Cometabolic biodegradation processes are potentially useful for the bioremediation of hazardous waste sites. In this study the potential application of phenol-oxidizing and nitrifying bacteria as "priming biocatalysts" was examined in the degradation of polycyclic aromatic hydrocarbons (PAHs), aryl ethers, and aromatic ethers. We observed that a phenol-oxidizing Pseudomonas strain cometabolically degrades a range of 2- and 3-ringed PAHs. A sequencing batch reactor (SBR) was used to overcome the competitive effects between two substrates and the SBR was evaluated as a alternative technology to treat mixed contaminants including phenol and PAHs. We also have demonstrated that the nitrifying bacterium Nitrosomonas europaea can cometabolically degrade a wide range polycyclic aromatic hydrocarbons (PAHs), aryl ethers and aromatic ethers including naphthalene, acenaphthene, diphenyl ether, dibenzofuran, dibenzo-p-dioxin, and anisole. Our results indicated that all the compounds are transformed by N. europaea and that several unusual reactions are involved in these reactions. In the case of naphthalene oxidation, N. europaea generated predominantly 2-naphthol whereas other monooxygenases generate 1-naphthol as the major product. In the case of dibenzofuran oxidation, 3-hydroxydibenzofuran initially accumulated in the reaction medium and was then further transformed to 3-hydroxy nitrodibenzofuran in a pH- and nitrite-dependent abiotic reaction. A similar abiotic transformation reaction also was observed with other hydroxylated aryl ethers and PAHs. We also characterized the role of AMO in the degradation of aromatic ethers. Our results indicated that aromatic ethers including anisole were transformed by both 0-dealkylation or hydroxylation reactions. This research has led to the development of a rapid colorimetric assay to detect AMO activity. / Graduation date: 1998

Laboratory scale microcosm studies were conducted using site specific groundwater and aquifer solids to assess the feasibility of stimulating indigenous microorganisms in-situ to biologically transform Trichloroethylene (TCE) and its lesser chlorinated daughter products dichloroethylene (DCE) and vinyl chloride (VC). Three different treatments were conducted to determine the best approach for biologically remediating TCE under site specific conditions: anaerobic reductive dechlorination, aerobic cometabolism and sequential anaerobic/aerobic stimulation. Studies were conducted in batch serum bottles containing aquifer solids, groundwater and a gas headspace. Long-term (302 days) TCE anaerobic reductive dechlorination studies compared lactate, benzoate and methanol as potential anaerobic substrates. Site characteristic sulfate concentrations in the microcosms averaged 1,297 mg/L and TCE was added to levels of 2.3 mg/L. Substrates were added at one and a half times the stoichiometric electron equivalent of sulfate. Nutrient addition and bioaugmentation were also studied. Both benzoate and lactate stimulated systems achieved complete sulfate-reduction and prolonged dechlorination of TCE to VC and ethylene. Dechlorination was initiated between 15 to 20 days following lactate utilization and sulfate-reduction in the presence of approximately 300 mg/L sulfate. Benzoate amended microcosms did not initiate dechlorination until 120 to 160 days following the complete removal of available sulfate. After 302 days of incubation lactate and benzoate amended microcosms completely transformed TCE to VC with 7 to 15% converted to ethylene. Re-additions of TCE into both systems resulted in its rapid transformation to VC. The dechlorination of VC to ethylene was very slow and appeared to be dependent on VC concentration. Hydrogen addition at 10����� and 10������ atmospheres had no effect on the transformation of VC. Rapid methanol utilization resulted in its nearly stoichiometric conversion to methane and carbon dioxide without significant sulfate-reduction or dechlorination occurring. Nutrient addition slightly enhanced dehalogenation with lactate but inhibited it with benzoate. Bioaugmentation with a TCE dechlorinating culture from a previous benzoate amended Point Mugu microcosm effectively decreased lag-times and increased overall dechlorination. Aerobic cometabolism studies evaluated methane, phenol and propane as cometabolic growth substrates. Methane and phenol amended microcosms were able to remove only 50 to 60% of the added TCE after four stimulations, while propane utilizers were unable to cometabolize any TCE. Primary substrate utilization lag-times of 4 to 5 days, 0 to 0.5 days and 40 to 45 days were observed for methane, phenol and propane, respectively. Cometabolism of VC was possible in the presence of methane. Complete removal of 210 ��g/L VC was achieved after 2 stimulations with methane under strictly aerobic conditions. Methane utilization and VC oxidation required nitrate addition, indicating that the system was nitrate limited. A sequential anaerobic/aerobic microcosm study failed to achieve methane utilization and VC transformation likely due to oxygen being utilized to re-oxidize reduced sulfate in the system. / Graduation date: 1999

Graduation date: 1995

No description available.

