Dissertation (PhD) -- University of Stellenbosch, 2002. / ENGLISH ABSTRACT: More than sixteen isolates of iron-oxidizing bacteria belonging to the genus Leptospirillum were included in this study, with the finding that they were clearly divisible into two major groups. Group I leptospirilla had mol% G+C ratios within the range 49-52%, three copies of rrn genes and based on 16S rRNA sequence data, clustered together with the Leptospirillum ferrooxidans type strain (DSM2705or LI5). Group II leptospirilla had mol% G+C ratios of 55-58%, two copies of rrn genes and based on 16S rRNA sequence form a separate cluster. Genome DNA-DNA hybridization experiments indicated that three similarity subgroups were present amongst the leptospirilla tested with two DNA-DNA hybridization similarity subgroups being found within group I. The two groups could also be distinguished based on the sizes of their 16S-23SrRNA gene spacer regions. We propose that the group II leptospirilla should be recognized as a new species with the name Leptospirillum ferriphilum sp. nov. Members of the two species can be rapidly distinguished from each other by amplification of their 16S rRNA genes and carrying out restriction enzyme digests of the products. Several but not all isolates of the group II leptospirilla, but none from group I (L. ferrooxidans) were capable of growth at 45°C. Plasmid DNA was isolated from strain ATCC49879 (L. ferrooxidans). Restriction endonuclease mapping of what appeared to be about 60 kb of plasmid DNA, established that two plasmids of approximately 30.0 kb and 27.0 kb were present. These were named p49879.1 and p49879.2 respectively. Attempts to isolate the plasmids separately were not successful. Partial sequencing of the two plasmids was carried out and sequence analysis of p49879.1 and p49879.2 indicated that the plasmids shared regions of homology. Total plasmid DNA was DIG-labelled and used as a probe in Southern hybridization experiments with genomic DNA from all sixteen original leptospirilla isolates as the target DNA. All leptospirilla belonging to Group I gave a positive signal, little or no homology to Group II leptospirilla was obtained. The region of homology present in all L. ferrooxidans strains was localized to an area on plasmid p49879.2 showing high amino acid identity to a transposase/putative transposase of Methanosarcina acetivorans and plasmid CPl from Deinococcus radiodurans Rl respectively. Whether these regions of homology indicate that complete, functional transposons are present in all L. ferrooxidans isolates still remains to be determined. Preliminary sequence analysis of both plasmids resulted in the identification of regions with amino acid sequence identity to the TnpA and TnpR of the Tn2l-like transposon family, and the mobilization regions of IncQ-like plasmids (particularly that of pTFl from At. ferrooxidans). Another potentially interesting ORF was identified in p49879.2 with high amino acid sequence identity to an ArsR-like protein that belongs to a second atypical family of ArsR transcriptional regulators. Whether this protein is functional in the regulation of arsenic resistance genes has not yet been determined, nor have other arsenic resistance genes been identified. Future work includes further sequence analysis of these plasmids to better understand their contribution to the isolates in which they are found. / AFRIKAANSE OPSOMMING: Meer as sestien isolate van die yster-oksiderende bakterieë, wat aan die genus Leptospirillum behoort, is in die studie ingesluit en die resultate het getoon dat dié groep verder in twee hoof groepe verdeel kan word. Groep I het "n mol% G+C van tussen 49% en 52% gehad, sowel as drie kopieë van die ribosomale gene (rrn). Hiermeesaam het die 16SrRNA volgorde data getoon dat hierdie isolate groepeer saam met Leptospirillum ferrooxidans (DSM2705T en LI5). Groep II leptospirilla het "n mol% G+C van tussen 55% en 58% gehad sowel as twee kopieë van die rrn gene en saam met die 16SrRNA volgorde data het hierdie isolate "n