Dissertationen zum Thema „Hazardous wastes Biodegradation“

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.

Le stockage est la méthode de traitement des déchets non dangereux la plus communément utilisée dans le monde entier car elle est un moyen simple et économique pour leur élimination. Malgré une volonté nationale et européenne pour réduire le stockage de déchets biodégradables, une quantité non négligeable est encore enfouie entrainant la mise en place de modes de gestion spécifique. Autrefois exploitées comme de simples fosses de remplissage, les installations de stockage de déchets non dangereux (ISDND) sont aujourd’hui des ouvrages complexes dont l’objectif est de réduire l’impact environnemental et de valoriser énergétiquement le biogaz produit par la biodégradation de la matière organique. Afin d’accélérer les processus de biodégradation un casier de déchets peut être exploité en mode bioréacteur en réinjectant des lixiviats pour augmenter la teneur en eau des déchets. Le défi des années 2000 était de dimensionner et valider les systèmes de réinjection de lixiviat pour garantir une distribution optimale de la teneur en eau dans les massifs de déchets. Aujourd’hui, la question est de de suivre l’évolution de la biodégradation en tous points d’un massif pour notamment comprendre l’effet de ce mode de gestion. Les méthodes géophysiques en plus d’être non-destructives et spatialisantes sont utilisées depuis des années sur les ISDND et pourraient être sensibles à l’évolution de la biodégradation d’un massif de déchets, comme cela a été démontré pour la biodégradation d’autres milieux. Ainsi la problématique de cette thèse est d’évaluer la capacité de certaines méthodes géophysiques pour suivre l’évolution des paramètres bio-physico-chimiques d’un massif de déchets au cours de sa biodégradation. Un premier travail bibliographique a permis d’identifier quatre méthodes électriques parmi les méthodes géophysiques disponibles pour répondre à cette question :• La résistivité électrique• Le potentiel spontané• La polarisation provoquée• La polarisation provoquée spectraleAprès cet état de l’art, le travail de thèse a été séparé en trois parties. La première a été consacrée à la mise en place du suivi de ces quatre méthodes à l’échelle du laboratoire dans des conditions contrôlées, la seconde à analyser le suivi géophysique long terme sur le site industriel de la SAS Les Champs Jouault et la dernière a évaluée les observations à ces deux échelles. Enfin, la conclusion présente le potentiel de la méthode de mesure du potentiel provoquée comme la plus pertinente pour suivre l’évolution de la biodégradation d’un déchet non dangereux au cours du temps et aborde son utilisation dans un cadre industriel
Storage is the most commonly used waste treatment method in the world because it is a simple and economical way to dispose of solid waste. Despite a national and European desire to reduce the storage of biodegradable waste, a significant amount is still buried, leading to the implementation of specific management methods. Formerly exploited as mere filling pits, MSWL are today complex structures whose objective is to reduce the environmental impact and energetically valorize the biogas produced by the biodegradation of organic matter. In order to accelerate biodegradation processes, a waste cell can be operated in bioreactor mode by reinjecting leachates to increase the water content of the waste. The challenge of the 2000s was to size and validate leachate re-injection systems to ensure optimal distribution of water content in the waste mass. Today, the question is to monitor evolution of the biodegradation in all points of a waste mass in particular to understand the effect of this management mode. Geophysical methods in addition to being non-destructive and spatializing have been used for years on MSWLs and could be sensitive to the evolution of a waste mass biodegradation, as has been demonstrated for the biodegradation of others environments. Thus the problematic of this thesis is to evaluate the capacity of certain geophysical methods to monitor the evolution of the bio-physicochemical parameters of a waste mass during its biodegradation. A first bibliographic work identified four electrical methods among the geophysical methods available to answer this question:• Electrical resistivity• Self potential• Time domain induced polarization• Spectral induced polarizationAfter this state of the art, the thesis work was separated into three parts. The first one was devoted to the implementation of the monitoring of these four methods at the laboratory scale under controlled conditions, the second to analyze the long-term geophysical monitoring at the industrial site of SAS Les Champs Jouault and the last one to evaluate the observations at these two scales. Finally, the conclusion presents the potential of time domain induced polarization method as the most relevant to monitor the evolution of a waste mass biodegradation over time and discusses its use in an industrial setting

