Thesis (MSc)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: Scientists and biochemical engineers alike are very interested in the control and regulation of free-energy metabolism in micro-organisms, whether the findings purely satisfy scientific curiosity or translate into the meeting of biotechnology company deadlines. We used a rather fundamental approach to investigate experimentally the control and regulation of yeast free-energy metabolism in anaerobic chemostat cultures using supply-demand analysis. This conceptually simple, quantitative framework, however, may lead to insight into the control properties of various metabolic pathways to be used in biotechnological applications. Supply-demand analysis is based on the theoretical framework of metabolic control analysis (MCA).Sections (of arbitrary size) of a metabolic pathway are grouped together around a linking metabolite. Those steps that produce the intermediate are combined into the supply block while the reactions that remove/consume the intermediate are grouped together as the demand. The elasticity coefficients of the supply and demand blocks (with regard to the linking metabolite concentration) can be used to determine the flux and concentration control coefficients by using the traditional MCAsummation and connectivity theorems. Supply and demand rate characteristics are a powerful visual approach to determine and display the control structure of the pathway under consideration and sets supply-demand analysis apart from traditional top-down analysis. Our first tool of analysis was a structured kinetic model of yeast growing in a chemos tat, constructed by using methods developed in our research group for modelling systems with variable volumes. Independent perturbations of the linking metabolite concentration resulted in a control profile where the control resided mainly in the demand (flux control coefficient of 0.92), as a result of a large negative supply elasticity. This elasticity, however, varied greatly under different conditions, leading to increased flux control by the supply in some cases. We extended our research to an experimental setup of Saccharomyces cerevisiae growing in a glucose-limited chemos tat supplemented with yeast extract as a source of carbon intermediates. This allowed glucose to act solely as the freeenergy source, as confirmed by balancing the glucose flux with the fluxes towards the fermentation products, ethanol and carbon dioxide. We obtained the supply rate characteristic by perturbing the ATPdemand through the addition of benzoate, which uncouples the proton gradient across the cell membrane. The demand rate characteristic was obtained by perturbing the ATP supply through changes in the dilution rate and thus the residual glucose concentration in the fermentor. The concentrations of ATPand ADPwere measured using a luciferase bioluminescence assay, while the fermentation products were measured with HPLCand C02 with an acoustic off-gas analyser. For our experimental conditions the flux-control of energy metabolism resided predominantly in the supply with respect to the linking metabolite [ATP]/[ADP](chosen as an indication of the free-energy state of the cell), i.e. a flux control coefficient of 0.90. Further, the [ATP]/[ADP]was under strong homeostatic control, as evidenced by the low [ATP]/[ADP]control coefficients of ± 0.12. We adjusted the structured kinetic model by varying strategic parameters, so that the results resembled the experimental observations more closely. However, the kinetics of our core model seem to be too simplistic to capture fully the extent of regulation displayed by the experimental system. The model did, however, reveal the regulatory importance of glucose transport into the cell. We conclude that the control and regulation of free energy metabolism in yeast strongly depend on the culturing conditions and on the steady state being analysed. / AFRIKAANSE OPSOMMING: Wetenskaplikes sowel as biochemiese ingenieurs is dikwels geïnteresseerd in die beheer en regulering van vry-energie metabolisme in mikro-organismes, hetsy die bevindinge suiwer wetenskaplike nuuskierigheid bevredig of die haalbaarheid van biotegnologie-maatskappy-mikpunte beteken. Ons het 'n redelik fundamentele benadering gevolg om die beheer en regulering van vry-energie metabolisme in gis eksperimenteel te bepaal in anaerobiese chemostaatkulture met behulp van aanbod- aanvraag analise. Dit is 'n konseptueel eenvoudige, kwantitatiewe raamwerk met die potensiaal om insig te gee in die beheereienskappe van verskeie metaboliese paaie wat nuttig kan wees in biotegnologiese toepassings. Aanbod-aanvraag analise is gebaseer op die teoretiese onderbou van metaboliese kontrole-analise (MKA).Dele (van arbitrêre grootte) van 'n metaboliese pad word gegroepeer rondom 'n verbindingsmetaboliet. Die stappe wat die intermediaat produseer word gekombineer as die aanbod terwyl die reaksies wat die intermediaat verbruik, saamgegroepeer word as die aanvraag. Die elastisiteitskoëffisiënte van die aanbod en aanvraag blokke (met betrekking tot die verbindingsmetabolietkonsentrasie) kan gebruik word om die fluksie en konsentrasie kontrolekoëffisiënte te bereken met behulp van die sommasie en konnektwiteit teoremas van MKA.Aanbod en aanvraag snelheidskenmerkgrafieke is 'n treffende visuele benadering om die kontroleprofiel van die betrokke metaboliese pad te bepaal en te vertoon. Hierdie kenmerk onderskei aanbod-aanvraag analise van bo-na-onder analise. Die eerste deel van ons ondersoek het behels 'n gestruktureerde kinetiese model (van gis wat groei in 'n chemostaat) met behulp van metodes wat in ons groep ontwikkel is om sisteme met variërende volumes te modelleer. Onafhanklike perturbasies van die verbindingsmetaboliet konsentrasie het gelei tot 'n kontroleprofiel waar die kontrole hoofsaaklik in die aanvraag gesetel was (fluksie kontrolekoëffisiënt van 0.92), as gevolg van 'n groot negatiewe aanbod-elastisiteit. Hierdie elastisiteit kan egter grootliks varieer tydens verskillende kondisies, wat lei tot 'n toenemende fluksle-beheer deur die aanbod in sommige gevalle. Ons het ons navorsing uitgebrei na 'n eksperimentele opstelling van Saccharomyces cerevisiae wat groei in 'n glukose-gelimiteerde chemostaat, aangevul met gisekstrak as 'n bron van koolstof-Intermediate. Dit bring mee dat glukose slegs as energiebron dien; dit is wel bevestig deur balanse op te stel van die koolstoffluksie vanaf glukose na koolstofdioksied en etanol as die fermentasieprodukte. Die aanbod snelheidskenmerkgrafiek is gegenereer deur die aanvraag van ATP te manipuleer deur middel van toevoeging van bensoaat, wat die protongradiënt oor die selmembraan ontkoppel. Die snelheidskenmerkgrafiek Vir die aanvraag is gegenereer deur die aanbod van ATP te manipuleer deur middel van 'n variasie in die verdunningstempo en sodoende die residuele glukose konsentrasie in die fermentor. Die konsentrasies van ATPen ADPis bepaal deur middel van 'n lusiferase bioluminessensie-essai, terwyl die fermentasieprodukte met 'n HPLCen CO2 met 'n akoestiese aflaatgasanaliseerder gemeet is. Vir die betrokke eksperimentele toestande was die flukste-kontrole van energiemetabolisme oorwegend in die aanbod met betrekking tot die verbindingsmetaboliet, [ATP]/[ADP](gekies as aanduiding van die vrye-energiestatus van die sel), naamlik 'n fluksie kontrolekoëffisiënt van 0.90. Verder was die [ATP]/[ADP]onder sterk homeostatiese beheer soos duidelik blyk uit die lae [ATP]j[ADP] kontrolekoëffisiënte van ± 0.12. Ons het die gestruktureerde kinetiese model aangepas deur strategiese parameters te verander om sodoende die eksperimentele gedrag te probeer naboots. Die kinetika van ons kernmodel blyk egter te simplisties te wees om die volle omvang van die regulering van die eksperimentele sisteem te vertoon. Die model het egter die belang van glukose transport oor die selmembraan aan die lig gebring. Ons kom tot die gevolgtrekking dat die beheer en regulering van vrye-energie metabolisme in gis sterk afhang van die groeitoestande sowel as die spesifieke bestendige toestand wat ondersoek word.

