Theses and dissertations (Applied Sciences)

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Dietary modulation of the human colonic microbiota through plant-derived prebiotic compounds
(2007) Kassim, Muhammad Arshad
The human gut microbiota play a major role in host health, and attempts are being made to manipulate the composition of the gut microbiota-increase the composition of bacterial groups, such as lactobacilli and bifidobacteria that are perceived as exerting health promoting properties. These bacteria defined as food supplements (probiotics) beneficially affect the host by improving the intestinal microbial balance, and have been used to change the composition of the colonic microbiota. However, such changes may be transient, and the implantation of exogenous bacteria therefore becomes limited. In contrast, prebiotics are naturally occurring carbohydrates that are classified as non-digestible oligosaccharides present in edible plants. These carbohydrates enter the colon as intact compounds, elicit systemic physiological functions and act as fermentable substrates for colonic microflora-influencing the species composition and metabolic characteristics of intestinal microflora providing important health attributes. Currently, a widely marketed prebiotic, inulin is extracted from plants of the family Asteraceae. There are many unexploited plants that are regularly consumed and that may have a prebiotic effect or can have very high levels of inulin which could make them commercially viable. In this study, we investigated prebiotic compounds, especially inulin from locally growing, non-commercialised leafy plants. The aqueous extracts of 22 plants from the families Asparagaceae, Alliaceae, Asteraceae, Solanaceae, Cucurbitaceae, Amaranthaceae, Acanthaceae, Polygonaceae, Portulaceae, Fabaceae, Chenopodiaceae, Pedaliaceae and Apiaceae from Kwa-Zulu Natal were investigated for a prebiotic effect using a modified batch-culture technique with Lactobacillus bulgaricus, Lactobacillus lactis, Lactobacillus reuteri and Bifidobacterium longum, four common probiotics and the inulin content of the plants was determined using high performance liquid chromatography. Of the 22 plants studied, Solanum nigrum, Amaranthus spinosus, Amaranthus hybridus, Asystasia gangetica, Senna occidentalis, Cerathoteca triloba, Asparagus sprengeri, Tulbaghia violacea, Sonchus oleraceus and Taraxacum officinale exhibited a prebiotic effect. The prebiotic effect of the Taraxacum officinale, Sonchus oleraceus and Asparagus sprengeri extracts on L. lactis and L. reuteri was higher than or equivalent to inulin-a commercial prebiotic. In this study, Sonchus oleraceus exhibited the best prebiotic effect-was the only plant to stimulate all the probiotics including B. longum. Of all the plants analysed, Asparagus sprengeri tuber contained the highest amount of inulin (3.55%).
Spirulina production in brine effluent from cooling towers
(2007) Choonawala, Bilkis Banu
Spirulina is a blue-green, multicellular, filamentous cyanobacterium that can grow to sizes of 0.5 millimetres in length. It is an obligate photoautotroph and has a pH growth range from 8.3 to 11.0.The large-scale production of Spirulina biomass depends on many factors, the most important of which are nutrient availability, temperature and light. These factors can influence the growth of Spirulina and the composition of the biomass produced by changes in metabolism. Brine effluent from cooling towers of electricity generating plants may provide an ideal growth medium for Spirulina based on its growth requirements, i.e. high alkalinity and salinity. The aim of this research was to optimise brine effluent from cooling towers by supplementing it with salts, in order to use this optimised effluent in a small open laboratory raceway pond in an attempt to increase the biomass production of Spirulina.