aparte groep gevorm. Genoom DNA-DNA hibridisasie eksperimente het gewys dat daar drie subgroepe onder die Leptospirillum wat getoets was is, met twee naverwante groepe wat onder Groep I val. Daar kan ook tussen die twee hoof groepe onderskei word op grond van die grootte van hul 16S- 23SrRNA intergeniese gebiede. Ons stel dus hier voor dat die Groep II leptospirilla as "n nuwe spesie beskou word naamlik, Leptospirillum ferriphilum sp, nov. Die twee spesies kan maklik onderskei word deur die PKR amplifikasie produk van die 16SrRNA te verteer met restriksie ensieme. Vele, maar nie al van die Groep II isolate kan by 45°C groei nie, terwyl geen van die Groep I leptospirilla (L.ferrooxidans) kan nie. Plasmied DNA was geisoleer uit Leptospirillum ferrooxidans ATCC49879. Aanvanklike analise het gedui op die teenwoordigheid van een 60.0 kb plasmied. Verdere restriksie ensiem kartering het wel getoon dat hierdie, in teen deel, twee plasmiede van ongeveer 30.0 kb en 27.0 kb in grootte is: p49879.1 en p49879.2. Pogings om die twee plasmiede apart te isoleer was onsuksesvol. Totale plasmied DNA is gemerk met die Random primed DNA labelling kit (Roche diagnostics) en gebruik as peiler in Southern klad eksperimente met genoom DNA, van al sestien isolate, as teiken. Alle leptospirilla wat aan Groep I behoort het "n positiewe sein gegee terwyl geen sein teen Groep II DNA opgemerk was nie. Die area wat, tussen die plasmiede en Groep I homologie getoon het, is gelokaliseer tot "n area op plasmied p49879.2 wat hoë amino suur identiteit toon aan "n transposase geen van Methanosarcina acetivorans, en "n voorgestelde transposase geen op plasmied CPI van Deinococcus radiodurans Rl. Dit moet nog vasgestel word of hierdie area van homologie dui op die teenwoordigheid van "n volledige, funksionele transposon in alle L. ferrooxidans isolate. Gedeeltelike DNA volgorde bepalings van beide plasmiede het gelei tot die identifikasie van areas met hoë amino suur volgorde identiteit aan die TnpA en TnpR gene van die Tn21-tipe transposon familie, sowel as aan die mobilisasie gene van IncQsoortige plasmiede (veral die van pTFI uit Acidithiobacillus ferrooxidans). "n Oop lees raam van belang, wat op plasmied p49879.2 geidentifiseer was, het hoë amino suur volgorde identiteit aan "n ArsR-tipe geen getoon wat aan "n tweede atiepiese familie van ArsR transkripsionele reguleerders behoort. Op die stadium is dit nog onbekend of hierdie protein funksioneel is in die regulering van arseen weerstandbiedenheidsgene.

Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Plasmids are an integral part of the horizontal gene pool and, therefore, are the main vectors for the spread of antibiotic and heavy metal resistance genes in the environment. Functional and taxonomic characterization of novel plasmids is, therefore, central to our general understanding of plasmid biology and their contribution to microbial evolution. Two 14-kb mobilizable plasmids, p31T1 and p36T2, conferring resistance to tetracycline were isolated from the opportunistic fish pathogens Aeromonas sobria and Aeromonas hydrophila and were found to have indistinguishable restriction fragment length polymorphism (RFLP) patterns (Marx, MSc Thesis). DNA sequence analysis of the two isogenic plasmids (only p36T2 was sequenced) revealed the presence of 18 putative open reading frames (ORFs), of which the tetAR tetracycline resistance genes, associated with a truncated Tn1721, were the only ORFs with significant similarity to known sequences within the NCBI database. Putative functions were assigned to 10 of the ORFs based on their distant homology with proteins of known function. Six of the 18 ORFs, spanning 5.7-kb, were found to comprise the minimal region required for replication (minimal replicon) by means of deletion analysis using derivatives of p31T1. Of the six ORFs, ORF2 and ORF4 were found to be essential for plasmid replication. Inactivation of ORF3 resulted in an increase of plasmid copy number (PCN) from ~3 to ~7 plasmids per chromosome and a decrease