"April 2002" / Includes bibliographical references (leaves 193-211) / xi, 211 leaves : ill., plates ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Plant Science, 2002

Glucoamylase, a saccharifying enzyme, is applied in the brewing industry to hydrolyse the dextrins of malted barley into simple sugars which can then be fermented by brewer's yeast. In order to establish the potential of glucoamylase from Lactobacillus amylovorus for application in the brewing industry, the main objectives of this study were: (1) to determine the cultural conditions for growth and glucoamylase production, (2) to purify the enzyme to homogeneity using chromatography and electrophoretic techniques, (3) to study biochemical properties of the purified enzyme, and (4) to clone the gene coding for glucoamylase, and characterize the recombinant glucoamylase. / The actively amylolytic Lactobacillus amylovorus ATCC 33621 produced an intracellular glucoamylase activity. Conditions for growth and glucoamylase production were maximized by using dextrose free MRS medium supplemented with 1% dextrin, at pH 5.5 and 37$ sp circ$C. Enzyme production was maximal during the late logarithmic phase of growth from 16-18 h. Crude cell extract showed optimal activity at pH 6.0 and 55$ sp circ$C. / Native and SDS-PAGE of the purified enzyme showed a monomeric protein of 47 kD. Glucoamylase activity was confirmed by activity staining using a starch/polyacrylamide gel where a zone of clearing was visible on a blue/black background stained with Kl/I$ sb2.$ Optimal pH, pl and temperature of purified glucoamylase were 4.5, 4.39 and 45$ sp circ$C, respectively. The enzyme was rapidly inactivated by temperatures above 55$ sp circ$C and was inhibited by heavy metals, e.g. Pb$ sp{2+}$ and Cu$ sp{2+}$ at 1.0 mM. EDTA did not inhibit the enzyme activity at a final concentration of 10 mM. Enzyme inhibition by 1 mM of p-chloromercuribenzoic acid (pCMB) and iodoacetate suggested that a sulfhydryl group was present in the enzyme active site. Kinetic studies of glucoamylase confirmed that the enzyme reacted preferentially with polysaccharides. HPLC analyses of the end products of enzyme action showed that glucose was the major end product of enzyme action and this glucose was responsible for end product inhibition. / The gene coding for glucoamylase was cloned into Escherichia coli using the STA2 glucoamylase gene of Saccharomyces diastaticus as a probe. Three glucoamylase producing transformants were identified as the insert sizes of about 5.2 Kb, 6.4 Kb and 5.9 Kb, respectively. When the characteristics of both recombinant and purified wild type glucoamylases were compared, both enzymes showed a similar pH range of 3.0-8.0, and temperature optimum of 45$ sp circ$C. The recombinant enzyme pH profiles were broader than that of the wild type and an optimum pH of 6.0 was obtained. This study has shown that glucoamylase from Lb. amylovorus is less heat stable than other bacterial glucoamylases and thus may be suitable for application in the brewing industry. Successful cloning of this gene coding for glucoamylase in brewer's yeast, Saccharomyces cerevisiae, would reap the advantageous properties of the enzyme while eliminating the costs of adding commercial enzymes.

