavy metals on the indigenous soil microorganisms, resulting in up to 87.26% and 69.58% decreases in activities, respectively. In both the biostimulated and bioaugmented soil microcosms, dehydrogenase activity appeared biphasic with an initial decrease followed by an increase in the treated soils over time. Results from this study provide relevant information on some alterations that could be introduced to overcome a critical bottle-neck of the application of bioremediation technology. In conclusion, the bioremediation strategies adopted in this study may be used as a rational methodology for remediation of sites co-contaminated with 1,2-DCA and heavy metals, subject to a thorough understanding of the microbial ecology and physico-chemical parameters of the site. / Thesis (M.Sc.)-University of KwaZulu-Natal, 2009.