Molecular characterization of filamentous bacteria isolated from full-scale activated sludge processes
(2007) Marrengane, Zinhle
Activated sludge flocs are responsible for flocculation, settling and dewaterability. It is important to maintain the growth off loc-forming bacteria for efficient sludge settleability and compaction for good quality effluent. Filamentous bacteria on the other hand are believed to provide rigid support network or backbone upon which floc-forming bacteria adhere to form stable activated sludge flocs (Wilderer et al., 2002; Ramothokang et al., 2003). Filamentous bacteria can also be detrimental to the process when they outgrow floc-forming bacteria. Morphologically filamentous bacteria are at an advantage as they have higher outward growth velocity and can extend freely to bulk liquid substrate. Proliferation of filamentous bacteria causes foaming and bulking (Martins et al., 2004). Although chemical alleviation measures to circumvent bulking are present, they are symptomatic (Chang et al., 2004). Eikelboom (1975) developed the first identification keys for the classification of filamentous bacteria that is primarily based on morphological characteristics and microscopic examination. Although very useful, this type of identification has its limitations. For instance some filamentous bacteria can change morphology in response to changes in the environment and although some of them can be morphologically similar they may vary considerably in their physiology and taxonomy (Martins et al., 2004). A vast number of filamentous bacteria are still very poorly understood which could be due to the problems of cultivation due to their slow growing nature and maintenance of cultures (Rossetti et al., 2006). This limitation necessitates a molecular approach to resolve the taxonomy of filamentous bacteria as it is a culture-independent technique which is highly accurate. This project was undertaken to verify the identity of pure cultures of filamentous bacteria isolated previously through the application of molecular techniques. The 16S rDNA are conserved regions in bacterial cells and they can be extracted and specific nucleic acid fragments amplified. Denaturation gradient gel electrophoresis enabled the separation of fragments of identical length but different size and served as an indication of purity (Muyzer et al., 1993).
Microbial degradation of polychlorinated biphenyls
(2007) Mustapha, Shubnum
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).
The evaluation of liquid hydrocarbon contamination of soil around petrochemical tank farms at a Durban refinery
(2004) Ramluckan, Krishan
The primary objective of this study was to determine the levels of liquid hydrocarbon contamination present in the soil within the SAPREF refinery. The secondary objective arising from this was the development of a gas chromatographic (GC) method using a fifty metre PONA (Paraffin, Olefin, Naphthalene and Aromatic) capillary column and the split injection technique, for the analysis. Toluene was the solvent selected, with the Standard method for the BÜCHI extraction system, to extract the hydrocarbons present in the soil samples taken at the five different tank farm sites in the refinery. The main purpose of the analysis and evaluation was to enable the refinery to establish environmental control measures to reduce the contamination of the soil in the area.
AB initio studies of a pentacyclo-undecane cage lactam
(2003) Singh, Thishana
The purpose of this study is to utilize computational techniques in the determination of the mechanistic pathways for the one-pot conversion of a pentacyclo-undecane (PCU) dione 1.1 to a pentacyclo-undecane cage lactam 1.2.
Application of thermostable a-Amylase from Thermomyces lanuginosus ATCC 58157 to nutritionally enhance starch based food
(2006) Padayachee, Thiriloshani
In Sub-Saharan Africa there is an urgent need to sustain and improve the quality of its food resources. Poverty eradication features high on the agenda of a number of world health organisations, while the number of underweight children in Africa continues to increase (Pellet, 1996). Providing nutritionally enhanced foods to the poor will help towards achieving this objective. Protein-energy malnutrition has been identified as one of the most important problems facing Africa, with maize as the staple diet (Nkama et al., 1995). However, a combination of several factors limits availability and the nutritional quality of maize. During starvation, energy and protein intakes decrease by 20-30%, with most of the children in Africa having an average protein intake of only 20 g per day (Igbedioh, 1996). Energy availability also affects protein utilization because of interrelationships of protein and energy metabolism (Elwyn, 1993). The diets of inhabitants in developing regions depend mainly on cereals (maize) for both protein and dietary energy which lacks indispensable amino acids, minerals, vitamins and carbohydrates. In light of these growing concerns an attempt was made to devise a scientific strategy to combat the nutritional shortfalls of maize meal. A multidisciplinary and concerted approach was followed within this project aimed at designing an improved thermostable amylase and applying the enzyme to nutritionally enhance maize meal. It was envisaged that the manipulation of maize meal, by the application of enzyme technology will improve the nutritional status of this staple food. The consequences is that an alternate solution for the eradication of an ailing, poverty stricken and malnourished African population is achievable. It is possible that the boundaries defining the limits of life will extend to even greater extremes through the application of novel technologies.