Cette thèse porte sur une étude de l'évolution biomécanique de déchets non dangereux selon différentes conditions de prétraitement et d'opération à différentes échelles. Après une introduction aux enjeux et aux données majeures concernant la gestion des déchets, leur stockage et les évolutions en cours (Chapitre I), la caractérisation du milieu déchet est abordée (Chapitre II). Ce milieu, triphasique et donc généralement non saturé en eau, nécessite une étude et des moyens d'investigation particuliers. De nombreuses études antérieures ont démontré la nécessité d'études couplées dédiées aux déchets. Pour ce faire, il est également nécessaire d'adapter un certain nombre de méthodes métrologiques aux déchets (Chapitre III). Ainsi, des méthodes de métrologie spécifique (humidité, masse volumique. . . ) sont étudiées et validées pour leur application à ce milieu, de l'échelle du laboratoire jusqu'à celle du site. Une plateforme d'essai constituée de quatre pilotes semi-industriels conçus avec Veolia Environnement Recherche & Innovation a été utilisé au LTHE pour une étude biomécanique à une échelle suffisante et en conditions très proches de celles rencontrées sur site (compression, température, humidité. . . ) (Chapitre IV). Les résultats obtenus permettent de caractériser la biodégradation en termes de suivis et de bilans, ainsi que d'en identifier les leviers principaux en fonction des conditions de prétraitement et d'opération. Enfin, une exploitation de ces résultats ainsi que d'autres résultats à l'échelle du laboratoire et du site ont permis de démontrer le couplage biomécanique et de proposer un modèle du tassement des déchets (Chapitre V)
This research addresses the biomechanical evolution of municipal solid waste subject to different pretreatment and operational conditions at different scales. After an introduction to the major stakes and figures related to waste management, waste landfilling and its evolution (Chapter I), the characterization of the waste medium is addressed (Chapter II). This triphasic unsaturated medium requires a dedicated scientific approach as well as specific investigation experiments. Several past investigations have demonstrated the need for specific coupled studies of waste. To do so, it seems also essential to adapt measurement methods (moisture, density. . . ) to the waste medium (Chapter III). Hence, measurement techniques have been studied in detail and validated for their use in this medium at scales ranging from the laboratory to the site. An experimental platform consisting of four pilot cells at a semi-industrial scale, designed with Veolia Environnement Recherche & Innovation, has been used at LTHE to investigate waste biomechanics at a sufficient scale and under site-near conditions (compression, temperature, moisture. . . ) (Chapter IV). The results enable to characterize biodegradation in terms of daily monitoring as well as final budgets, but also to identify the major drivers of biodegradation depending on the pre-treatment and operational conditions. Finally, these results and other laboratory- and site-scale results have been used to demonstrate the biomechanical coupling and to propose a model for waste settlement (Chapter V)

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

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

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.