This study was carried out to gain knowledge of the degradation processes in an anaerobic environment of pre-treated waste for different degrees of pre-treatment and the evolution of waste pre-treatment by forced aeration. Pre-sorted MSW (MSW) was pretreated by composting for 16 weeks in a laboratory scale using forced aeration. Oxygen concentrations were maintained at 15-18% of oxygen in air for the first 8 weeks and 10-15 % for the later 8 weeks. The ambient temperature was kept constantly between of 20-35 QC. Representative samples of waste from the reactor were collected every fortnight wherein analysis and full characterisation on the solid matter (C/N ratio, TS and VS, R17, Biogas) and on the eluate (BOO, COD, TOC, TKN, Conductivity, pH, NOx and NH3)) were conducted. The process showed a sharp increase in temperature in the first 6 weeks, ranging from 30- 70 QC indicating a period of high biological activity, a decrease from day 30 to day 50 from 70 to 30 QC and a consistent decrease throughout the later days of the process from 35-25 QC. The sharp increase in temperature signifies a period of maximum biological activity, where readily biodegradable material decomposes as well as some of the resistant materials pointing out the success and efficiency of the forced aeration process. For the first 25 days in an anaerobic environment, waste pre-treated for four weeks was the most active, indicated by a large volume of gas produced. For the MSW pre-treated for 8, 10, 12 and 16 the volume of gas produced remained basically similar throughout the length of the experiments. CH4 production in an anaerobic reactor shows an increasing trend for all degrees of stabilisation up to 6 weeks, after that the gas production and quality deceases and is comparable to the remaining degrees of treatment. A gradual decrease in concentration of key parameters (organics) analogous to the European limit in Europa (1998), were observed after 5 weeks pre-treatment. The study highlighted that the highest efficiency of pre-treatment is achieved in 6 to 8 weeks and, therefore it is not recommended to prolong the treatment any further. / Thesis (M.Sc.Eng.)-University of KwaZulu-Natal, Durban, 2007.