Assessing milk quality using the electronic nose
(2007) Govender, Samantha
There are many ways for milk and dairy products to develop flavour defects. Sensory evaluation, has been the traditional approach to characterize off flavours. The need for odour sensing devices becomes greater when volatile and semi-volatile organic compounds are present in the product in parts per billion or even in the parts per trillion concentration range that cause off flavours. Today, sophisticated, sensitive instrumental tests such as electronic nose technology coupled with gas chromatography are capable of detecting, identifying and quantifying the specific chemical agents responsible for off flavours. This study focused on the use of the electronic nose as a novel technology for the detection and monitoring of milk quality by testing the effects of heat treatment at 63˚C and shelf life. Microbiological testing, sensory evaluation and gas chromatographic analysis were carried out together with aroma profiling using the electronic nose to determine milk quality.
Evaluation of bacteriological techniques, sensory evaluation, gas chromatography, and electronic nose technology for the early detection of Alicyclobacillus acidoterrestris in fruit juices
(2007) Harrichandparsad, Zeenat
Alicyclobacillus acidoterrestris is a spore-forming spoilage micro-organism found in fruit juices whose spores are not destroyed by typical pasteurisation. Once its spores outgrow and multiply in finished juice products, they produce two volatile taint compounds namely guaiacol and 2,6-dibromophenol. In the food industry margins for errors are small and monitoring of products to avert such errors is crucial. Conventional microbiological monitoring is one such technique for spoilage micro-organisms another being automated systems which can detect taints. Both these categories were evaluated in this study with the electronic nose and gas chromatograph being the specific automated systems being assessed. Sensory evaluation was also assessed as a diagnostic tool in the detection of taints. Isolation and identification of what was thought to be A. acidoterrestris was a laborious and expensive exercise which eventually proved inconclusive. A pure culture was purchased and juices were then inoculated with two levels of A. acidoterrestris spores and incubated. Juices from each level of inoculation were evaluated at different time intervals via the above-mentioned monitoring techniques. Of the three media assessed in the microbiological method, Bacillus acidoterrestris medium (BAM) was found to be the most effective for enumerating A. acidoterrestris followed by K-medium (KM) then Orange Serum Agar (OSA). While BAM was still indicating the presence of A. acidoterrestris KM and OSA were not (counts of <10cfu/g). This illustrated that this micro-organism could be easily overlooked if KM or OSA were being used to enumerate them. Considering that many workers actually do use KM and OSA as their media of choice in enumerating A. acidoterrestris (perhaps because BAM is very tedious to prepare) the cause for concern is a real one. Assessment of the resultant taints via sensory evaluation after inoculation and incubation reveals the inability of many panellists to detect taints at levels (as assessed by GC) far above their documented threshold values. While GC is an extremely useful and powerful tool, the level of expertise and skill required to use such an instrument cannot be overlooked nor can the expense involved. With regard to the electronic nose assessment for the presence of the volatile taint compounds, an important finding was that the electronic nose indicated significant differences between test and control samples when panellists performing sensory evaluation did not. This also correlated to an interval when enumeration on OSA illustrated no A. acidoterrestris after several days of inoculation and incubation and BAM and KM did. Without implying that the electronic nose has no drawbacks, it has proved, in this instance to be a simple and easy piece of equipment to use. It can be used to detect taints produced under simulated spoilage conditions at reduced analysis times, levels of expertise, cost and energy.
