Theses and dissertations (Applied Sciences)

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Assessment of a biological nutrient removal process for the remediation of edible oil effluent
(2002-05-10) Mkhize, Sandile Psychology; Bux, Faizal
Eutrophication is a natural process that is greatly aggravated by the action of man in the natural environment. Deterioration of South Africa's natural water resources results directly or indirectly from the discharge of industrial effluent rich in nutrient nitrogen and phosphorus. The South African edible oil refmeries generally discharge poor quality effluent which impacts negatively on the water resources and wastewater treatment installations. The main aim of this study was to assess the capacity of a laboratory scale effluent treatment process that will produce final effluent of acceptable quality with regards to organic load and phosphate concentration prior to its discharge into the municipal sewerage system. The study was conducted in three stages: wastewater characterization, treatability studies, and laboratory scale treatment investigations. After analysing various effluent parameters, treatability studies were conducted using an aerobic-anaerobic sequencing batch reactor with a total hydraulic retention time of 24 hours. The results showed an average of 75 % reduction of COD and more than 90 % removal of fats, oils and grease (FOG). Based on the results of effluent characterisation and treatability studies, a laboratory scale activated sludge effluent treatment process was designed and operated with two bioreactors (aerobic and anaerobic) in series. The system was operated for a period of one-month resulting in 70 % removal of COD and 4% reduction in phosphate (P04-P). After some structural and operational changes from the original design configuration, the system was the operated continuously for the duration of the study period. An optimum COD removal of 75 % and 107 mgll P04-P reduction was achieved during the last operational phase of the system. More than 95 % reduction in fats, oils and grease (FOG) had been achieved in both semi-continuously and continuously operated systems.b.7
Identification of polyphosphate accumulating bacteria from pilot- and full scale nutrient removal activated sludges
(1999) Atkinson, Blaise William; Bux, Faizal
General removal of phosphorus (P) from wastewater was introduced in Scandanavia in the late 1960's. At that time it was believed that P alone was limiting to algal growth and that the sole removal of P would solve the problem of eutrophication. However, we now know that both P and nitrogen (N) contribute to this deleterious effect and as such, much research has been conducted concerned with both the biological and chemical removal of these nutrients from sewage effluents. Enhanced biological phosphorus removal (EBPR), which is basically the biological accumulation of soluble P (as polyphosphate or poly-P) from the bulk liquid in excess of normal metabolic requirements, still tends to be sensitive to many external parameters and, as such, is subject to fluctuations. This makes it extremely difficult for wastewater treatment installations to achieve and maintain full compliance with strict discharge regulations. A more comprehensive understanding of the microbial community within the mixed liquor of a wastewater treatment system is therefore required which will ultimately assist in improving system design and performance. Chemical and civil engineers, when designing biological wastewater treatment systems, consider only the processes (biological or chemical) taking place within the reactor/s with little or no regard for the individual microbial species or the entire microbial community involved. Process design appears to be tackled empirically from a 'black box' approach; biological reactions or processes occurring within a system such as wastewater treatment are all lumped together and attributed to a single surrogate organism ie., the response of the surrogate to certain stimuli accounts for the total system response. This is similar to an analogy which Professor George Ekama (Dept of Civil Engineering, UCT), a leading scientist in wastewater treatment and process design, refers to where engineers, if, for example, are confronted with modelling the dynamics of carbon dioxide utilisation ofa forest, would recognise the accumulative system response and not give cognisance to each individual tree's contribution. It is true that if one had to consider every microbial species present in a highly organised community such as activated sludge, process models, designed to make quantitative and qualitative predictions as to the expected effluent quality from a particular design, would become increasingly complex and superfluous. It is evident from the countless accomplishments that engineers have succeeded, to a certain degree, in modelling wastewater treatment systems. One only has to consider the tremendous success of biological P (bio-P) removal and nitrification/denitrification processes at full-scale. However, there are limitations to this empirical approach and EBPR processes occasionally deteriorate in phosphate removal efficiency. In order to further optimise biological processes, whether they be organics oxidation, bio-P removal, nitrification or denitrification, biological