The brewing industry constantly experiences problems in trying to maintain the quality of beer produced. Unfavourable conditions during fermentation may alter the performance of the yeast strain Saccharomyces cerevisiae, resulting in a "poor" end-product. It has been established that high concentrations of extracellular ethanol, when added to the fermentation medium inhibit yeast activity. It has been recently suggested that increased carbon dioxide pressure could inactivate the yeast activity adding to further brewing problems. The aim of this study was to investigate the effect of extracellular carbon dioxide pressure and ethanol addition, on yeast performance when added to a fermentation medium, and to establish whether an inhibitory relationship existed between ethanol and carbon dioxide pressure, when combined and added to the fermentation medium. Dissolved C0₂ in the medium, medium pH and substrate utilisation were analysed daily during a fermentation, as were membrane fatty acid composition. These parameters were used to assess the effect of ethanol and carbon dioxide on the yeast performance and consequently the final end-product. Supplementing the medium with extracellular ethanol, even as low as 5%, was shown to inhibit yeast performance during fermentation. This effect was even more marked as the ethanol concentration was increased, with almost total inhibition of yeast activity occuring after the addition of 15% ethanol (v/v). A similar effect was observed when elevated C0₂ pressures were applied to the medium, and although low C0₂ pressures initially induced the synthesis of saturated yeast membrane fatty acids, elevated C0₂ pressures (greater than 1,0 atm.) was shown to follow a similar inhibitory trend, if not as dramatic, as ethanol. A combination of both ethanol and C0₂ pressure showed a further increase in the level of yeast inhibition, although the low C0₂ pressure appeared to initially inhibit the toxicity of ethanol on the yeast. Increasing the levels of the C0₂/ethanol treatment (1,0 atm.), showed a synergistic effect on yeast performance. The results of this study indicate that both extracellular ethanol and carbon dioxide do appear to inhibit yeast performance and affect membrane fatty acid composition of the cells by inhibiting the synthesis of the respective fatty acid. This affect has a significant bearing on the general metabolism of the yeast cell.

No description available.

"May 2003" / Includes bibliographical references (leaves 216-225) / 225 leaves : ill., plates (col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, School of Agriculture and Wine, 2003

"May 2003" / Includes bibliographical references (leaves 216-225) / 225 leaves : ill., plates (col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, School of Agriculture and Wine, 2003

Thesis (MSc)--University of Stellenbosch, 2005. / ENGLISH ABSTRACT: In the brewing industry yeast cells are re-used in successive fermentations. Consequently, the state of the cells at the end of each successive fermentation could impact on the quality of the subsequent fermentations. The use of markers to evaluate the fermentative ability of yeast to resist stress enables brewers to select populations of yeast for brewing. Yeasts are typically exposed to osmotic-, ethanol- and cold-stress during the high-gravity brewing process. In this study the vitality of the yeast cells was monitored during and after each successive high-gravity brewing fermentation. This was done by measuring the cell metabolites, which included glycerol, trehalose and glycogen. Others markers that were evaluated for yeast viability were the number of budding scars, the levels of activity of the enzymes neutral trehalase and esterase and the expression level of the heat shock protein Hsp12p. Coupled to these evaluations, the growth of the yeast and the utilisation of the sugars glucose, fructose, maltose and maltotriose were monitored during the fermentations. The experiments were conducted in 2-litre E.B.C. tubes at either 14 oC or at 18oC using standard techniques. Comparable growth patterns were obtained for different re-pitching fermentations, with fermentation 1 at 18ºC and 5 and 6 at 14°C being the most active fermentations. The higher temperature encouraged more rapid growth and a greater numbers of cells. The wort attenuation was more rapid at 18°C than at 14°C. Glucose and fructose in wort were utilised prior to maltose and maltotriose. At 18°C the yeast consumed the sugars faster, with mean utilisation values of 97.3% glucose, 100% fructose, 59.9% maltose and 65.6% maltotriose. At the lower temperature of 14°C high concentrations of residual sugars remained at the end of the fermentation. All re-pitching fermentations revealed lower viabilities at 18°C in comparison to the 14°C fermentations. Simultaneously, a number of other markers