Evaluation of traditional South African leafy plants for their safety in human consumption
(2007) Mudzwiri, Mashudu; Reddy, Lalini; Odhav, Bharti
Eighteen traditionally leafy vegetables consumed as food or medicinal compounds by a majority of people in the KwaZulu Natal province of South Africa were analysed for the presence of potentially harmful chemicals (antinutrients) and for their toxicity and mutagenicity. The purpose of the study was to determine whether leafy vegetables were safe for human consumption. Chemical analysis showed that none of the vegetables contained cyanogenic glycosides, however all the vegetables contained oxalic acid ranging from 24.1 mg/ml to 798.2 mg/ml with Solanum nigrum, Portulaca oleracea and Mormodica balsamina showing the highest concentrations. Most of the vegetables contained negligible amounts of phytic acid and saponins, except for Momordica balsamina (3.01 mg/ml and 1.83 mg/ml, respectively). Fourteen of the plants contained alkaloids with Portulaca oleracea having the highest content (1.53 g total alkaloids/5 g leaf material). Eight of the plants were found to inhibit trypsin activity. These chemical analyses were carried out in duplicate and the mean and standard deviation were used. The Ames test revealed that none of the leafy vegetables produced a mutagenic frequency above 1, except 10 000 µg/ml organic extract of Senna occidentalis (mutagenecity considered at mutagenic frequency above 2), thus none were considered mutagenic. All 18 organic extracts did not kill off more than 50% brine shrimp and were thus considered non-toxic. On the other hand the aqueous extracts of seven vegetables, namely, Physalis viscosa, Amaranthus dubius, Justicia flava, Bidens pilosa, Senna occidentalis, Chenopodium album and Ceratotheca triloba, killed more than 50% of the shrimp and are thus considered toxic above 100 µg/ml. The MTT assay carried out on the organic extracts indicated that 17 vegetables did not kill off more than 50% of HepG2 cells and were thus considered non-cytotoxic. The aqueous extracts of four vegetables, namely, Justicia flava, Asystasia gangetica, Momordica balsamin and Senna occidentalis, however killed more than 50% of the shrimp and were thus considered cytotoxic above 1 000 µg/ml. It may be concluded from the antinutrient analyses and the bioassays on the 18 vegetables that caution needs to be maintained with the consumption of certain leafy vegetables included in this study, especially Senna occidentalis.
Characterisation of the microbial communities present in an anaerobic baffled reactor utilising molecular techniques
(2005) Lalbahadur, Tharnija; Bux, Faizal
The provision of safe and sanitary water is a constitutional right and above all, a necessity of life. As a result of the rapid urbanisation and the past policies of apartheid, a large population of South Africa dwell in informal settlements, where there is very little hope of development, as the government does not possess the resources that are necessary for a full-scale sanitation programme. Therefore, on-site treatments have been considered to provide sanitation in these dense peri-urban areas. The anaerobic baffled reactor (ABR) is one such sanitation system. This reactor utilises the phenomenon of anaerobic digestion to degrade substrates. One of the major disadvantages of any anaerobic treatment processes is the extreme sensitivity of the bacterial communities, thus inducing slow recovery rates following toxic shocks. Therefore, an understanding of these microbial consortia is essential to effectively control, operate and optimise the anaerobic reactor. Fluorescence in situ hybridization, 4’,6-diamidino-2-phenylindole (DAPI) staining and DNA sequencing techniques were applied to determine the microbial consortium, as well as their reactions to daily operating conditions. With an understanding of these populations and their responses to perturbations within the system, it is possible to construct an anaerobic system that is successful in its treatment of domestic wastewater. In situ hybridizations were conducted for three operating periods, each characterised by specific flow rates. Results showed Eubacterial population dominance over the Archaeal population throughout both of the operating periods investigated. However, these cells cumulatively consisted of 50% of the total biomass fraction, as determined by DAPI staining. Group-probes utilised revealed a high concentration of fermentative acidogenic bacteria, which lead to a decrease in the pH values. It was noted that the ABR did not separate the acidogenic and methanogenic phases, as expected. Therefore, the decrease in pH further inhibited the proliferation of Archaeal acetoclastic methanogens, which were not present in the second operating period. DNA sequencing results revealed the occurrence of the hydrogenotrophic Methanobacterium and Methanococcus genera and confirmed the presence of Methanosarcina. Sequencing of the bacterial DNA confirmed the presence of the low G+ C Gram Positives (Streptococcus), the high G+C Gram Positives (Propionibacterium) and the sulfate reducing bacteria (Desulfovibrio vulgaris). However, justifications were highly subjective due to a lack of supportive analytical data, such as acetate, volatile fatty acids and methane concentrations. Despite this, findings served to add valuable information, providing details on the specific microbial groups associated with ABR treatment processes.