community analyses will have to play a more significant role in design. The better microbial community structure and function is understood, the better the control and management of the system. With the advent of improved microbial identification and enumeration (to a certain extent) techniques (in situ), it was considered significant to investigate the mechanism ofbio-P removal and to elucidate which bacteria are actively responsible for this process. To this end, experimental work was conducted in two phases: \xAE laboratory, where samples of mixed liquor were obtained from a full-scale wastewater treatment facility exhibiting biological nutrient removal (BNR) characteristics and @ pilot plant, where an enhanced culture ofpolyphosphate accumulating organisms (PAO's) was developed and probed using molecular identification and enumeration techniques (as well as a cultivation-dependent approach). During phase \xAE of experimentat
Extraction, characterisation and metal biosorption of extracellular polysaccharides from activated sludge
(1998) Zondo, Raynold Mduduzi; Swalaha, Feroz Mahomed
Waste activated sludge is a biological adsorbent whose potential to remove metals from solution and effluent has been demonstrated. Extracellular polysaccharides (EPS) as components of activated sludge are thought to contribute to activated sludge metal biosorption. During the present study characterisation and determination of the metal biosorptive capabilities of domestic and industrial extracellular polysaccharides (EPS) revealed similarities both in terms of chemical composition and metal adsorption potential. Extracellular polysaccharides were extracted from activated sludge, obtained from domestic and industrial sludge treatment plants, using chemical techniques which involved sodium hydroxide extraction and solvent precipitation. A purification technique, which involved precipitation of protein with chloroform and removal of nucleic acids was developed. To assess the efficiency of the purification method, the ratio of extracted polysaccharide to the amount of protein present was determined. This provided an indication of the magnitude of EPS extracted in relation to the degree of cellular disruption. The type of activated sludge being treated was shown to be of particular importance. The quantity of EPS present in the original sample was found to be higher in domestic sludge than in industrial sludge. Purified EPS was fractionated in a column of DEAE-Sepharose CL-6B using stepwise pH gradient elution. Molecular weight distribution was conducted on a column of Sepharose CL-4B. Component monosaccharides were identified by paper chromatography. Monomers identified were glucose, fructose, glucuronic acid and galactosamine. Ion-exchange chromatography results demonstrated the presence of a number of different polysaccharide fractions while gel filtration results indicated a wide molecular weight distribution range of EPS from both domestic and industrial activated sludge. This indicated potential for variety in the EPS content of the activated sludge. Metal adsorption studies were conducted to determine the capabilities of EPS to adsorb metals
Evaluation of anaerobic sludges as metal biosorbents and development of a biotechnological process for metal ion removal from selected wastewater
(1997) Bux, Faizal; Kasan, Hamanth C.
As a result of rapid expansion of the industrial sector and increasing population, the environment has been under phenomenal stress. The volume of sewage and other effluents has increased tremendously in the last century. Globally, approximately .12 million tonnes of dry sludge biomass is produced and discarded of by landspreading, landfilling, incineration or dumping in lagoons and oceans. The discharge of industrial effluents into receiving waters has been documented to be the cause of severe environmental contamination. Heavy metals have been the cause of particular environmental concern. Their toxic and carcinogenic potentials at low concentrations, as well as the large quantities disposed to the environment, have prioritised them as leading contaminants. Current technologies of remediating heavy metal containing effluents are expensive and, in most cases, ineffective. Locally, most industries are merely diluting their effluents, thus resulting in the loss of valuable water resources. Waste sludges have shown the ability to adsorb heavy metals from their aqueous environment. Therefore, the current study attempted firstly, to compare biosorptive capacities of various waste sludges for a range of heavy metal ions, and secondly, to establish a relationship, if any, between biosorptive capacity and sludge surface charge. Finally, a laboratory scale biosorption process, encompassing desorption and recovery of metal ions from sludge surfaces, would have to be developed. Effluents used included pure, metal solutions of divalent zinc, cadmium, copper, nickel, trivalent and hexavalent chromium. In addition, synthetic effluents comprising a cocktail of the above-mentioned metal ions as well as an industrial effluent from a metal plating company were used. Five waste digested sludges were prepared and challenged against pure metal solutions to determine and compare their respective biosorptive capacities. Mechanisms of biosorption were elucidated using the