in plasmid stability from ~80 % to 16 % over approximately 127 generations (7 days). Furthermore, by means of β-galactosidase promoter fusion assays it was shown that ORF3 autoregulated its own promoter. These results, therefore, suggested that although ORF3 was not essential for replication, it may be involved in plasmid copy number regulation and control. Host range analysis indicated that p31T1 was able to replicate in two other members of the γ-proteobacteria group (Escherichia coli and Pseudomonas putida) but was unable to do so in an α-proteobacterium strain, thus suggesting a limited host range. Furthermore, p31T1 was mobilized only at low frequencies (5.4 x 10-5 transconjugants per donor) by an IncP-1 conjugative system though it is possible that the mobilization system of these plasmids is adapted to function optimally with alternate conjugative systems. Given the unique PCN, stability, host range and mobilization characteristics determined for p31T1 and that no other plasmid replication and mobilization systems with significant sequence similarity to these plasmids have yet been identified, it is likely that these two plasmids are the first representative members of a new family of plasmids found within aquacultureassociated Aeromonas species and which are involved in the spread of tetracycline resistance. / AFRIKAANSE OPSOMMING: Plasmiede vorm ‘n integrale deel van die horisontale geen poel en vorm daarom die hoof vektore vir die verspreiding van antibiotika- en swaarmetaal-weerstandbiedende gene in die omgewing. Funksionele en taksonomiese karakterisering van nuwe plasmiede is belangrik in die begrip van plasmied biologie en hul bydrae tot mikrobiese evolusie. Twee 14-kb mobiliseerbare plasmiedes, p31T1 en p36T2, met tetrasiklien weerstandigheid was vanaf die opportunistiese vis patogene Aeromonas sobria en Aeromonas hydrophila geïsoleer en het identiese restriksie fragment lengte polimorfisme (RFLP) patrone. DNA volgorde analise van die twee isogeniese plasmiede (slegs die volgorde van p36T2 was bepaal) het die teenwoordigheid van 18 moontlike oop leesrame (OLR) getoon. Die tetAR tetrasiklien weerstandbiedende gene, wat met ‘n verkorte Tn1721 transposon geassosieerd is, was die enigste OLR wat beduidende volgorde ooreenkoms met bekende volgordes binne die NCBI databasis getoon het. Moontlike funksies was toegeken aan 10 van die OLRe en was gebasseer op vêrlangse homologie met proteïene met bekende funksies. Ses van die 18 OLRe strek oor ‘n 5.7- kb minimale replikon fragment wat benodig word vir replisering en is deur middel van delesie analises van p31T1 derivate gevind. Van hierdie ses OLRe, word OLR2 en OLR4 benodig vir plasmied replisering. Inaktivering van OLR3 het ‘n toename in plasmied kopiegetal (PKG) vanaf ~3 tot ~7 plasmiede per kromosoom en ‘n afname in stabiliteit vanaf ~80% tot 16% oor 127 generasies (7 dae) tot gevolg gehad. Verder kon daar deur middel van β-galaktosidase fusie analises getoon word dat OLR3 sy eie promotor outoreguleer. Hierdie resultate stel dus voor dat alhoewel OLR3 nie benodig was vir replikasie nie, mag dit dalk by plasmied kopiegetal regulering en beheer betrokke wees. Bakteriële gasheer analises het getoon dat p31T1 in 2 addisionele lede van die γ-proteobakterieë groep (Escherichia coli en Pseudomonas putida) kon repliseer, maar nie in ‘n α-proteobacterium nie. Verder kon p31T1 teen ‘n lae frekwensie (5.4 x 105) gemobiliseer word deur ‘n IncP-1 konjugasie sisteem, maar dit mag wees dat die mobilisering eerder optimaal kan plaasvind met ‘n alternatiewe konjugasie sisteem. Na aanleiding van die unieke PKG, stabiliteit, gasheer en mobilisering eienskappe wat vir p31T1 bepaal is en die feit dat geen ander replisering en mobilisering sisteme met noemenswaardige volgorde homologie tot hierdie plasmiede gevind kon word nie, blyk dit dat hierdie van die eerste lede van ‘n nuwe familie van plasmiede binne die akwakultuur-geassosieerde Aeromonas spesies is, wat betrokke is by die verspreiding van tetrasiklien weerstandbiedendheid.