were evaluated. The intracellular trehalose concentration per cell varied considerably with each fermentation. Trehalose levels at 18°C gradually increased in concentration from 48h until the end of the stationary phase. Much lower trehalose concentrations were observed in fermentations conducted at 14°C. Higher and more consistent glycerol concentrations were found in fermentations at 14°C with mean concentrations of 12 mg/g dry weight at pitching. The expression of the heat shock protein Hsp12p level increased during the fermentation but no sharp increase was detected in any particular fermentation. No increase in yeast budding scar number was observed during re-pitching fermentations. Neutral trehalase and esterase activities in fermentations at 18°C were especially high at pitching. Neutral trehalase activities at 14°C were all generally lower than in the case of fermentations at 18°C. The fermentation ability of flocculated yeast in slurry and yeast suspended in beer was investigated after exposure to various stresses. The aged yeast present in the slurry was generally found to be more resistant to stress, in particularly to osmotic stress, throughout the serial re-pitching process. The fermentation rates of both yeast types were especially sensitive to prior exposure to ethanol stress. / AFRIKAANSE OPSOMMING: In die broubedryf word gisselle herhaaldelik gebruik vir agtereenvolgende fermentasies. Derhalwe kan die toestand van die gisselle teen die einde van elke agtereenvolgende fermentasie ‘n invloed hê op die kwaliteit van die daaropvolgende fermentasies. Deur gebruik te maak van merkers om die fermentasievermoë van gis om stres te weerstaan te evalueer, stel dit bierbrouers in staat om gispopulasies te selekteer. Gedurende die hoëdigtheid brouproses word giste tipies aan osmotiese-, etanol- en koue-stres blootgestel. In hierdie studie, gedurende hoë-digtheid fermentasies, is die lewensvatbaarheid van die gisselle gedurende en na elke agtereenvolgende fermentasie gemonitor deur die volgende selmetaboliete te bepaal: gliserol, trehalose en glikogeen. Bykomende merkers vir gis lewensvatbaarheidsbepalings was: die aantal botselletsels, die vlakke van aktiwiteit van die neutrale trehalose en esterase ensieme, en die uitdrukkingsvlak van die hitteskokprotein Hsp12p. As aanvullende evaluasies is die groei van die gis en die gebruik van die suikers glukose, fruktose, maltose en maltotriose gedurende fermentasies gemonitor:. Die proewe is in 2-liter E.B.C. buise uitgevoer, by ‘n temperatuur van 14oC of 18oC, deur van standaard tegnieke gebruik te maak. Die groeipatrone van die verskillende herhaaldelike-inokulasie gistings was ongeveer dieselfde. Fermentasie 1 by 18ºC en fermentasies 5 en 6 by 14°C was die mees aktiewe fermentasies. Die hoër temperatuur het vinniger groei en ‘n groter aantal selle begunstig. Die wortattenuasie was vinniger by 18°C as by 14°C. Glukose en fruktose in mout is voor die maltose and maltotriose opgebruik. By 18°C het die gis die suikers vinniger opgebruik. Gemiddelde gebruikswaardes vir die sewe reeksgewyse fermentasies was die volgende: 97.3% glukose, 100% fruktose, 59.9% maltose en 65.6% maltotriose. Teen die einde van fermentasie by 14°C was daar hoë konsentrasies van die oorblywende suikers, hoofsaaklik na fermentasie 1. Alle herhaaldelike inokulasie fermentasies het lae lewensvatbaarheid by 18°C in vergelyking met 14°C fermentasies getoon. Ander merkers is ook gelyktydig gebruik. In die verskillende fermentasies was daar ‘n groot verskil in die intrasellulêre trehalose konsentrasie per sel. Trehalose konsentrasies by 18°C het geleidelik toegeneem, vanaf 48 uur tot aan die einde van die stationêre fase. Baie laer trehalose konsentrasies is gemeet vir fermentasies by 14°C. In fermentasies by 14°C was die gliserolkonsentrasies hoër en meer konstant. Gemiddelde konsentrasies was 12mg/g 14°droë gewig by inokulasie. Die uitdrukking van die hitteskokproteien Hsp12p vlak het gedurende fermentasie toegeneem, maar daar was geen skerp toename vir die afsonderlike fermentasies nie. Die bepaling van die aantal botselletsels per sel het daarop gewys dat die gemiddelde aantal nie toegeneem het met die veroudering van die gis gedurende reeksgewyse herhaaldelike inokulasie nie. Neutrale trehalase aktiwiteite in fermentasies by 18°C was besonders hoog, veral by inokulasie. Die neutrale trehalase aktiwiteite in die fermentasies by 14°C was in die algemeen laer as die by 18°C. Die fermentasievermoë van die geflokkuleerde gis in die sediment en gesuspendeerde gis in die bier is ondersoek na blootstelling aan verskeie tipes stres. Die verouderde gis teenwoordig in die sediment was in die algemeen meer bestand teen stres, veral aan osmotiese stres, dwarsdeur die reeksgewyse herhaaldelike inokulasie proses. Etanolstres het die gistingstempo van beide giste dieselfde geaffekteer.