Chemoprotective action of natural products on cultured human epithelial cells exposed to aflatoxin B1
(2005) Reddy, Lalini; Odhav, Bharti
Previous studies indicate that a mutation in the non-oncogenic p53 gene is epidemiologically linked to human HCC (Ozturk, 1991; Chan et al., 2003). Hsu et al. (1991) found this link in Chinese, South African and Asian patients and Hollstein et al. (1993) found the same gene mutation in Taiwanese patients. The incidence of these aberrations is reported to be about 20- 50% in HCC’s (Kishimoto et al., 1997). There is sufficient evidence to indicate that carotenoids in addition to their well known antioxidant properties (Paiva and Russel, 1999), also affect intercellular communication, immune responses, neoplastic transformations and growth control, and cellular levels of enzymes that detoxify carcinogens (Zhang et al., 1991; Brockman et al., 1992; Pryor et al., 2000). To date studies carried out have used the rat (Foote et al., 1970; Gradelet et al., 1998) and the mule duckling model (Cheng et al., 2001) to show the protective effect of these carotenoids against AFB1 exposure. Of the well known carotenoids, lycopene and beta- carotene occur in abundance in fruits and vegetables and are safe for human consumption. Aflatoxin B1 frequently induces mutations of the p53 gene which is linked to HCC. Although there is much evidence from epidemiological studies linking the beneficial aspects of carotenoids to the prevention of cancer, the cellular and molecular mechanisms need to be understood in order to implement large scale intervention strategies to prevent AFB1 induced carcinoma. The use of chemical or dietary interventions to alter the susceptibility of humans to the actions of carcinogens and to block, retard or reverse carcinogenesis is an emerging chemoprotective strategy for disease prevention (Abdulla and Gruber, 2000; Kensler et al., 2003; Bingham and Riboli, 2004). Chemoprotection by natural products involves maintaining cellular integrity, preventing DNA alterations, activation of p53 suppressor protein and apoptosis. The aim of this study was thus to investigate the cellular and molecular mechanisms by which beta-carotene and lycopene may prevent the AFB1-induced toxic changes in human hepatocytes. In order to achieve this aim, the following objectives were set out: i. To optimise an in vitro system for the evaluation of AFB1 damage to cultured hepatocytes. ii. To determine the biochemical protection offered by beta-carotene and lycopene to AFB1-exposed hepatocytes, by measuring the mitochondrial activity, cell viability and ROS levels using appropriate enzyme assays and flow cytometry. iii. To determine the cellular protection offered by beta-carotene and lycopene to AFB1-exposed hepatocytes, by studying the morphological changes at the structural and ultrastructural levels using phase contrast light and electron microscopy respectively. iv. To determine the molecular protection offered by beta-carotene and lycopene to AFB1-exposed hepatocytes, by detecting apoptotic bodies as genomic markers and measuring the levels of p53 protein and AFB1-N7-guanine adducts produced.
Comparative analysis of genetically modified maize by implementation of a half-seed extraction technique
(2007) Pienaar, Fernando; Permaul, Kugen
The development of transgenic plants resulted in the need to utilize the various molecular methods (e.g., ELISA, real - time PCR etc.) for the detection or analysis of the presence or absence of a specific trait in a particular plant (Bt in this study). The overall aim of this study was to optimize a half – seed extraction technique as part of a laboratory protocol for transgenic maize plants and to explore the possibility of using the following molecular techniques: horizontal isoelectric focusing, real - time PCR and ELISA, as methods for detection of the Bt trait for incorporation into the half – seed extraction protocol.