Langmuir adsorption isotherm model. Sludge surface charge was determined using the millivolt quantification method. Upscaling of bioreactor trials to fully mixed laboratory scale was also investigated. These experiments encompassed the use of three sludges showing the greatest potential for biosorption and desorption using the selected mineral acid, H2S04, In addition, a simultaneous fully mixed biosorption and desorption process was designed and optimised. Subsequent trials involved comparing the latter process with a packed bed configuration whereby biomass was immobilised using poly sulfone resin. The overall comparative adsorptive capacities of the sludges (SI-SS) for metal ions in single solutions was S3 > S2 > S4 > SS > SI. Surface charge determination showed S3 to contain the most electronegative charge, with other sludges following in the same descending order as mentioned above. These findings supported the theory of a direct correlation between sludge surface charge and biosorptive potential. The affinity series of the sludges for metal ions followed the descending order of Cd2+ > Cu2+ > Ni2+ > Zn2+ > Cr6+ > Cr3+. Fully mixed studies, using mixed synthetic effluents, resulted in lower biosorptive capacities being recorded by the three selected sludges ie., S2, S3 and S4, as compared to single solution experiments. Biosorption studies with industrial effluent, containing Zn2+ as the most prevalent metal at 119.4 mg.F'. resulted in S3 biosorbing a maximum of 4.5 mg.g' of the cation. Sulphuric acid (H2S04) at O.2N, hydrochloric acid (HCI) at O.2N and acetic acid (CH3COOH) at O.4N were tested for their desorptive efficiencies. Sulphuric acid proved to be the most effective desorbing agent. Using S3 as biosorbent and O.2N H2S04 as desorbent, the manipulation and operation of a simultaneous process proved to be successful since both biosorption and desorption occurred concurrently, thus reducing time required for successful remediation considerably. Immobilised biomass, in a packed bed configuration, produced acceptable final effluent regarding standards as stipulated by the Durban Municipality for trade effluents. However, biosorption capacity of the sludge was compromised, with subsequent reductions in desorption being recorded, when the process was compared to fully mixed trials. Affinity series determined for the packed bed process wasC~+ >Cd2+>Zn2+>Cu2+>Cr6+ >Ni2+. Waste digested sludge has shown potential as metal biosorbent on an industrial scale. The present findings have succeeded in demonstrating a novel laboratory scale biotechnological process for the remediation of metal laden industrial effluents.
Expression of anti-HIV peptides in tobacco cell culture systems
(2009) Moodley, Nadine; Odhav, Bharti; Chikwamba, Rachel
Nearly half of all individuals living with HIV worldwide at present are woman and the best current strategy to prevent sexually transmitted HIV is antiretrovirals (ARVs). Microbicides are ARV’s which directly target viral entry and avert infection at mucosal surfaces. However, most promising ARV entry inhibitors are biologicals which are costly to manufacture and deliver to resource-poor areas. Microbicides formulated as simple gels, which are currently not commonly used in ARV therapy, show immense potential for use in prevention and treatment of multidrug-resistant viral infections in developing countries. Among the most potent HIV entry inhibitory molecules are lectins, which target the high mannose N-linked glycans which are displayed on the surface of HIV envelope glycoproteins. Of the microbicides, the red algal protein griffithsin (GRFT) has potent anti-HIV inhibitory activity and is active by targeting the terminal mannose residues on high mannose oligosaccharides. It has a total of 6 carbohydrate binding sites per homodimer, which likely accounts for its unparalleled potency. The antiviral potency of GRFT, coupled with its lack of cellular toxicity and exceptional environmental stability make it an ideal active ingredient of a topical HIV microbicide. v Scytovirin (SVN) is an equally potent anti-HIV protein, isolated from aqueous extracts of the cyanbacterium, Scytonema varium. Low, nanomolar concentrations of SVN have been reported to inactivate laboratory strains and primary isolates of HIV- 1. The inhibition of HIV by SVN involves interactions between the protein and HIV-1 envelope glycoproteins gp120, gp160 and gp41. Current recombinant production methods for GRFT and SVN molecules are unfortunately hampered by inadequate production capacities. This project therefore aimed to determine if these molecules can be produced in plant cell culture systems. The transgenic tobacco cell culture system was evaluated to determine if it can be an alternative, cost effective production system for these molecules. Results of the study show that the microbicide genes can be cloned into plant transformation vectors, used to successfully transform SR1 tobacco cell lines and adequately produce 3.38ng and 10.5ng of GRFT and SVN protein respectively, per gram of SR1 tobacco callus fresh weight. The promising results attained in this study form the basis for further work in optimising plant cell based production systems for producing valuable anti-HIV microbicides, a possible means to curbing the elevated HIV infection rates worldwide.