Thesis (PhD)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: This study describes: · Development of analytical tools to investigate the light struck flavour (LSF) in beer by Gas chromatography mass spectrometry (GCMS) and by liquid chromatography mass spectrometry/mass spectrometry (LCMS/MS). Development of a high performance liquid chromatography (HPLC) method to analyse carbohydrates in beer. · The efficiency a pilot scale ultraviolet (UV-C) system at 254 nm to inactivate spoilage microorganisms spiked in commercial beer. Bacteria test were Lactobacillus brevis, Acetobacter pasteurianus and Saccharomyces cerevisiae · A pilot scale UV treatment of commercial and non-commercial lager beers at UV dosage of 1000 J/L. Following the UV treatment, the correlation between chemical analyses and sensory tests conducted by consumers’ tasters were investigated. · A pilot scale UV treatment of non-commercial beer brewed with reduced hops iso-α-acids (tetrahydro-iso-α-acids) at UV dosage of 1000 J/L. Sensory changes and chemical properties were investigated. · The development and optimisation of an UV light emitting diodes (UV-LED) bench scale apparatus. Chemical and microbiological tests were conducted to investigate the effect of UV-LEDs on beer at 250 nm and 275 nm wavelengths. / AFRIKAANSE OPSOMMING: Hierdie studie beskryf: · Die ontwikkeling van analitiese toerusting om die invloed van lig op die smaakontwikkeling in bier te bestudeer m.b.v gaschromatografie massa spektrometrie (GCMS) en vloeistofchromatografie massa spektrometrie/massa spektrometrie, asook die ontwikkeling van ‘n hoë druk vloeistofchromatografiese metode vir die analise van koolhidrate in bier. · Die doeltreffendheid van ‘n toetsskaal ultraviolet (UV-C) sisteem om die nadelige mikroorganismes waarmee die bier geïnnokuleer was, by 254 nm te inaktiveer.. Toetse is uitgevoer met die volgende bakterieë, Lactobacillus brevis, Acetobacter pasteuriants en Saccharomyces cerevisiae. · ‘n Toetsskaal UV behandeling van kommersiële en nie-kommersiële lager biere by ‘n UV dosering van 1000 J/L. Na UV behandeling is die verwantskap tussen chemiese analises en ‘n reeks sensoriese toetse deur vebruikers proeërs ondersoek.. · ‘n Toetsskaal UV behandeling van ‘n nie-kommersiële bier gebrou met verlaagde hops-iso-α-sure (tetrahidro-iso-α -sure) by UV dosering van 1000 J/L. Sensoriese veranderinge asook chemiese eienskappe is ondersoek. · Die ontwikkeling en optimalisering van ‘n UV-lig emissie diodes bankskaal apparaat. Chemiese en mikrobiologiese toetse is uitgevoer om die effek van UV lig op bier by 250 nm en 275 nm te ondersoek.