The genus Bacillus is comprised of Gram-positive, rod-shaped, spore-forming bacteria which are well known for their ability to produce a diverse array of antimicrobial compounds. Ofparticular interest is the ability of certain strains to produce antifungal compounds. Such organisms have the potential for application in agriculture where they can be used as biocontrol agents against selected plant pathogenic fungi. A study was undertaken to further characterize selected Bacillus isolates that exhibit broad spectrum antifungal activity. Dual culture bioassays were used to screen seven selected Bacillus isolates for activity against four plant pathogenic fungi in vitro. All isolates were able to inhibit the pathogens to varying degrees. Two isolates, R29 and B81, were selected for further testing and characterization. Further bioassays were performed on five complex nutrient media which were adjusted to pH S.S and 7, and both incubated at 2SoC and 30°C" respectively. It was found that pH and media composition showed significant influences on the antifungal activities of the isolates tested, but that a SoC temperature difference in incubation temperature did not. Tryptone soy agar was found to give rise to the largest inhibition zones. Both isolates were tentatively identified using standard biochemical and morphological tests. Based on its phenotypic characteristics, R29 was identified as a strain of B. subtilis. B81 proved to be more difficult to assign to a specific group or species of Bacillus, though B. subtilis and B. licheniformis were considered to be the nearest candidates. Genomic DNA was extracted from both isolates and a portion of each of their 16s rDNA genes were amplified and sequenced for homology testing against the GeneBank database. Homology testing confirmed that both isolates were members of the genus Bacillus and most probably strains of B. subtilis. The DNA fragment used for sequencing proved to be too small to give conclusive identification of the isolates. Isolate R29 was selected for further characterization of its antifungal compound/so Growth curve studies using a defined synthetic medium showed that antifungal activity arose during the stationary phase and appeared to be closely linked to sporulation. The antifungal component of cell free culture supematant was extracted using various methods including thin layer chromatography, acid precipitation, hydrophobic interaction chromatography and methanol extractions. High performance liquid chromatography (HPLC) analysis of extracts from acid precipitation and hydrophobic interaction chromatography revealed two active peaks indicating that at least two antifungal compounds were produced. Methanol extracted samples produced the cleanest sample extract but only revealed one active peak from the HPLC fraction . Nuclear magnetic resonance analysis of purified samples indicated that the antifungal compound/s have aromatic complex and peptide structures. The extracted antifungal compounds were Protease K resistant and found to be thermostable at temperatures ranging 80-121oC, and, were active at pH ranges of 3-13. The antifungal compounds were found to exhibit similar properties to known antifungallipopeptides i.e. iturin A and fengycin A and B. Further characterization and identification of the active compounds is recommended usmg methods such as liquid chromatography mass spectrometer and matrix-assisted laser desorption ionisation time-of- flight. The results presented in this dissertation provide a basis from which antifungal compounds produced by strains ofBacillus can be further characterized. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2004.

Microbial leaching plays a significant role in the natural weathering of silicate containing ores such as diamond-bearing kimberlite. Harnessing microbial leaching processes to pre-treat mined kimberlite ores has been proposed as a means of improving diamond recovery efficiencies. The biomineralization of kimberlite is rarely studied. Therefore, this study investigated the feasibility of exploiting both chemolithotrophic and heterotrophic leaching processes to accelerate the weathering of kimberlite. Preliminary investigations using mixed chemolithotrophic leaching cultures were performed on four finely ground kimberlite samples (<100μm) sourced from different mines in South Africa and Canada. Mixed chemolithotrophic cultures were grown in shake flasks containing kimberlite and inorganic basal media supplemented either with iron (Fe2+, 15g/l) or elemental sulfur (10g/l) as energy sources. Weathering due to dissolution was monitored by Inductive Coupled Plasma (ICP) analyses of Si, Fe, K, Mg and Ca in the leach solutions at known pH. Structural alterations of kimberlite after specified treatment times were analyzed by X-ray Powder Diffraction (XRD). The results of the preliminary investigation showed that weathering can be accelerated in the presence of microbial leaching agents but the degree of susceptibility and mineralogical transformation varied between different kimberlite types with different mineralogical characteristics. In general, the results showed that the kimberlite sample from Victor Mine was most prone to weathering while the sample from Gahcho Kue was the most resistant. It was therefore deduced that kimberlite with swelling clays as their major mineral component weathered relatively more easily when compared to kimberlite that consisted of serpentine and phlogopite as their major minerals. Gypsum precipitates were also distinguished indicating