A final cover on a waste impoundment is the main physical barrier between the waste impoundment and the environment designed to protect against physical, chemical and biological factors isolating the waste from the atmospheric environment. Since the early 1990‟s regulators in the United States have started accepting vegetation covers in lieu of the prescriptive covers. Currently in South Africa, data that provide field performance comparisons of alternative vegetation covers are few or non-existent; hence a research program was undertaken by an industrial corporation in South Africa to determine the potential use of vegetation covers. In proposing a practical way forward, the Company (AECI Limited) reached an understanding with the Regulators that a vegetated evapotranspiration (ET) cover, would be acceptable provided that its performance in limiting surface water infiltration (and subsequent leaching) could be quantitatively demonstrated. The overall object of this research was to determine the capability of vegetation cover to limit effluent leaching from a waste impoundment. Analysis of the following sub-objectives were required to address and give answers to this study (1) determine, as accurately as possible a climatic water balance on the vegetation covers, (2) determine the geohydrological properties of the material of the waste impoundment, (3) determine the fate of the water i.e. proportion reused via evapotranspiration as opposed to the proportion infiltrating the waste body beneath the root zone and (4) determine the leaching potential below the waste. The study identifies and evaluates the climatic (above ground) and geohydrological (sub-surface) parameters used to estimate the water balance of the materials for a waste impoundment. The study then utilizes these parameters at the respective sites in a finite-element model, called the HYDRUS-2D model, to simulate the water balance of the material. The simulated water balance results were then compared against collected field data, which provide the evidence of the efficiency of a vegetation cover to limit effluent from the impoundment.
Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2009.

Thesis (M.Tech.: Biotechnology)-Dept. of Biotechnology, Durban University of Technology, 2007 xxi, 117 leaves
The aromatic compounds Polychlorinated Biphenyls (PCBs) are one of the largest groups of environmental pollutants. The greatest concern is the release of PCBs in the water systems by industrial effluent, accidental spillages or leaks. PCBs are able to bioaccumulate in the fatty tissues of animals, fish and humans. The impact on human health due to PCBs has prompted interest in their degradation. The application of microbial degradation of PCBs can transform many PCB metabolites. There are a wide variety of microorganisms that can degrade PCBs or utilise them as sole carbon sources. This study focused on isolating microrganisms from industrial wastewater capable of aerobic degradation of PCBs. The degradation potential of the selected isolates were investigated by using different analytical techniques viz. ultra violet or visible spectrophotometer (UV/Vis), thin layer chromatography (TLC) and gas chromatography electron capture detector (GC-ECD).

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.