Thesis (MScAgric.)--University of Stellenbosch, 2001. / ENGLISH ABSTRACT: Changes in the bacterial population throughout the malting process of two barley cultivars, i.e. Clipper (local cultivar) and Prisma (imported cultivar), malted at Southern Associated Maltsters (SAM), Caledon, South Africa, were studied. Samples were taken from four individual runs of each cultivar at ten different stages, i.e. dry barley before steep, water from the first steep water-stand, barley after draining the first steep, water from the second steep water-stand, barley from the second steep water-stand, barley after draining of the second steep, barley from the first, second and third days of germination in the germination vessels (GV), and malt after kilning. Emphasis was placed on the taxonomy and composition of the lactic acid bacteria (LAB) isolated from the ten different phases. The LAB were identified to species level by using numerical analysis of total soluble cell protein patterns, RAPD-PCR banding patterns and 16S rRNA sequencing. The Gram-negative bacteria were identified to genus level by using the API 20E system and included Citrobacter spp., Enterobacter spp., Pantoea spp., Proteus spp., Seratia spp., Kluyvera spp., Klebsiella spp., Vibrio spp. and Escherichia coli. The number of viable bacteria throughout the malting process of the two cultivars did not differ significantly, although the LAB counts in the barley before steep and on the kilned malt were higher in Prisma than in Clipper. Leuconostoc argentinum, Leuconostoc laetis and Weissella confusa were the most predominant in both cultivars. A few strains of Weissella paramesenteroides, Lactobacillus casei, Lactococcus laetis and Lactobacillus rhamnosus were also isolated. Lb. casei and Lb. rhamnosus were not isolated from the Prisma cultivar, whilst W paramesenteroides and Le. laetis were absent in the Clipper cultivar. Kilned malt of the Clipper cultivar contained predominantly Le. argentinum, whereas the Prisma cultivar contained mainly Le. lactis. The effect of these bacteria on the fermenting ability of the brewer's yeast Saccharomyces cerevisiae SAB 05, was also studied. Fermentations were conducted in wort prepared from Clipper and Prisma malt. Yeast in combination with the different bacteria were used in the fermentation studies. Wort with only yeast was used as control. Emphasis was placed on the effect the bacteria has on the gravity, pH, yeast- and bacterial- counts and the different volatile aroma compounds produced throughout the fermentations. The presence of LAB and Gram-negative bacteria had no effect on the yeast to reduce the gravity of the fermenting wort, whilst the LAB caused a decrease in the pH of the fermentations in both Clipper and Prisma wort. The cell numbers of the Gram-negative bacteria decreased throughout the fermentations, whilst the LAB cell numbers remained constant. Comparisons could be drawn between the volatile aroma compounds produced in the control fermentation and fermentations with yeast and Gram-negative bacteria, yeast and Lactobacillus spp. and yeast and Weissella spp. Leuconostoc spp. had a much greater influence on the aromatic composition of fermented malt, with much more clear variations between Prisma and Clipper. No major differences were recorded in the aroma profiles of Prisma and Clipper malt fermented in the presence and absence of Lactococcus spp. The Gram-negative bacteria had no significant effect on the volatile aroma compounds produced by the yeast, whilst the LAB had a definite effect on aroma composition in both cultivars. The levels of four of the five principle aroma compounds, present in beer, were in the acceptable concentration range on the fmal day of fermentation. The compounds with the highest concentrations were iso-amyl alcohol, acetic acid and acetoin, with acetic acid being present in the highest concentration in all the fermentations. / AFRIKAANSE OPSOMMING: Veranderinge in die bakteriese