Graduation date: 2006

No description available.

There is potential for the anaerobic baffled reactor (ABR) to be implemented on-site for pre-treatment of coloured wastewaters. The implementation of waste minimisation and cleaner production strategies in industry will result in the production of smaller volumes of concentrated wastewaters. With implementation of the ABR, the concentrated waste stream could be pre-treated, with an acclimated biomass, which should facilitate sufficient degradation such that the effluent could be discharged to sewer for further treatment. The ABR is a high-rate compartmentalised anaerobic bioreactor, the design of which promotes the spatial separation of microorganisms. The use of molecular teclmiques to characterise the microbial populations and the dynamics of these populations with time and/or changing operating conditions will add to the current understanding of the process, which is based on the biochemical pathways and chemical analyses. This knowledge will allow for optimisation of the design of the ABR. The hypothesis of the horizontal separation of acidogenesis and methanogenesis through the ABR was proven. Changes in the HRT affected the operation of the reactor, however, recovery from these upsets was almost immediate and operation of the reactor was stable. Two synthetic dye waste streams, one food dye (tartrazine) and one textile dye (Cl Reactive Red 141), and a real industrial dye wastewater, were treated in separate laboratory-scale ABRs. These investigations showed that successful treatment of a highly coloured wastewater is possible in the ABR. The design of the ABR facilitates efficient treatment of concentrated dye wastewaters by protecting the sensitive methanogens from the inhibitory dye molecules and promoting efficient colour and COD reduction. The molecular-based method, fluorescent in situ hybridisation, allowed the direct identification and enumeration of microbial populations active in the ABR. In all of the reported investigations, there was a definite shift in the microbial populations through the ABR, with a predominance of eubacteria in the first compartments (acidogenesis) and archaea (methanogenesis) in the later compartments. The number of compartments involved in each depended on the strength of the substrate (organic loading rate - OLR). A combination of FISH probing, and the analysis of 98 archaeal l6S rDNA clone inserts provided useful descriptions of the methanogens actively involved within each compartment. These showed a predominance of the Methanosaeta spp., particularly in the last compartments of the reactor. Methanogens present in the first four compartments consisted of species of Methanobacterium and Methanospirillum, a relatively unstudied methanogen Methanomethylovorans hollandica, and an unidentified short filamentous species. / Thesis (Ph.D.)-University of Natal, Durban, 2002.