Overexpression and partial characterization of a modified fungal xylanase in Escherichia coli
(2009) Wakelin, Kyle; Permaul, Kugen; Singh, Suren
Protein engineering has been a valuable tool in creating enzyme variants that are capable of withstanding the extreme environments of industrial processes. Xylanases are a family of hemicellulolytic enzymes that are used in the biobleaching of pulp. Using directed evolution, a thermostable and alkaline stabl xylanase variant (S340) was created from the thermophilic fungus, Thermomyces lanuginosus. However, a host that was capable of rapid growth and high-level expression of the enzyme in large amounts was required. The insert containing the xylanase gene was cloned into a series a pET vectors in Escherichia coli BL21 (DE3) pLysS and trimmed from 786 bp to 692 bp to remove excess fungal DNA upstream and downstream of the open reading frame (ORF). The gene was then re-inserted back into the pET vectors. Using optimized growth conditions and lactose induction, a 14.9% increase in xylanase activity from 784.3 nkat/ml to 921.8 nkat/ml was recorded in one of the clones. The increase in expression was most probably due to the removal of fungal DNA between the vector promoter and the start codon. The distribution of the xylanase in the extracellular, periplasmic and cytoplasmic fractions was 17.3%, 51.3% and 31.4%, respectively. The modified enzyme was then purified to electrophoretic homogeneity using affinity chromatography. The xylanase had optimal activity at pH 5.5 and 70°C. After 120 min at 90°C and pH 10, S340 still displayed 39% residual activity. This enzyme is therefore well suited for its application in the pulp and paper industry.
Expression of a modified xylanase in yeast
(2009) Mchunu, Nokuthula Peace; Permaul, Kugen; Singh, Suren
Protein engineering has provided a key for adapting naturally-occurring enzymes for industrial processes. However, several obstacles have to be overcome after these proteins have been adapted, the main one being finding a suitable host to over-express these recombinant protein. This study investigated Saccharomyces cerevisiae, Pichia pastoris and Escherichia coli as suitable expression hosts for a previously modified fungal xylanase, which is naturally produced by the filamentous fungus, Thermomyces lanuginosus. A xylanase variant, NC38, that was made alkaline-stable using directed evolution was cloned into four different vectors: pDLG1 with an ADH2 promoter and pJC1 with a PGK promoter for expression in S. Cerevisiae, pBGP1 with a GAP promoter for expression in P. pastoris and pET22b(+) for expression in E. Coli BL21 (DE3). S. Cerevisiae clones with the p DLG1-NC38 combination showed very low activity on the plate assay and were not used for expression in liquid media as the promoter was easily repressed by reducing sugars used during production experiments. S. cerevisiae clones carrying pJC1-NC38 were grown in media without uracil while P. Pastoris clones were grown in YPD containing the antibiotic, zeocin and E. Coli clones were grown in LB with ampicillin. The levels of xylanase expression were then compared between P. Pastoris, S. cerevisiae and E. coli. The highest recombinant xylanase expression was observed in P. Pastoris with 261.7U/ml, followed by E.coli with 47.9 U/ml and lastly S. cerevisiae with 13.2 U/ml. The localization of the enzyme was also determined. In the methylotrophic yeast, P. Pastoris, the enzyme was secreted into the culture media with little or no contamination from the host proteins, while the in other hosts, the xylanase was located intracellularly. Therefore in this study, a mutated alkaline stable xylanase was successfully expressed in P. Pastoris and was also secreted into the culture medium with little or no contamination by host proteins, which favours the application of this enzyme in the pulp and paper industry.