populasie van die gars kultivars, Clipper (plaaslik) en Prisma (ingevoer), vermout by Southern Associated Maltsters (SAM), Caledon, Suid Afrika, is ondersoek. Monsters is van vier individuele lopies van elke kultivar en tydens tien verskillende fases van die vermoutingsproses geneem. Die tien verskillende stadia het die volgende ingesluit: Droë gars voor benatting, water van die eerste benattingsfase, gars nadat water van die eerste benattingsfase gedreineer is, water van die tweede benattingsfase, gars van die tweede benattingsfase, gars na die dreinering van water in die tweede benattings fase, gars na die eerste, tweede en derde dag van ontkieming binne die ontkiemingstenke, en mout na droging. Klem is geplaas op die taksonomie en samestelling van melksuurbakterieë (MSB) wat tydens die tien verskillende fases geïsoleer is. Die MSB is tot spesievlak geïdentifiseer deur gebruik te maak van numeriese analise van totale oplosbare selproteïen bandpatrone, RAPD-PKR bandpatrone en 16S rRNA volgorde-bepaling. Gram-negatiewe bakterieë is tot op genusvlak geïdentifiseer deur gebruik te maak van die API 20E toetssisteem. Spesies van die genera Citrobacter, Enterobacter, Pantoea, Proteus, Seratia, Kluyvera, Klebsiella, Vibrio asook Escherichia coli is geïdentifiseer. Tydens die vermoutingsproses van die twee kultivars is geen beduidende verskille in die lewensvatbare bakterietellings gevind nie, alhoewel die MSB-tellings in die gars voor benatting en mout na droging in Prisma hoër was as in Clipper. Leuconostoc argentinum, Leuconostoc laetis en Weissella confusa het die meeste voorgekom in beide kultivars. Kleiner hoeveelhede van Weissella paramesenteroides, Lactobacillus casei, Lactococcus laetis en Lactobacillus rhamnosus is ook geïsoleer. Lb. casei en Lb. rhamnosus het nie in die Prisma-kultivar voorgekom nie, terwyl W paramesenteroides en Le. laetis nie in die Clipper-kultivar teenwoordig was nie. Le. argentinum het meestal in die gedroogde mout van die Clipper-kultivar voorgekom, terwyl Le. laetis meestal in die Prisma-kultivar waargeneem is. Die effek van hierdie bakterieë op die fermentasievermoë van die brouersgis Saccharomyces cerevisiae SAB 05 is ook bestudeer. Die fermentasies is in Clipper- en Prisma- wort gedoen. Vir die fermentasiestudies is gis in kombinasie met verskillende bakterieë gebruik. Wort met slegs gis het as kontrole gedien. Klem is geplaas op die effek van die bakterieë op die digtheid, pH, gis- en bakterietellings en die verskillende vlugtige komponente wat tydens die fermentasies geproduseer is. Die teenwoordigheid van MSB en Gram-negatiewe bakterieë het geen effek gehad op die vermoë van die gis om die digtheid van die gefermenteerde wort te verlaag nie. Die MSB het wel 'n verlaging van die pH in beide Clipper- en Prisma- wort teweeggebring. Tydens die fermentasie het die Gramnegatiewe bakterietellings verminder, terwyl die MSB-tellings konstant gebly het. 'n Verband is gevind tussen vlugtige komponente geproduseer in die kontrole-fermentasie en fermentasies met gis en Gram-negatiewe bakterieë, gis en Lactobacillus spp. en gis en Weissella spp. Leuconostoc spp. het groter veskille in die samestelling van die gefermenteerde wort teweeg gebring met duidelike verskille tussen Clipper en Prisma. Die teenwoordigheid van Lactococcus spp. het nie groot verskille in die samestelling van die gefermenteerde wort getoon nie. Op die laaste dag van die fermentasies was die vlakke van vier uit die vyfbelangrikste vlugtige aroma komponente wat in bier voorkom in die kontrole fermentasies in aanvaarbare konsentrasies teenwoordig. Die Gramnegatiewe bakterieë het geen beduidende invloed gehad op die vlugtige aroma komponente wat deur die gis geproduseer is nie, terwyl die MSB 'n besliste effek in die aroma-samestelling van beide die kultivars gehad het. Die komponente met die hoogste konsentrasies was, isoamiel-alkohol, asynsuur en asetoin. Asynsuur was in al die fermentasies in die hoogste konsentrasie teenwoordig.