Microorganisms have the potential to remove heavy metals from polluted waters and effluents and may be used in clean-up processes. Microbial associations were enriched for and adapted to grow in nutrient solutions containing various concentrations of different metals. As immobilised cells are known to be more stable and more efficient in metal uptake than are corresponding planktonic or free-living cells the attachment of the microbial associations was investigated using a model stream and it was found that biofilm development was better on rough surfaces such as ground glass and polystyrene than on smooth surfaces such as unetched glass plates and glass beads. When comparing metal uptake by planktonic and attached microorganisms, attached populations were found to have a greater metal-uptake capacity. The uptake of individual metals from various metal combinations was tested with various proportions of pregrown metal-adapted microbial populations as inoculum and it was found that a particular metal was taken up more readily by microbial associations which had previously been exposed to that metal. Lead (Pb2+) appeared to be taken up more readily than copper (Cu2+) or cadmium (Cd2+) while Cd2+ was more actively removed than Cu2+ from solution. pH also affected metal uptake and the optimum range for Cu2+ uptake by the Cu2+ -adapted microbial association was found to be between 5.8 and 7.0. Dead microbial biomass was investigated and found to have efficient metal uptake capacity. Living mycelium from an isolated Aspergillus species showed poor uptake of Cu2+ initially, but when this fungus was pregrown and subsequently killed by moist heat treatment the non-living mycelium was efficient in removal of Pb2+ and Cu2+ ions. The optimum mycelial biomass concentration for metal uptake was also determined. The mechanism of metal uptake by this Aspergillus species was determined, using electron microscopy and EDX techniques, to be metabolism-independent biosorption onto the hyphal surface. Thus the microbial associations and fungal cultures used in this study were shown to have the potential for use in the removal of heavy metals from polluted waters. / Thesis (Ph.D.)-University of Natal, Pietermartizburg, 1995.

With the exceptions of mixing and heating mechanisms, and the recycling of settled solids, no radical changes or improvements have been made to conventional anaerobic digesters treating municipal sewage. These digesters usually function with a hydraulic retention time of 30 to 60 days and at a total solids concentration of 2.6 %(m/v). Volumetric loading is limited since high loadings effect the displacement of the slow growing methanogens. Thus, the hydraulic retention time is coupled to the solids retention time. A crossflow microfiltration unit has been constructed at Northern Waste Water Treatment Works, Durban, to concentrate sludge from a conventional anaerobic digester and, thus, facilitate operation with a higher solids concentration. In addition, this process should result in the retention of the active biomass which would otherwise be lost as a waste product of the treatment process. The solids retention time is, thus, decoupled from the hydraulic retention time. The net result could be higher volumetric loadings, increased microbial activity and increased volatile solids destruction and, hence, improvement in the efficiency of anaerobic digestion of sewage sludge. To test these, different experiments were conducted to specifically determine the effect of higher solids loads. Preliminary experiments were undertaken to determine the biodegradability of primary sludge from the Northern Waste Water Treatment Works. Results showed that primary sludge of 76% VS could be reduced to approximately 48 to 50% VS during an experimental period of 85 days. Reduction of the first 20% VS was rapid if conditions were optimum but subsequent reduction from 55 to 50% VS was slow. It was calculated that approximately 0.88 l gas was produced for every g volatile solids catabolised. Further experiments were conducted to investigate the effects of different solids concentrations on microbial activity. The results showed that the volume of gas produced increased as the solids concentration increased from 2 to 6%(m/v). Digesters with solids concentrations of 6 to 13%(m/v) produced similar volumes of gas. Digesters with solids concentrations of 6 to 13%(m/v) TS produced approximately 300 ml more gas than the control during the 20 days experimental period. The rate of gas production also increased as the solids concentration increased. However, digesters containing 11%(m/v) and 13%(m/v) TS produced similar rates. These results indicate that the introduction of concentrated sludge into the digester improves digestion efficiency. Finally, a semi-continuous digester was operated at a 30 days retention time and at optimum temperature to investigate the efficacy of digesters with increased solids concentrations. The results showed that the rate of gas production increased as the solids concentration increased from 2%(m/v)(control) to 3.8%(m/v). However, the digester operated with 4.7%(m/v) TS produced gas at a rate lower that the digester with 3.8%(m/v) TS. The volatile solids concentrations of all four digesters were similar, indicating neither favourable nor unfavourable effects from increased solids concentrations. The digesters operated with 3.8%(m/v) and 4.7%(m/v) TS produced higher concentrations of volatile acids than the control. The alkalinity concentrations (>_4000 mg t-1 ) were similar for all four digesters. / Thesis (M.Sc.)-University of Natal, Durban, 1995.