The essential oil from Cymbopogon validus
(2007) Naidoo, Nelisha; Odhav, Bharti; Baijnath, Himansu
The chemical and biological properties of the essential oil from Cymbopogon validus were investigated. Hydro-distillation was used to extract the oil from C. validus, the flower-heads, leaves, culms and rhizomes. The percentage oil yields obtained from the plant organs varied from 0.05 to 1.23%, with the greatest concentration found in the flower-heads and rhizomes, 1.23 and 1.12% respectively. A sensory evaluation of the oil revealed that the essential oil was slightly murky, pale yellow in colour, had a strong turpentine-like smell and remained liquid at room temperature. The oxidative stability of C. validus oil was evaluated by determining its Rancimat induction period (negative), peroxide value (60.56 meq/kg), iodine value (84.55), percentage free fatty acids (0.19%) and percentage cholesterol (3.03%). These results indicated that the oil was highly susceptible to oxidation. Chromatographic profiles of the oils from C. validus, as well as the plant organs were generated using gas chromatography-mass spectroscopy (GC-MS). Predominant compounds present in the oil included alpha-Cubebene, Camphene, Geraniol, Limonene, Myrcene, Palmitic acid and Sabinene. C. validus essential oil was also investigated for its antimicrobial (disk diffusion), antioxidant (1, 1-Diphenyl-1-Picrylhydrazyl (DPPH) assay), anti-inflammatory (5-lipoxygenase assay), anti-mosquito properties (insecticidal, larvicidal and repellency assays) and toxicity profile (Brine shrimp and Ames assays). The oil showed poor antimicrobial activity and inhibited the growth of only Gram positive bacteria with a minimum inhibitory concentration (MIC) of 0.0625 (vol/vol) for Bacillus, Micrococcus and Staphylococcus species. The oil also exhibited excellent antioxidant activity, scavenging more than 80% of DPPH free radicals and possesses anti-inflammatory activity (IC50=190 ppm). C. validus oil showed good adulticidal activity (53.7% mortality) and excellent larvicidal (100% mortality) and repellent activity (100% repellency) against Anopheles arabiensis mosquitoes. At high concentrations, the oil was toxic to brine shrimp larvae. However, when diluted it was safe and the minimum inhibitory concentration was 0.0001(vol/vol). The absence of revertant colonies at all essential oil concentrations in the Ames test suggest that the oil is not mutagenic. These results lead the way for exploiting C. validus oil as a multi-functional agent that has antibacterial, anti-oxidant, anti-inflammatory, and anti-mosquito properties.
Elucidation of the microbial community structure within a laboratory scale activated sludge process using molecular techniques
(2006) Padayachee, Pamela; Bux, Faizal
The microbial community present in a laboratory-scale modified Ludzack-Ettinger activated sludge system was investigated using a combination of novel molecular techniques. The parent system was investigated for a duration of one year and samples were taken at regular intervals to determine the profile and structure of the microbial community present within the anoxic and aerobic zones of the MLE system. The combination of molecular techniques included fluorescent in situ hybridisation (FISH) and denaturing gradient gel electrophoresis (DGGE). FISH was performed using oligonucleotide probes, which were complementary to conserved regions of the rRNA for the alpha, beta and gamma subclasses of the gram negative family Proteobacteria as well as a group-specific HGC oligonucleotide probe as a representative of the gram positive actinomycetes branch. The total eubacteria present was determined using the EUB oligonucleotide probes, EUB388, EUB388-II and EUB388-III. The DGGE analysis of PCR-amplified 16S rDNA gene segments was used to examine the microbial community profile in the anoxic and aerobic zones. The profile for each of the zones revealed a number of consistent bands throughout the duration of the laboratory-scale process. However, the profiles obtained suggested that a diverse microbial community existed within the aerobic and anoxic zones. The bands also indicated the presence of dominant and less dominant species of bacteria. Hybridisations obtained from the FISH analyses indicated that the alpha and gamma subclasses were predominant within the anoxic zone and the aerobic zone showed a dominance of the beta subclass of Proteobacteria. The steady state behaviour of the MLE system was confirmed with the results obtained from COD, TKN, nitrates and OUR analytical tests. COD and nitrogen mass balances were conducted to confirm the acceptance of the results obtained for each batch as an indication of the system performance for the MLE model. Nitrogen mass balances indicated an upset in the nitrogen levels for batches two and seven.