While the production of mead, a fermented honey beverage, has declined in popularity around the world in recent centuries, a substantial mead industry continues to exist in Africa with an estimated annual production of 1 to 1.7 billion litres. This is largely an ‘invisible industry’, and has functioned outside the formal economy due to proscription of indigenous beverages during colonial times. The traditional African mead industry is, however, also now under pressure due to the environmental degradation of scarce natural ingredients, urbanisation and loss of indigenous knowledge systems (IKS) and, with time, the beverage will likely follow the declining trend of mead consumption observed elsewhere. An analysis of early reports of African mead production suggested that the Khoi-San, among the earliest inhabitants of the continent, are the originators of the mead making techniques which use fibrous plant materials derived from specific plant species, to facilitate mead fermentation in some way. The Eastern Cape represents a region with a large body of Khoi-San IKS preserved in their descendants among the Afrikaans and Xhosa populations. A survey to establish a baseline of mead-making technology in the Eastern Cape was undertaken, and involved interviewing traditional mead makers across an area of roughly 100 000 km2, showing that the mead, iQhilika(Xhosa) Kari (Khoi-San/Afrikaans), is produced using a very similar process throughout the region. This involves the roots of a Trichodiadema sp. plant (imoela – Xhosa, karimoer – Afrikaans/Khoi-San), honey, extract of brood and/or pollen and water. Various other fruit sugar sources were also found to be added at times producing seasonal beverages with unique organoleptic properties. A model traditional iQhilika production operation was investigated in order to describe the main features of the process. Biomass immobilised on Trichodiadema root segments was found to be distributed evenly through the profile of the bioreactor resulting in a well mixed fermentation and a productivity of 0.74 g EtOH/l/h. In the initial stages of fermentation, the ethanol yield was highest in the mid-regions of the bioreactor, but with time the regions closer to the surface, which had atmospheric contact had a higher yield. This phenomenon was attributed to aerobic fatty acid synthesis which allowed the yeast close to the surface to function more efficiently despite rising ethanol concentrations. The mead contained 44.25 g/l (7 % volume) ethanol produced in a fermentation time of 43.5 h. Yeast biomass in the traditional process was either immobilised in the form of flocs or attached to the Trichodiadema intonsum support. Electron microscopy revealed that the cells were covered in a layer of extra-cellular polymeric substance apparently assisting the immobilization, and which was populated by a consortium of yeasts and bacteria. Yeasts isolated from iQhilika brewed in two regions separated by 350 km were found to be very closely related Saccharomyces cerevisiae strains as determined by molecular genetic analysis. The traditional beverage was found to contain populations of Lactic acid bacteria (LAB), which are known spoilage organisms in other beverages. Spoilage characteristics of these organisms matched descriptions of spoilage provided by the IKS survey. Other possibly beneficial LAB, which may contribute useful flavour compounds, were also found to be present in the system. The basic functional aspects of the traditional process were used to design a continuous bench-scale tower bioreactor and process development was based on the IKS survey. This consisted of a packed bed bioreactor, consisting of 2 mm3 T. intonsum root segments, immobilising a novel Saccharomyces cerevisiae strain isolated from a traditional batch of iQhilika. The bioreactor performed well with a yield of close to the theoretical maximum and an ethanol productivity of 3.45 g EtOH/l/h. The parameters of the 5.6 l/d bench-scale bioreactor were used to design a full-scale production bioreactor with a planned maximum output of 330 l/d. This bioreactor had a productivity of 0.19 g EtOH/l/h. The organoleptic properties of the product produced were considered by a taste panel to be better than those of the product of the bench-scale tower bioreactor. This research was based on the development of IKS which imposed a number of constraints and obligations on the project to ensure environmental, and social, in addition to financial viability of the scale-up operation. Makana Meadery was established in partnership with Rhodes University as an empowerment company which, in addition to undertaking the commercialisation of the iQhilika process, would also develop methods for the production of scarce ingredients traditionally unsustainably sourced from fragile ecosystems, provide beekeeping training and the manufacture of beehives.