that a partial alteration in the kimberlite mineralogical structure occurred. Both energy sources positively influenced the dissolution process, with sulfur producing superior results. This was attributed to the generation of sulfuric acid which promotes cation dissolution and mineral weathering. Success in the preliminary investigations led to further experimental testing performed to determine the effect of particle size and varying energy source concentrations on the biotransformation of kimberlite. It was observed that although weathering rates of the larger kimberlite particles (>2mm<5mm) were lower than that of the finer particles, slight changes in their mineralogical structures represented by the XRD analyses were seen. Optimisation studies of energy source concentration concluded that although the highest concentration of elemental sulfur (20% w/w) and ferrous iron (35% w/w) produced the most pronounced changes for each energy source tested, the leaching efficiency at these concentrations were not drastically greater than the leaching efficiency of the lower concentrations, as expected. Following the success of batch culture shake flasks weathering tests, the effect of continuous chemolithotrophic cultures on the biotransformation of larger kimberlite particles (>5mm<6.7mm) was investigated. A continuous plug-flow bioleach column was used to model the behaviour of chemolithotrophic consortia in a dump- or heap leaching system. Two sequential columns were setup, in which the first consisted of kimberlite mixed with sulfur and the second purely kimberlite. Inorganic growth medium was pumped to the first column at a fixed dilution rate of 0.25h-1 and the leachate from the first column dripped into the second. After an 8 week investigation period, the ICP and XRD data showed that weathering did occur. However, the pH results showed that the leaching process is governed by the amount of acid produced by the growth-rate independent chemolithotrophic consortia. Data from pH analyses also showed that the leaching bacteria reached ‘steady state’ conditions from day 45 onwards. The pH also remained higher in the second column than in the first column highlighting the alkaline nature of the kimberlite ores and its ability to act as a buffering agent and resist weathering. This important factor, as well as further optimisation studies in process operating conditions and efficiency, needs to be considered when establishing heap-leaching technology for these kimberlite ores. In the preliminary heterotrophic investigation, Aspergillus niger was used to produce organic metabolites to enhance kimberlite mineralization. The results demonstrated that the organic acid metabolites generated caused partial solubilization of the kimberlite minerals. However, it was deduced that for more significant changes to be observed higher amounts of organic acids need to be produced and maintained. The results obtained in this study also showed that the type of kimberlite presents a different susceptibility to the dissolution process and the presence of the fungal cells may improve the leaching efficiency. The results in this study provided an optimistic base for the use of microbial leaching processes in accelerating the weathering of kimberlite. These findings may also serve to supply data to formulate recommendations for further and future column microbial leach tests as well as validation and simulation purposes. / Thesis (M.Sc.) - University of KwaZulu-Natal, Pietermaritzburg, 2008.

The indiscriminate use of fossil fuels has led to global problems of greenhouse gas emissions, environmental degradation and energy security. Developments of alternative and sustainable energy resources have assumed paramount importance over the past decades to curb these challenges. Biohydrogen is emerging as an alternative renewable source of energy and has received considerable attention in recent years due to its social, economic and environmental benefits. It can be generated by dark fermentation on Organic Fraction of Solid Municipal Waste (OFSMW). These OFSMW exist abundantly and poses disposal challenges. This study models and optimizes the production of biohydrogen on a mixture of agro-municipal wastes; it examines a semi-pilot scale production on these substrates and the feasibility of generating bioelectricity from the process effluents and reviews the prospect of enhancing fermentative biohydrogen development using miniaturized parallel bioreactors. The fermentation process of biohydrogen production on agro-municipal wastes was modelled and optimized using a two-stage design. A mixture design was used for determination of optimum proportions of co-substrates of Bean Husk (BH), Corn Stalk (CS) and OFSMW for biohydrogen production. The effects of operational setpoint parameters of substrate concentration, pH, temperature and Hydraulic Retention Time (HRT) on hydrogen response using the mixed substrates were modelled and optimized using box-behnken design. The optimized mixtures were in the ratio of OFSMW: BH: CS = 30:0:0 and OFSMW: BH: CS = 15:15:0 with yields of 56.47 ml H2/g TVS and 41.16 ml H2/g TVS respectively. Optimization on physico-chemical parameters using the improved substrate suggested optimal setpoints of 40.45 g/l, 7.9, 30.29 oC and 86.28 h for substrate concentration, pH, temperature and HRT respectively and hydrogen yield of 57.73 ml H2/g TVS. The quadratic polynomial models from the mixture and box-behnken design had a coefficient of determination (R2) of 0.94 and 0.79 respectively, suggesting