Strategies to control bacteriophage infection in a threonine bioprocess
(2009) Cele, Nolwazi; Permaul, Kugen; Snyman, Francisca
Production of numerous biotechnologically-important products such as threonine is based on cultivation of bacterial cultures. Infection of these bacterial cultures by bacteriophages has a detrimental effect in the production of these bioproducts. Despite this, most people controlling these bioprocesses do not recognize the early signs of bacteriophage infection. SA Bioproducts (Ply) Ltd was no exception and has suffered tremendous loss of production time after bacteriophages infected threonine producing E. coli strain B. This study was aimed at developing assays to control and prevent bacteriophage infection at this company. These included determining the source of phages by monitoring the process plant environment, optimising the detection and enumeration methods so as to monitor the levels of bacteriophages in the environment, identification of bacteriophages in order to determine the number of bacteriophages capable of infection threonine producing E. coli strain B, treatment and of phages, and possible prevention of phage infection. Adam's DAL method was very efficient at detecting phages in the samples collected at various areas (sumps, odour scrubber, process water, and soil) around the plant for 16 weeks. High levels of phages were found in the sumps and this was identified as the source of infection. Samples collected were grouped together according to their source. The samples were enriched and purified in order to characterise them. The prevalent phage in all samples was identified as a T1-like phage. Bacterial strains that grew on the plate in the presence of phages were assumed to be resistant to phages or contained lysogenic phages which would explain the new lytic cycles that were observed whenever these resistant strains were used for production. UV light, green v indicator plates, and a mutagen (Mitomycin C) were used to detect Iysogens. Mitomycin C at 1 IJg/ml was found to be most effective in detecting lysogenic phages. This was shown by new plaque forming units that were visible on the DAL plates. Temperature (heat), chemicals, and inhibitors (vitamins) were investigated as strategies for prevention and treatment of bacteriophage infection. Bacteriophage samples were exposed to 70, 80, 100, and 120°C. At these temperatures pfu counts in the samples were reduced significantly. At 120°C there was a complete inactivation of bacteriophages within 30 minutes. Chemicals investigated such as sodium hydroxide and Albrom 100T were capable of complete deactivation of bacteriophages at a very low concentration (0.1%). Therefore, these chemicals can be used to clean the plant area and sumps. Vitamins C, K and E solutions were investigated to determine their inhibitory effect on bacteriophages. Vitamin C, K and E reduced pfu counts by 3, 2, and 4 logs, respectively. Therefore vitamin C and E solutions were mixed and to determine if mixing them would enhance their inactivation capabilities. This resulted in a reduction greater than 9 logs of phage in the sample (from 7.7 x 109 to 3 pfu/ml). The host bacterium was also exposed to this mixture to determine effect of the vitamin mixture on its growth. It was found that there was no effect exerted by this mixture on the host bacteria. This proved to be an ideal mixture for combating phages during fermentation. However, vitamin E is not cost effective for co-feeding in 200 m' fermenters, and therefore vitamin C solution was a cost-effective alternative. It was concluded that bacteriophage contaminated bioprocessing plant should be properly cleaned using a combination of heat and chemicals. Bacteriophage infection should be prevented by employing inhibitors.