Graduation date: 1994

Bibliography: leaves 177-191.

Bibliography: leaves 177-191. / xxi, 191 leaves : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Soil and Water, 2001

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.

Bioremediation is the process whereby the degradation of organic polluting compounds occurs as a result of biochemical activity of macro- and microorganisms. Bioremediation of hydrocarbon contaminated soils can be practised in situ or ex situ by either stimulating the indigenous microorganisms (biostimulation) or introducing adapted microorganisms which specifically degrade a contaminant (bioaugmentation). This investigation focused on ex situ remediation processes with special attention to the processes and microbiology of landfarming and thermal bioventing. Landfarming was investigated at pilot-scale and full-scale, and thermal bioventing at laboratory and pilot-scale. This study indicated that pilot-scale bioremediation by landfarming was capable of effecting a total petroleum hydrocarbon concentration (TPHC) reduction of 94% (m1m) from an initial concentration of 320 gkg-I soil to 18 gkg-I soil over a period of 10 weeks. Reactors receiving biosupplements showed greater rates of bioremediation than those receiving nutrients. Promotion of TPHC catabolism by addition of a commercial or a site-specific microbial biosupplement was similar. Seedling experiments proved that bioremediation did not necessarily leave the soil in an optimal condition for plant growth. The full-scale landfarming operation reduced the TPHC concentrations from 5 260 - 23 000 mgkg- I to 820 - 2335 mgkg- I soil over a period of 169 days. At full-scale, the larger fraction of more recalcitrant and weathered petroleums. and the less intensive treatment resulted in a slower rate of TPHC reduction than was found in the pilot-scale study. Three distinct decreases in the TPHC were observed during the full-scale treatment. These presented an ideal opportunity to investigate the microbiology of the soil undergoing treatment. The dominant culturable microorganisms were isolated and identified. The bioremediation process was dominated by Bacillus and Pseudomonas species. The method used to study the population was, however, biased to culturable, fast growing microorganisms which represent a small portion of the total microbial population. For this reason, a method to study the total eubacterial population in situ with rRNA targeted oligonucleotide probes was adapted and found to be a valuable technique. Soil microorganisms respiratory activity was investigated at different times in the full-scale treatment. A clear correlation between activity and degradation was recorded. The effect of a supplement. anaerobically digested sludge, was also assessed by this method. Thermal bioventing was investigated as an ex situ in-vessel treatment technology for small volumes of highly contaminated soils. This proved to be a viable technique for the bioremediation of petroleum hydrocarbons at laboratory-scale. Volatilisation contributed to at least 40% of the reduction. Of the two supplements evaluated. dried sludge promoted degradation to a greater extent than chicken manure. The pilot-scale study proved that a chemical contaminant reduction of at least 50% could be achieved in 13 weeks by thermal bioventing. Of the supplemented reactors. the presence of dried sludge and commercial biosupplement etfected the largest contaminant decrease. As a possible supplement to increase the rate of bioremediation. dried anaerobically digested sludge was more effective than chicken manure. A parallel laboratory-scale experiment gave similar results. Gravimetric analyses were found to be conservative indications of the remediation process. The results of this study shed some light on our. still. limited understanding of bioremediation. The gap between the technology in the laboratory and field was narrowed and a better understanding of the soil microbiology was achieved. Due to the limited control of environmental parameters in the case of landfarming. thermal bioventing was investigated and proved to be an effective alternative. The latter technology is novel in Southern Africa. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1996.