Improvement of thermostability of a fungal xylanase using error-prone polymerase chain reaction (EpPCR)
(2007) Pillay, Sarveshni; Permaul, Kugen; Singh, Suren
Interest in xylanases from different microbial sources has increased markedly in the past decade, in part because of the application of these enzymes in a number of industries, the main area being the pulp and paper industry. While conventional methods will continue to be applied to enzyme production from micro-organisms, the application of recombinant DNA techniques is beginning to reveal important information on the molecular basis and this knowledge is now being applied both in the laboratory and commercially. In this study, a directed evolution strategy was used to select an enzyme variant with high thermostability. This study describes the use of error-prone PCR to modify the xylanase gene from Thermomyces lanuginosus DSM 5826, rendering it tolerant to temperatures in excess of 80°C. Mutagenesis comprised of different concentrations of nucleotides and manganese ions. The variants were generated in iterative steps and subsequent screening for the best mutant was evaluated using RBB-xylan agar plates. The optimum temperature for the activity of xylanases amongst all the enzyme variants was 72°C whilst the temperature optimum for the wild type enzyme was 70°C. Long term thermostability screening was therefore carried out at 80°C and 90°C. The screen yielded a variant which had a 38% improvement in thermostability compared to the wild type xylanase from pX3 (the unmutated gene). Successive rounds of error-prone PCR were carried out and in each round the progeny mutant displayed better thermostability than the parent. The most stable variant exhibited 71% residual activity after 90 minutes at 80˚C. Sequence analysis revealed four single amino acid residue changes that possibly enhanced their thermostabilities. This in vitro enzyme evolution technique therefore served as an effective tool in improving the thermostable property of this xylanase which is an important requirement in industry and has considerable potential for many industrial applications.
Functional characterisation of heterotrophic denitrifying bacteria in wastewater treatment systems
(2005) Ramdhani, Nishani; Bux, Faizal
Atmospheric nitrogen pollution is on the increase and human activities are directly or indirectly responsible for the generation of the various nitrogen polluting compounds. This can lead to the two major problems of eutrophication and groundwater pollution. Therefore, the removal of nutrients such as nitrogen and phosphorus from wastewater is important. Nitrogen removal from wastewater is achieved by a combination of nitrification and denitrification. Thus, there is a need to identify and characterise heterotrophic denitrifying bacteria involved in denitrification in wastewater treatment systems. The aim of this study, therefore, was to characterise heterotrophic denitrifying bacteria through detailed biochemical and molecular analysis, to facilitate the understanding of their functional role in wastewater treatment systems. Drysdale (2001) isolated heterotrophic denitrifiers to obtain a culture collection of 179 isolates. This culture collection was used to screen for nitrate and nitrite reduction using the colorimetric biochemical nitrate reduction test. The isolates were thereafter Gram stained to assess their gram reaction, cellular and colonial morphology. Based on these results identical isolates were discarded and a culture collection of approximately 129 isolates remained. The genetic diversity of the culture collection was investigated by the analysis of polymerase chain reaction (PCR)-amplified 16S ribosomal DNA (rDNA) fragments on polyacrylamide gels using denaturing gradient gel electrophoresis (DGGE). Thus DNA fragments of the same length but different nucleotide sequences were effectively separated and microbial community profiles of eight predominant isolates were created. Batch experiments were conducted on these eight isolates, the results of which ultimately confirmed their characterisation and placed them into their four functional groups i.e. 3 isolates were incomplete denitrifiers, 2 isolates were true denitrifiers, 2 isolates were sequential denitrifiers and 1 isolate was an exclusive nitrite reducer.
Production of enzymes for application on animal feeds
(2007) Godana, Busiswa; Singh, Suren; Mitra, R.