that the models were adequate to navigate the optimization space. The feasibility of a large-scale biohydrogen fermentation process was studied using the optimized operational setpoints. A semi-pilot scale biohydrogen fermentation process was carried out in 10 L bioreactor and the potential of generating bioelectricity from the process effluents was further assessed using a two-chambered Microbial Fuel Cell (MFC) process. The maximum hydrogen fraction of 46.7% and hydrogen yield of 246.93 ml H2/g TVS were obtained from the semi-pilot process. The maximum electrical power and current densities of 0.21 W/m2 and 0.74 A/m2 respectively were recorded at 500 Ω and the chemical oxygen demand (COD) removal efficiency of 50.1% was achieved from the MFC process. This study has highlighted the feasibility of applying agricultural and municipal wastes for large-scale microbial production of hydrogen, with a simultaneous generation of bioelectricity from the process effluents. Furthermore, the potential of generating an economical feasible biohydrogen production process from these waste materials was demonstrated in this work. Keywords: Biohydrogen production, Organic Fraction of Solid Municipal Waste (OFSMW), Modelling and optimization, Fermentation process, Renewable energy, Bioenergy / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2013.

Thesis (doctoral)--University of Oulu, 2007. / Includes bibliographical references. Also available on the World Wide Web.

Thesis (MTech (Chemical Engineering))--Cape Peninsula University of Technology, 2016. / Microbial spoilage has been reported in various food products and this has led to increased food, fruit and beverage losses, thereby threatening economic growth, food safety and security. Furthermore, statistics have shown that more than 30% of agricultural produce in developing countries, mostly in Africa, is lost owing to microbial spoilage. Beverages, food and fruits are predominant contributors to the South African export market. In recent years, contamination of these products resulting in spoilage has been a problem, although partial spoilage control has been achieved using chemical preservatives such as dimethyl dicarbonate, sodium benzoate, potassium sorbate, and sulphur dioxide (SO2). However, prolonged exposure to these chemical preservatives can cause human health problems such as skin and/or eyesight damage, muscle and stomach pain, cardiovascular disease and the impairment of brain function. To mitigate such health concerns, biologically benign alternatives are deemed suitable, providing the rationale for this study.

Diversity of micro-organism communities in activated sludge have been analyzed by culture -dependent methods, which exclude the majority of endogenous microbes due to the selective nature of the media. Molecular and biochemical techniques have been evaluated, but they are time - consuming, complex and the results are difficult to interpret. Methods such as community level carbon source utilization patterns (i.e. Biolog) are easy to use and detect different patterns, which could be related to diversity and function, in this and other studies. Our aim was not to try and detect each and every metabolic reaction of all the individuals in the community, but the collective pattern for a specific community. Since, 1) a high species diversity should lead to a higher relative number of substrates utilized, because there are more possibilities and 2) upon dilution, some organisms will be lost (causing a decrease in species diversity) from the community, depending on their abundance and the relative contribution (perhaps only one metabolic reaction in the system), reducing the number of possibilities. The extent of the reduction of the possibilities upon dilution, should theoretically reflect something about the community structure. The key, therefore, lies in the interpretation of the results. The Biolog system unlike traditional culture - dependent methods, which are generally selective for the component of the community that has to be cultured, can reflect the activities of a broad range of bacteria. In this study the Biolog system was not considered as a culture - dependent method, but rather as a collection of metabolic tests (database) used for the purpose of generating a recognizable pattern for a specific community. Our hypothesis was that microbial community level carbon source utilization could be used to determine diversity and evenness in activated sludge systems. In our study we used activated sludge systems representative of an environment with a high species diversity and uneven distribution of species, indicated that upon dilution some of the substrates where no longer utilized due to the loss of some of the species. / Dissertation (MSc Agric (Microbiology))--University of Pretoria, 2006. / Microbiology and Plant Pathology / unrestricted

Thesis (D.Tech.: Biotechnology)-Dept. of Biotechnology, Durban University of Technology, 2008. xxiv, 322 leaves / Activated sludge models now in use worldwide for the design and operation of treatment systems use hypothetical concentrations of active organisms. In order to validate and calibrate model outputs, concentrations and activities of organisms responsible for nitrification and denitrification need to be reflected by actual measurements. This research has been initiated by the observation of an increasing gap of suitable techniques that exist in the direct measurement and separation of active biomass components, responsible for COD removal and denitrification.