In the following study, the potential role that microorganisms play in the removal of Mn (II) and Fe (II) was assessed using biofilter sand and water samples collected from a biofiltration system (operated by Umgeni Water in KwaZulu-Natal, Nottingham Road, at the Nottingham combined school, South Africa) treating borehole water containing manganese and iron. Initially the presence of Mn (II) and Fe (II) oxidizing bacteria was demonstrated in the biofiltration system. Thereafter, the contribution of individual microorganisms to the overall removal of manganese and iron was assessed in the laboratory by determining the difference in metal oxidation in the presence and absence of active bacteria at neutral pH, simulating conditions in the biofilter. Controls were run to verify the elimination via physiochemical reactions occurring within the biofiltration system. Finally a diversity snapshot of the bacteria present within the biofilter matrix was established via analysis of a clone library. Viable bacterial counts for the biofiltration system were established using MSVP (minimal salts vitamins pyruvate) medium - plus added manganese sulfate or iron sulfate targeting Mn (II) and Fe (II) oxidizing bacteria - and R2A for heterotrophic bacteria. In the first experimental chapter, batch tests using MSVP were employed to determine manganese oxidation, by measuring the pH and ORP (oxidation reduction potential) in experimental flasks and controls over time. There was a clear drop in pH and a concomitant increase in ORP when an isolated manganese oxidizing strain (designated LB1) was grown in MSVP plus added manganese sulfate, indicating manganese oxidation. Based on physiological characteristics established by the VITEK-2 system as well as by 16S rRNA gene sequence analysis and MALDI-TOF (Matrix assisted laser desorption ionization-time of flight mass spectrometry) mass spectrometry of cell extracts, the isolate was identified as a member of the genus Acinetobacter. EDX (energy dispersive X-ray analysis) analysis of crystals formed in batch culture tests, containing MSVP plus either added manganese or iron sulfate, confirmed the ability of the isolate to oxidize both Mn (II) and Fe (II). The leucoberbelin blue colorimetric assay and batch tests using MSVP both demonstrated that in the presence of the isolated strain, Acinetobacter sp. LB1, the rate of Mn (II) oxidation at neutral pH was enhanced as compared to abiotic controls. In the second experimental chapter the difference in Fe (II) oxidation between biological and abiological systems at neutral pH was determined using batch tests run with Acinetobacter sp. LB1 and Fe (II) in saline. In addition, the rate of Fe (II) oxidation was also determined at acidic pH and at alkaline pH in experimental and control flasks. To determine Fe (II) removal under conditions simulating those in the biofiltration system, batch tests were set up using borehole water freshly collected from the biofiltration system. In order to verify the contribution of native microorganisms in the borehole water to Fe (II) oxidation, these flasks were spiked with bacterial strains isolated from the biofiltration system - Acinetobacter sp. LB1 and Burkholderia sp. strain LB2 - and two known iron oxidizing strains Leptothrix mobilis (DSM 10617) and Sphaerotilus natans (DSM 565) were used to determine the contribution of reference iron oxidizers to Fe (II) oxidation. A separate set of the same flasks with the addition of filter sand was used to qualitatively demonstrate iron oxidation as it would occur within the biofiltration system. The ferrozine assay was employed to quantify the amount of Fe (II) in batch tests employing saline medium and in batch tests employing borehole water. EDX analysis was employed to confirm the presence of Fe (II) in oxidation products in the batch test flask with filter sand spiked with Acinetobacter sp. LB1. In the presence of Acinetobacter sp. LB1 at neutral pH in saline medium, the rate of Fe (II) oxidation was very similar to that in the abiological controls thus demonstrating that the presence of metabolically active microorganisms does not per se enhance the oxidation of Fe (II) like in the case of Mn (II) at neutral pH. Surprisingly, in the heat inactivated control, apparently the highest amount of Fe (II) was oxidized. As expected, at acidic pH very little oxidation of Fe (II) took place and at alkaline pH almost all Fe (II) in the flasks was removed and small amounts oxidized as determined by the amount of Fe (III) produced. Batch tests using borehole water proved that native microorganisms within the biofiltration system were more efficient in the oxidative removal of Fe (II) from the system, in comparison to the reference iron oxidizing strains. In the final experimental chapter, the presence of biofilms with actively metabolizing cells was examined on a pooled sample of biofilter matrix from the manganese and iron filter using CLSM (confocal laser scanning microscopy) image analysis. DNA was extracted from the biofilm material associated with biofilter matrix to establish a diversity snapshot of the bacteria present within the biofilter matrix. ARDRA (amplified “rDNA” restriction analysis) analysis of the clone library revealed the presence of 15 unique OTU’s (operational taxonomic unit) based upon restriction patterns of amplified 16S rRNA genes of a total of 100 randomly selected clones. The majority of the clones were closely related to the genera Nitrospira and Lactococcus. Overall, 42% of the clones were assigned to the phylum Proteobacteria, 13% to the phylum Actinobacteria, 24% to the phylum Firmicutes and 21% to the phylum Nitrospirae. Overall, the results demonstrate that bacteria present within an established biofiltration system at neutral pH can contribute to the oxidative removal of Mn (II) and, apparently only to a smaller degree, to that of Fe (II) present in borehole water and that species within the proteobacterial genus Acinetobacter are potentially involved in the geochemical cycling of these two metals. Keywords: Biofiltration, iron and manganese oxidation, Acinetobacter sp. LB1, batch tests, 16S rRNA, MALDI-TOF MS analysis, Mn (II) and Fe (II) colorimetric assays, EDX analysis, biofilm formation, CLSM image analysis, 16S rRNA clone library Abbreviations: MSVP (minimal salts vitamins pyruvate), ORP (oxidation reduction potential), EDX (energy dispersive X-ray analysis), MALDI-TOF MS (Matrix assisted laser desorption ionization-time of flight mass spectrometry), rRNA (ribosomal RNA), ARDRA (amplified “rDNA” restriction analysis), CLSM (confocal laser scanning microscopy), OTU (operational taxonomic unit)
Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2013.