Sulphate-reducing bacteria (SRB) mediate the reduction of metals/metalloids directly or indirectly. Bioremediation of arsenic contaminated water could be a cost-effective process provided a cheap carbon source is used. To this end, molasses was tested as a possible source of carbon for the growth of sulphate-reducing bacteria (SRB). Its chemical composition and the tolerance of SRB toward different arsenic species [As (III) and As (V)] were also investigated. Batch culture studies were carried out to assess 1, 2.5 and 5 g l-1 molasses as suitable concentrations for SRB growth. The results indicate that molasses does support SRB growth, the level of response being dependent on the concentration; however, growth on molasses was not as good as that obtained when lactate, the usual carbon source for SRB, was used. The molasses used in this study contained several metals including Al, As, Cu, Fe, Mn and Zn in concentrations ranging from 0.54-19.7 ìg g-1, but these levels were not toxic to the SRB. Arsenic tolerance, growth response and sulphate-reducing activity of the SRB were investigated using arsenite and arsenate solutions at final concentrations of 1, 5 and 20 mg l-1 for each species. The results revealed that very little SRB growth occurred at concentrations of 20 mg l-1 As (III) or As (V). At lower concentrations, the SRB grew better in As (V) than in As (III). Batch cultures of sulphate-reducing bacteria (SRB) in flasks containing pine bark, sand and polystyrene as support matrices and Postgate medium B were used to study formation of biofilms. The effects of the support matrices on the growth of the organisms were evaluated on the basis of pH and redox potential change and the levels of sulphide production and sulphate reduction. Characterisation of the matrix surfaces was done by means of environmental scanning electron microscopy (ESEM). A consortium of SRB growing on polystyrene caused a 49% of original sulphate reduction whereas on sand a 36% reduction occurred. Polystyrene was further examined for its durability as a long-term support material for the growing of SRB in the presence of As(III) and/or As(V) at concentrations of 1, 5 and 20 mg l-1. Both sulphate reduction and sulphide production were greater in this immobilised system than in the matrix-free control cultures. With pine bark as support matrix no significant sulphate reduction was observed. The kinetics of sulphate reduction by the immobilised cells were compared with those of planktonic SRB and found to be superior. The leaching of organic compounds, particularly phenolic substances, from the pine bark had a detrimental effect on the growth of the SRB. Different proportions of pine bark extract were used to prepare media to investigate this problem. Growth of SRB was totally inhibited when 100% pine bark extract was used. Analysis of these extracts showed the concentration of phenolics increased from 0.33 mg l-1 to 7.36 mg l-1 over the extraction interval of 15 min to 5 days. Digested samples of pine bark also showed the presence of heavy metals. The effects of nitrate, iron and sulphate and combinations thereof were investigated on the growth of a mixed culture of sulphate-reducing bacteria (SRB). The addition of 30 mg l-1 nitrate does not inhibit the production of sulphide by SRB when either 50 or 150 mg l-1 sulphate was present. The redox potential was decreased from 204 to -239 mV at the end of the 14 day batch experiment in the presence of 150 mg l-1 sulphate and 30 mg l-1 nitrate. The sulphate reduction activity of the SRB in the presence of 30 mg l-1 nitrate and 100 mg l-1 iron was about 42% of original sulphate, while if no iron was added, the reduction was only 34%. In the presence of 20 mg l-1 either As(III) or As(V), but particularly the former, growth of the SRB was inhibited when the cells were cultured in modified Postgate medium in the presence of 30 mg l-1 nitrate. The bioremoval of arsenic species [As(III) or As(V)] in the presence of mixed cultures of sulphate-reducing bacteria was investigated. During growth of a mixed SRB culture adapted to 0.1 mg l-1 arsenic species through repeated sub-culturing, 1 mg l-1 of either As(III) or As(V) was reduced to 0.3 and 0.13 mg l-1, respectively. Sorption experiments on the precipitate produced by batch cultured sulphate-reducing bacteria (SRB-PP) indicated a removal of about 77% and 55% of As(V) and As(III) respectively under the following conditions: pH 6.9; biomass (2 g l-1); 24 h contact time; initial arsenic concentration,1 mg l-1 of either species. These results were compared with synthetic iron sulphide as adsorbent. The adsorption data were fitted to Langmuir and Freundlich isotherms. Energy dispersive x-ray (EDX) analysis showed the SRB-PP contained elements such as sulphur, iron, calcium and phosphorus. Biosorption studies indicated that SRB cell pellets removed about 6.6% of the As(III) and 10.5% of the As(V) from water containing an initial concentration of 1 mg l-1 of either arsenic species after 24 h contact. Arsenic species were precipitated out of synthetic arsenic-contaminated groundwater by reacting it with the gaseous biogenic hydrogen sulphide generated during the growth of SRB. The percentage removal of arsenic species was dependent on the initial arsenic concentration present. Lastly, laboratory scale bioreactors were used to investigate the treatment of arsenic species contaminated synthetic groundwater. A mixed culture of SRB with molasses as a carbon source was immobilised on a polystyrene support matrix. The synthetic groundwater contained either As(III) or As(V) at concentrations of 20, 10, 5, 1 or 0.1 mg l-1 as well as 0.1 mg l-1 of a mixture with As(III) accounting for 20, 30, 40, 60 and 80% of the total. More that 90% and 60% of the As(V) and As(III) respectively were removed by the end of the 14-day experiment. At an initial concentration of 0.1 mg l-1 total arsenic had been reduced to below the WHO acceptable level of 10 ìg l-1 when the proportion of As(III) was 20 and 30%, while at 40% As(III) this level was reached only when the treatment time was increased to 21 days. The efficiency of As(III) removal was increased by first oxidising it to As(V) using MnO2. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2008.