Thesis (MSc)--University of Stellenbosch, 2005. / ENGLISH ABSTRACT: Aspalathus linearis is a leguminous shrub native to the Cedarberg Mountains in the Western Cape, of which the leaves and stems are used for the preparation of rooibos tea. Over the past few decades, rooibos tea and other related products have gained popularity due to their health promoting properties. These beneficial properties can partly be ascribed to the phenolic constituents that are trapped within the cellulolytic plant material of the tea leaves as glycoconjugated aroma and phenolic compounds. Although many fungal species are known for their efficient hydrolysis of plant material, fungal enzymes have not been evaluated for the bioprocessing of rooibos tea to improve its commercial value. It was the objective of this study to identify a specific cocktail of microbial enzymes to enhance the maceration of the rooibos plant material, while retaining the antioxidant content. During this study, 11 fungal species known for the production of hydrolytic enzymes, as well as 12 species isolated from rooibos tea products, were screened for their potential to improve aroma development and/or increased extraction of soluble matter and/or antioxidants from rooibos tea material. After culturing in Potato Dextrose medium, the crude enzyme extracts of the 23 isolates were evaluated on spent rooibos tea for enhanced extraction of soluble solids (SS) and/or total polyphenols (TP). Nine strains increased the yield in SS (improvement varying from 3% to 42%), while 14 strains yielded higher levels of TP (increase varying from 1% to 36%). Little improvement in colour development from green (unfermented) rooibos tea was observed, but the enzyme extracts from Pleurotus ostreatus var. florida, Lentinula edodes, Aspergillus oryzae, Aspergillus tubingensis, Paecilomyces variotti and Trichoderma reesei improved the aroma development from green tea to some extent. Ten-fold concentrated enzyme extracts from four of these isolates were able to release at least an additional 10% in SS from the green tea. The crude enzyme extracts prepared from three food-grade strains, i.e. Aspergillus oryzae, Lentinula edodes and Pleurotus ostreatus var.florida, contained relatively high levels of endoglucanase, xylanase and pectinase activities. Eight different culture media were evaluated for optimal hydrolase and laecase production by these food-grade fungi. MYPG proved to be the best growth medium, while 1% spent grain, 1% wheat straw and 1% pineapple peel gave the best induction of xylanase, cellulase, pectinase and laecase activities for L. edodes. When cultured in the Yeast Peptone (YP) medium + 1% wheat straw, the L. edodes enzyme cocktail showed the best improvement in both the aroma and colour development of green tea and may be considered for shortening of the fermentation time required for green tea processing. Traditional open-air fermentation of rooibos tea can take up to -1-6hours, which results in a significant loss in antioxidants and therefore also in its pharmaceutical and nutraceutical value. The Rhizopus oryzae cocktail prepared in YP + 1% wheat straw showed potential for the development of a quick-draw fermented tea made by infusion, where there is improved colour release and more than 20% improved extraction of soluble solids without a loss in the TP content. When cultured in Potato Dextrose medium, the L. edodes cocktail can be used for aroma and colour development from green tea, while the R. oryzae cocktail can be used for increasing the antioxidant content in rooibos extracts from green or fermented tea. This was confirmed with small-scale industrial treatments of fermented tea where the L. edodes YP + wheat straw cocktail improved the release in SS by more than 10% and the R. oryzae yP + wheat straw cocktail increased the yield in SS by more than 