Most waste generated in South Africa is sent to landfills for disposal, and although it is confined in specific areas, it can potentially affect both above and below ground water resources, impacting environmental and public health. This is particularly relevant in a country where water supplies are limited and groundwater resources are prone to pollution. The primary objective of this study was to assess the performance of an upflow packed-bed bioreactor purposedesigned for the treatment of leachates produced by landfills in the Durban Metropolitan Area (DMA). The effect of parameters such as the nature of the biofilm support matrix, aeration rate and recycle rate on the efficacy of the system were investigated. Another major aim of the project was to develop a low maintenance technology that could, nonetheless, bioremediate leachate effectively at minimum cost. This aspect of process design is a crucial factor in areas where there is a shortage of both funds and skilled labour. The glass 132 l packed-bed upflow bioreactor was evaluated by measuring its efficiency in terms of chemical oxygen demand (COD) and biological oxygen demand (BOD) reduction and ammonia removal. The bioreactor could be configured as a batch-type system, which was useful for comparing operating conditions; or as a continuous cascade system, which was used to assess its overall performance. Different biofilm support matrices viz. various grades of pine bark, plastic bioballs and ceramic noodles were evaluated in 22 l batch-type reactors. Leachates from five landfill sites were remediated during the course of the study, and only the leachate from Shongweni landfill, which had a remarkably low BOD:COD ratio (0.05), was intractable and could not be successfully treated; even in flask trials designed to test strategies such as augmentation of microflora and biostimulation. The other leachates investigated were from the Umlazi, Marianhill, Bisarsar Road (all general sites) and Bul-Bul Drive (a semi-hazardous site) landfills, all of which were remediated to some degree. Originally, leachate from the Umlazi landfill site was used, but it became unavailable when the site closed enforcing the use of other leachates for the remainder of the investigation. Leachates from Marianhill, Bisarsar Road and Bul-Bul Drive were treated simultaneously in duplicate operating the six-chambered bioreactor in the batchtype configuration. The highest COD removal efficiency (49 %) was obtained in the chambers treating the Bul-Bul Drive leachate, which was therefore used for further investigations. This leachate had the highest BOD:COD ratio and was therefore expected to be the most suited to biological remediation. The bioreactor performed best when plastic bioballs were used as biofilm support matrix with a relatively low level of aeration, although the uncomposted form of pine bark was used initially as the support matrix because it is inexpensive and readily available in South Africa. However, although satisfactory COD reduction (30 – 61 %) and ammonia removal (87 – 98 %) was achieved when the Umlazi leachate was treated, the possibility of compounds leaching out of the bark and affecting the quality of the treated leachate was a concern. Also, pine bark would be prone to mechanical degradation in a full scale operation. Of the other solid support matrices tested using the Bul-Bul leachate, COD removal efficiencies were superior with plastic bioballs (60 %) than with pine bark chips (29 %). The former therefore became the preferred biofilm support matrix. Aeration level did influence bioremediation of the Umlazi landfill leachate since those chambers aerated with an aquarium pump (0.05 – 0.1 litres air/litre leachate/min; 60 % COD removal) performed better than those aerated with a blower (0.6 -0.7 litres air/litre leachate/min; 42 % COD removal) and those that remained unaerated (44 % COD removal). Recycle rate did not significantly affect bioremediation, but the performance of the system was higher when operated in batch mode (up to 60 % influent COD removal), rather than in continuous flow-through (cascade) mode when only 37 % of the influent COD in the Bul-Bul leachate was removed. Under the latter conditions, most of the reduction occurred in the first four chambers and very little biodegradation occurred in the final two chambers. The cascade-mode will require some refinement to enhance the COD removal efficiencies achieved. However, it did eliminate 89 % of the BOD present in the raw leachate, producing a treated effluent with a consistent BOD:COD ratio of 0.05. The COD removal efficiencies achieved covered a wide range from a minimum of 23 % with Marianhill leachate to a maximum of 63 % with leachate from Bul-Bul Drive. These results are comparable with many of those reported by other authors treating landfill leachate. Up to 98 % of the ammonia was removed when the Umlazi leachate was treated. However, ammonia removal from the other leachates tested was erratic. Although the treated leachate from this system could not be released into the environment without further remediation, the reduction in concentration of pollutants would allow its return to the local water supply via a wastewater treatment plant. This was achieved without temperature and pH regulation or addition of extraneous nutrient sources. A cost-effective, low maintenance technology such as this one would be a useful tool for the treatment of effluents such as landfill leachate in countries like South Africa where although water conservation is urgently required, resources for highly sophisticated effluent remediation are often not readily available.
Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2011.