Bioactivity of famine food plants from the family: Amaranthaceae
(2009) Singh, Alveera; Odhav, Bharti; Reddy, Lalini
Information regarding the nutritional value of wild food plants in Africa and current information varies from source to source. Prior to commercialization of wild foods the nutritional, ethnobotanical, medical, chemical, anthropological and toxicity requires investigation. Plants from the Amaranthaceae family were chosen because the family is characterized by several species which are used by indigenous communities as a source of nutrition in different plants of the world. The focus of this study was to investigate the nutritional and biological activities of three plants from the Amaranthaceae family viz. Achyranthes aspera, Alternanthera sessilis and Guilleminea densa that are considered famine plants. This study aimed to determine the nutritional value (proximate, minerals and vitamins), biological activity, toxicity and potential of a tissue culture system for three species from the family Amaranthaceae. Nutritional analysis comprised of determining moisture, ash, protein, fat, carbohydrate, dietary fibre and energy. Mineral analysis of calcium, copper, iron, magnesium, manganese, phosphorus, sodium and zinc was performed by microwave digestion and then analyzed by ICP Spectrophotometry. Vitamin A, Vitamin B1, Vitamin B2, Vitamin B3 and Vitamin C were also analyzed. For biological and safety analyses aqueous and methanolic extracts were prepared. Anti-oxidative and anti-inflammatory properties of the extracts were tested; antimicrobial activity was tested by evaluating the bactericidal, fungal effect and minimum inhibitory concentration on selected bacteria and fungi using the agar disk diffusion method. Anti mosquito potential was determined by setting up repellency, larvacidal assay and insecticidal assay. The safety and toxicity analysis was carried out by measuring cytotoxicity, toxicity and mutagenicity. The potential of an in vitro tissue culture system of A. aspera, A. sessilis and G. densa was determined using micropropagation. A. aspera indicated significant amounts moisture, ash, dietary fibre, protein, vitamin B1, vitamin B2, magnesium and manganese. Plant extracts of A. aspera had antibacterial activity against the Gram negative bacteria Esherichia coli, Pseudomas aeroginosa and Salmonella typhi; Gram positive bacteria Staphylococcus epidermis and Staphylococcus aureus. The methanolic extract had antifungal activity against Sacchromyces cerevisiae and exhibited significant free radical scavenging activity as well as 85% repellency against Anopheles arabiensis. The aqueous extract stimulated the growth of the K562 (Chronic Myclogenous Leukaemia) cell line and the plant extracts showed no mutagenicity or toxicity. A. sessilis indicated significant levels of ash, dietary fibre, protein, energy, vitamin A, vitamin B1, vitamin B2, vitamin B3, iron, magnesium and manganese present. Plant extracts of A. sessilis had antibacterial activity against Gram negative bacteria P. aeroginosa and Gram positive bacteria S. epidermis. The plant also showed antifungal activity against the yeasts S. cerevisiae and Candida albicans. The methanolic plant extract showed excellent antioxidant activity. The aqueous plant extract stimulated the growth of the K562 cell line and the plant extracts possessed no mutagenicity or toxicity. This plant grew well in a tissue culture system where it was propagated from callus to a fully grown plant able to survive in environmental conditions. G. densa has ash and dietary fibre, vitamin B2, vitamin B3 and iron. The plant extracts had antibacterial activity against Gram negative bacteria E. coli, P. aeroginosa and Klebsiella. oxytoca; Gram positive bacteria Baccilus stereathermophilus and S. aureus. The plant also has antifungal activity against C. albicans and significant repellency activity against A. arabiensis where it showed 100% repellency. This plant was not found to be mutagenic or toxic. The results obtained from this study show promising potential for the plants to be exploited as famine food plants. The nutritional value, biological activity and ability to micropropagate A. aspera, A. sessilis and G. densa indicates a good potential for purposes of harnessing biotechnological products.
Protein engineering of fungal xylanase
(2007) Stephens, Dawn Elizabeth
Protein engineering technologies, such as directed evolution and DNA recombination, are often used to modify enzymes on a genetic level for the creation of useful industrial catalysts. Pre-treatment of paper pulps with xylanases have been shown to decrease the amounts of toxic chlorine dioxide used to bleach pulp. This study was undertaken to improve the thermal and alkaline stabilities of the xylanase from the fungus Thermomyces lanuginosus using ep-PCR and DNA shuffling.

Theses and dissertations (Applied Sciences)

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