30% and the TP by more than 20%. / AFRIKAANSE OPSOMMING: Aspalathus linearis is 'n fynbosplant inheems aan die Sederberge in die Wes-Kaap, waarvan die blare en stingels vir die voorbereiding van rooibostee gebruik word. Die afgelope paar dekades het die gewildheid van rooibostee en verwante produkte aansienlik toegeneem weens die gesondheidsvoordele wat dit inhou. Hierdie voordelige eienskappe kan toegeskryf word aan die fenoliese komponente wat binne die sellulolitiese plantweefsel van die teeblare as gekonjugeerde geur- en fenoliese verbindings vasgevang is. Alhoewel verskeie swamspesies vir hul doeltreffende degradering van plantmateriaal bekend is, is fungale ensieme nog nie geëvalueer vir die prosessering van rooibostee om die kommersiële waarde daarvan te verbeter nie. Die doelwit van hierdie studie was om 'n spesifieke kombinasie van mikrobiese hidrolitiese ensieme te identifiseer wat die maserasie van rooibos plantmateriaal sal verhoog met behoud van die anti-oksidant inhoud. Tydens hierdie studie is 11 swamspesies wat bekend is vir die produksie van hidrolitiese ensieme, asook 12 swamspecies wat vanaf rooibostee produkte geïsoleer is, geëvalueer vir hul potensiaalom geurontwikkeling en/of ekstraksie van oplosbare stowwe en/of anti-oksidante vanuit rooibostee materiaal te verbeter. Die kru ensiemekstrakte van die 23 isolate, wat ID Aartappel-Dextrose medium opgegroei is, is op oorskot rooibostee geëvalueer vir verhoogde ekstraksie van oplosbare vastestowwe (SS) en/of totale polifenole (TP). Nege rasse het die opbrengs van oplosbare vastestowwe verhoog (verbetering tussen 3% en 42%), terwyl 14 rasse die totale polifenoliese vlakke laat toeneem het (tot so hoog as 36%). Baie min verbetering in kleurontwikkeling van groen (ongefermenteerde) rooibostee is waargeneem, maar ensiemekstrakte van Pleurotus ostreatus var. florida, Lentinula edodes, Aspergillus oryzae, Aspergillus tubingensis, Paecilomyces variotti en Trichoderma reesei, het wel die aroma ontwikkeling vanaf groen tee tot 'n mate verbeter. Tienvoudig gekonsentereerde ekstrakte van vier van hierdie isolate het 'n verbetering van meer as 10% in die ekstraksie van opgeloste vastestowwe uit groen tee tot gevolg gehad. Die ensiemekstrakte van drie swarnme bekend vir hul gebruik in die voedselindustrie, nl. A. oryzae, L. edodes and P. ostreatus var. florida, het relatief hoë vlakke van endoglukanase, xylanase en pektinase aktiwiteit getoon. Agt verskillende kultuur-media is vir die optimale produksie van hidrolitiese and lakkase ensieme vanaf hierdie voedsel-graad swarnme geëvalueer. MYPG was die beste groeimedium vir L. edodes, -terwyl 1% koringstrooi, 1% oorskot graan en 1% pynappelskil die beste induksie van xylanase, pektinase, endoglukanase en lakkase aktiwiteite vir hierdie organisme getoon het. Lentinula edodes opgegroei in YP medium + 1% koringstrooi, het die beste verbetering in aroma en kleur getoon vanaf groen tee getoon. Hierdie ekstrak kan dus moontlik gebruik word vir die verkorting van die fermentasietyd wat vir groen tee benodig word. Ope-lug fermentasie van groen tee duur gewoonlik tot 16 uur en lei tot 'n aansienlike verlies in antioksidant-inhoud. Die R. oryzae ekstrak het die beste potensiaal vir die vervaardiging van 'n "quick-draw" tee getoon met 'n goeie kleurvrystelling sonder enige verlies in SS en TP opbrengs. Wanneer die swamme in Aartappel-Dextrose medium opggegroei word, kan die L. edodes ensiemekstrak vir aroma en kleurontwikkeling van groen tee aangewend word, terwyl die R. oryzae ensiemekstrak vir die verhoging van die antioksidant-inhoud in rooibos ekstrakte van groen tee of gefermenteerde tee gebruik kan word. Dit is bevestig met die kleinskaalse behandeling van gefermenteerde tee waar die L. edodes YP + 1% koringstrooi ensiemekstrak die vrystelling van SS met meer as 30% en die TP met meer as 20% verbeter het.