The presence of heavy metal ions in waste streams is one of the most pervasive environmental issues of present times. A rotating biological contactor (RBC) was used to investigate the potential capacity of microbial biofilms in remediation of the metal ion species from a mixed metal contaminated effluent solution containing Cr+3 , Pb+2 and Cu+2 , each at a concentration of 200 mg r1 • In the first part of this study the effectiveness of various support materials for the development of microbial biofilms capable of removing heavy metals from a synthetic effluent was investigated. EDX analysis showed that none of the support matrices investigated, viz. gravel, polyester batting and sand, adsorbed metal ions on their surfaces; hence, metal adsorption was due purely to microbial activities. The biofilms attached more firmly and uniformly to polyester batting than to gravel and sand. The characteristics of polyester batting which made it a superior support matrix were its surface roughness and porous hydrophilic nature, which provided a larger surface area for the adhesion of microorganisms and attraction of nutrients during the biofilm development process. The selective accumulation of metal ion specIes by various microbial populations grown as biofilm using polyester batting as support matrix in separate compartments of a single-stage RBC bioreactor was examined. Lead ions were readily accumulated by almost all the microbial biofilms tested. Fungus-dominated biofilms selectively accumulated chromium ions whereas biofilms comprising mainly bacteria more readily accumulated copper ions from the mixed metal contaminated effluent solution. However, where interactions between the bacterial and fungal components were encouraged the mechanical stability of the biofilms was enhanced so that large amounts of all three metal ion species were removed by this biofilm. The combined effect of a series of bench-scale columns containing liquid humic acid and a three stage RBC bioreactor on the removal of metal ion species from a mixed metal contaminated effluent was investigated. After seven days of treatment the combined system had removed approximately 99% of the Cr+3, 98% of the Pb+2 and 90% of the Cu+2 ions from the mixed metal contaminated synthetic effluent. Complexation of the metal ions with humic acid was the predominant factor accounting for approximately 68-86% Cr+3 , 70-86% Pb+2 and 53-73% Cu+2 removal levels within the columns. A large proportion of the remaining Cr+3 and Pb+2, but not of the Cu+2, was removed in compartment 1 of the RBC. This suggested that the presence of the former two metals in solution might have reduced the removal of the Cu+2 ions from the system. The removal of substantially large amounts of the competing ions chromium and lead during the initial stages of the treatment process meant that copper was successfully taken up in the second and third RBC compartments. Hence, the economy of the treatment process was improved as larger quantities of the metal ions were removed in a shorter period of time than was possible when using the individual treatments (humic acid-metal complexation and biofilm adsorption) separately. More than 75%,92% and 86% of the adsorbed Cr+3 , Pb+2 and Cu+2 ions, respectively, were recovered from the three RBC bioreactor compartments following repeated washing of the biofilms with 0.1 M HCI. This relatively easy desorption suggested that the metal ions were simply adsorbed onto the surfaces of the biofilm cells rather than being taken into the cytoplasm of the cells.
Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2004.