Fischer-Tropsch Reaction Water (FTRW) is a high organic strength wastewater produced as a by-product in Sasol’s Fischer-Tropsch Reactors. Typically it has an organic load of 18000 mgCOD/L and is highly acidic with a pH of approximately 3.8. It is deficient in nutrients (N and P and other micronutrients). This dissertation deals with the biological and physico-chemical model development of a dynamic anaerobic digestion model, and explores two different approaches to representing the physico-chemical processes that complement and interact with the bioprocesses. The performances of the resultant two dynamic models (ADFTRW1 & AD-FTRW2) were compared in order to assess to what extent the more detailed and rigorous ionic speciation modeling in AD-FTRW2 addressed the shortcomings attributed to the simplified physicochemical modeling in AD-FTRW1. The ionic speciation model used in AD-FTRW2 uses a classic equilibrium formulation along the same lines as in the UCTADM2 model for anaerobic digestion of municipal wastewater sludges (Brouckaert et al., 2010), while AD-FTRW1 uses a simplification of the approach developed by Musvoto et al. (2000) in order to represent short chain fatty acid (SCFA) dissociation and the weak acid base chemistry of the inorganic carbon system. A 44 day extract from a 700 day laboratory-scale dataset (Van Zyl et al. 2008) was used as the basis for comparing the models. During this period the membrane bio-reactor was subjected to varying flow and load conditions. To validate the models, the experimentally measured and model predicted process variables of reactor alkalinity, reactor pH, biogas production and effluent SCFA concentration were compared. It was found that AD-FTRW2 provided superior agreement with pH data, but predictions of alkalinity, gas production rate and effluent short-chain fatty acids were not significantly improved in AD-FTRW2 relative to AD-FTRW1. This outcome was hypothesized since pH is strongly dependent on physico-chemical processes such as ionic interactions in solution and gas exchange which were the components to the models (AD-FTRW1 versus AD-FTRW2) which differed most significantly. Alkalinity, which is also highly influenced by physico-chemical model representations showed substantial improvement however statistical analysis could not show this improvement to be significant. The other two variables that were compared, biogas production and effluent SCFA concentration, displayed very similar agreement with experimental data. These variables depend more on mass balance effects and biological kinetics and were therefore not significantly altered by the more rigorous handling of aqueous chemistry in AD-FTRW2. It was concluded that AD-FTRW2 constitutes an improvement in model predictive power over AD-FTRW1 at a small cost in computing time. / Thesis (M.Sc.Eng.)-University of KwaZulu-Natal, Durban, 2013.

Thesis (M.S.)--University of Nevada, Reno, 2006. / "August, 2006." Includes bibliographical references (leaves xx-xx). Online version available on the World Wide Web.

Thesis (Ph.D.) - Carleton University, 2006. / Includes bibliographical references (p. 233-242). Also available in electronic format on the Internet.