Ruminants diets in most developing countries are based on fibrous feeds, mainly mature pastures and crop residues. These feeds are unbalanced and particularly deficient in protein, minerals and vitamins and are highly lignified with low digestibility. These characteristics result in low intake and productivity and only approximately 40% of energy in forage becomes available to the animal. Enzymes can be used as biological tool to enhance digestion through the action of cellulase, hemicellulase and lignase enzymes resulting in improved meat, milk and wool production. The development of feed additives holds great promise for the improvement of livestock growth and yield for both large commercial and smaller subsistence farmers. The aim of this study was to develop optimal media for the production of lignocellulolytic enzymes (laccases, xylanases, and cellulases) and to evaluate the application of these enzymes to improve the nutritional digestibility of high fibre feedstocks, such as veld grass.
Molecular analyses of pure cultures of filamentous bacteria isolated from activated sludge
(2005) Naidoo, Dashika
The activated sludge process is the mostl used biological treatment process. Engineers and microbiologists are constantly seeking ways to improve process efficiency, which can be attributed to the increasing demand for fresh water supplies and proper environmental management. Since the inception of the activated sludge process, bulking and foaming have been major problems affecting its efficiency. Filamentous bacteria have been identified as the primary cause of bulking and foaming. Numerous attempts have been made to resolve this problem. Some of these attempts were effective as interim measures but failed as long term control strategies. The identification of filamentous bacteria and the study of their physiology have been hampered by the unreliability of conventional microbiological techniques. This is largely due to their morphological variations and inconsistent characteristics within different environments. To fully understand their role in promoting bulking and foaming, filamentous bacteria need to be characterized on a molecular level. The aim of this study was, therefore, to identify filamentous bacteria in pure culture with the purpose of validating these findings to the physiological traits of the pure cultures when they were isolated. Fourteen different filamentous cultures were used for this study. The cultures were identified using specific oligonucleotide probes via fluorescent in situ hybridisation and nucleotide sequencing. Prior to sequencing, an agarose gel and a denaturing gradient gel Electrophoresis profile were determined for each isolate. The various techniques were optimised specifically for the filamentous isolates. The isolates were identified as Gordonia amarae, Haliscomenobacter hydrossis, Acinetobacter sp./Type 1863, Type 021N, Thiothrix nivea, Sphaerotilus natans and Nocardioform organisms.
Molecular characterization of aflatoxigenic and non-aflatoxigenic aspergillus isolates
(2007) Mngadi, Phakamile Truth
For decades the genus Aspergillus (of fungi) has been classified based on morphological and growth criteria. Members of the Aspergillus section Flavi are economically valuable and methods of differentiating them are thus very important. Several molecular methods have been developed to distinguish these strains. Also, a number of biochemical and genetic studies have been used in order to provide a better means of classification (Lee et al., 2004). Aflatoxins, the most frequently studied mycotoxins, are produced by certain Aspergillus species/strains/isolates of fungi. The aflatoxin biosynthetic pathway studies have led to a number of discoveries. Several structural and regulatory genes (and their enzymes) involved in the biosynthesis of aflatoxins have been discovered and purified (Trail et al., 1995). Aflatoxin production and contamination of agricultural crops are major causes of economic losses in agriculture. Thus, better methods of characterization/differentiation are required for both aflatoxigenic and non-aflatoxigenic isolates. Molecular biology is one of the current tools used to differentiate between these isolates. Polymerase Chain Reaction (PCR)-based randomly amplified polymorphic DNA (RAPD) analysis has been used successfully in the analysis of DNA relatedness of species of fungi, bacteria, plants and animals. Dendograms which evaluate/assess the likeness between different isolates has also been used (Martinez et al., 2001). Restriction fragment length polymorphism (RFLP) analysis has been applied to a number of studies to detect differences between fungi and to establish relationships between them. Therefore, the scope of this study was to investigate RAPD analysis (with dendograms) and detection of RFLPs by hybridization as molecular methods that can distinctly differentiate or characterize the aflatoxigenic and non-aflatoxigenic Aspergillus isolates.