The ability of the subsurface microbial population to degrade the major organic contaminants at three sites was assessed. The three sites included an abandoned wood creosoting facility in Conroe, Texas where the silty sand aquifer was contaminated primarily with polynuclear aromatic hydrocarbons; a sandy aquifer in Traverse City, Michigan where a spill of jet fuel polluted the ground water with benzene, toluene, and xylenes; and a site on the Texas Gulf Coast in which the fine sand aquifer was contaminated with benzene, toluene, naphthalene, and bis(2-chloroethyl) ether. Representative subsurface cores and ground water samples were collected for each site and used in the experiments to characterize the microbial population, to determine their ability to degrade the organic contaminants particular to the site, and to ascertain the factors limiting biodegradation of the contaminants at these sites. Laboratory experiments on the Conroe site revealed that the microbial population found in the contaminated zone could degrade the pollutants, but the microorganisms were not acclimated in the uncontaminated areas. The limiting factor at this site was the supply of dissolved oxygen. Field tests and modeling simulations confirmed these results. At the Traverse City site, few organisms were found that could degrade the test compounds in the uncontaminated zone. The microorganisms from the uncontaminated site could not respond as rapidly to a natural substrate as organisms in the contaminated zone which suggests their metabolic status was altered by the contamination. Oxygen, not inorganic nutrients, limited biodegradation at this site although anaerobic degradation of the contaminants may also occur. At the third site, the organisms in the uncontaminated zone were active against benzene, toluene, and to a lesser degree, naphthalene. In the contaminated zone, a toxicant may have reduced the amount of biodegradation occurring. This project demonstrated that microorganisms in the contaminated zones of two sites were active against the contaminants, but were limited in their ability to degrade these contaminants by the supply of dissolved oxygen. At the third site, a toxicant appeared to control biodegradation.

Batch microcosm studies were carried out to screen for microorganisms from the subsurface of Hanford DOE site that could cometabolically transform chloroform (CF) under aerobic conditions. The potential need for CF bioremediation at the Hanford site has resulted from the large release of carbon tetrachloride (CT) to the subsurface, of which a fraction anaerobically transformed to CF. Potential cometabolic substrates were screened for their ability to promote aerobic cometabolism of CF. The potential cometabolic substrates tested were isoprene, propene, octane, ammonia, methane, propane, and butane. Microcosms were constructed with 125 ml batch serum bottles filled with 25 g of aquifer solids, 50 ml of synthetic groundwater, and 60 ml of headspace air. Consumption of methane, butane, propane, and propene was slow, while the consumption of ammonia was very slow. Microorganisms stimulated on propene and octane showed no ability to transform CF. Limited CF was transformed in microcosms stimulated on ammonia and methane. Over 90% transformation of CF was observed in microcosms stimulated on either butane or propane during the initial incubation. Successive addition studies with methane, propane, and butane microcosms were conducted, because these substrates showed the most potential for driving CF cometabolism. The studies indicated that the most effective CF transformation was achieved by butane-utilizers. CF transformation was correlated with the consumption of the primary substrate. Propane- and butane-utilizers grown in the absence of CF showed transformation yields 3 times greater than those grown in the presence of CF. In butane fed microcosms, CF transformation was linked with butane and oxygen consumption, indicating that an oxygenase enzyme of the butane-utilizers was likely responsible for CF transformation. The butane-utilizers showed no ability to transform CT, which also suggests the possibility of CF transformation by an oxygenase enzyme. In butane microcosms, complete transformation of 55 pg of CF (1200 ��g/L of CF in aqueous solution) was observed. The maximum transformation yield of 0.03 g CF transformed/g substrates consumed was achieved by the butane-utilizers. A stoichiometric amount of chloride was released to solution from CF during CF transformation, indicating that complete dehalogenation of CF was achieved by butane-utilizers. In our knowledge, these were the first observations, demonstrating butane as a cometabolic substrate for CF transformation.
Graduation date: 1997