O método empregado na produção de fungos entomopatogênicos teve poucas alterações em mais de 40 anos de utilização no Brasil. A produção adotada nas biofábricas em larga escala é basicamente através da fermentação sólida em arroz com elevado uso de mão de obra e longo tempo de produção. Assim, o presente estudo consistiu em desenvolver estratégias que possam ser implementadas nas diferentes etapas do sistema de produção de Metarhizium anisopliae ESALQ1037, como adição ao arroz de materiais inertes (bagaço de cana-de-açúcar, maravalha, esponja vegetal e palha de arroz) e outros substratos (milheto, flocos de arroz e sagu) para separação dos grãos de arroz e aumento da aeração, uso de blastosporos como inóculo em sistemas bifásicos tendo como substrato sólido o arroz ou mistura de farelos (trigo, milho, aveia, arroz, soja e algodão) ou suportes de crescimento e esporulação (bagaço de cana-de-açúcar, vermiculita, tecido de lã, palha de arroz e maravalha). Foi testado o uso da esterilização por fervura em substituição à autoclavagem e recuperação de conídios por extração em água como substituto ao peneiramento, além de armazenamento em condições de alta e baixa temperatura e umidade: 97%UR e 22ºC; 97%UR e 4ºC; 0%UR e 22ºC; 0%UR e 4ºC. O meio líquido M11 (a base de 45g L-1 glicose e 36g L-1 extrato de levedura) foi o mais adequado para produção de blastosporos (3,0 x 108 blastosporos/mL) e biomassa. A inoculação de blastosporos em arroz resultou em produção maior e mais rápida de conídios (4,51 x 108 conídios/g) em 7 dias de crescimento fúngico. O farelo de trigo inoculado com blastosporos produzidos em meio M11 resultou em maior produção de conídios (1,2 x 109 conídios/g) quando comparado aos farelos e arroz inoculados com suspensão de conídios (máximo de 7,8 x 108 conídios/g). O bagaço de cana-de-açúcar autoclavado seco foi o suporte que resultou em maior produção do fungo quando associado ao meio líquido. A adição ao arroz de bagaço de cana-de-açúcar umedecido, maravalha e esponja vegetal não umedecidas promoveu incremento na produção de conídios (> 3,3 x 109 conídios/g) quando comparado ao arroz somente (1,00 x 109 conídios/g). A exposição do arroz à fervura por 10 e 15 minutos resultou em produção de conídios semelhante à produção em arroz autoclavado, sem a presença de contaminantes. Farelo de trigo foi o substrato mais promissor para produção do fungo especialmente quando misturado ao farelo de arroz (1,75 x 109 conídios/g) na fermentação sólida. Conídios extraídos do arroz por peneiramento ou lavagem em água e recuperados com ou sem a adição de argilas, terra de diatomácea e caulim apresentaram viabilidade superior a 92% após armazenamento por 60 dias a 22°C e 97% UR. A 22°C e 0% UR, somente os conídios extraídos do arroz por peneiramento e os extraídos em água e recuperados com a adição de argila preta e caulim apresentaram viabilidade superior a 92% após armazenamento por 60 dias. Estratégias testadas neste estudo para produção e extração de conídios de M. anisopliae são alternativas viáveis para serem incorporadas na produção em larga escala e reduzir o tempo de fermentação e mão de obra, aumentando a eficiência de conversão de substrato em conídios. / The method used in the production of entomopathogenic fungi had few changes in more than 40 years of use in Brasil. The production of large-scale biofactories is basically through solid fermentation in rice with high labor and long production time. Thus, the present study consisted in developing strategies that could be implemented in the different stages of the production system of Metarhizium anisopliae ESALQ1037, as addition to rice of inert materials (sugarcane bagasse, shavings, vegetable sponge, rice straw) and other substrates (millet, rice flakes and sago) for separation of rice grains and increased aeration, use of blastospores as inoculum in biphasic systems having as a solid substrate the rice or mixture of bran (wheat, corn, oats, rice, soybean and cotton) or growth and sporulation supports (sugarcane bagasse, vermiculite, wool, rice straw and shavings). The use of boiling sterilization to replace autoclaving and recovery of conidia by water extraction as a substitute for sieving was tested, as well as storage under high and low temperature and humidity conditions: 97% RH and 22 ºC; 97% RH and 4°C; 0% RH and 22°C; 0% RH and 4°C. The M11 liquid medium (the base of 45g L-1 glucose and 36g L-1 yeast extract) was the most suitable for the production of blastospores (3.0 x 108 blastospores / mL) and biomass. The inoculation of blastospores in rice resulted in larger and faster production of conidia (4.51 x 108 conidia / g) in 7 days of fungal growth. The wheat bran inoculated with blastospores produced in M11 medium resulted in higher conidia production (1.2 x 109 conidia / g) when compared to the bran and rice inoculated with conidial suspension (maximum of 7.8 x 108 conidia / g). The dried autoclaved sugarcane bagasse was the carrier that resulted in higher fungus production when associated with the liquid medium. The addition of moistened sugarcane bagasse and non-moistened shavings and vegetable sponges promoted increase in the production of conidia (> 3.3 x 109 conidia / g) when compared to rice only (1.0 x 109 conidia / g). Exposure of the boil rice for 10 and 15 minutes resulted in conidial production similar to the production in autoclaved rice without the presence of contaminants. Wheat bran was the most promising substrate for the fungus production especially when mixed with rice bran (1.75 x 109 conidia / g) in solid fermentation. Conidia extracted from rice by sieving or washing in water and recovered with or without the addition of clays, diatomaceous earth and kaolin showed viability higher than 92% after storage for 60 days at 22 °C and 97% RH. At 22 °C and 0% RH, only the conidia extracted from the rice by sieving and those extracted in water and recovered with the addition of black clays and kaolin had viability higher than 92% after storage for 60 days. Strategies tested in this study for the production and extraction of conidia of M. anisopliae are viable alternatives to be incorporated in the large scale production and to reduce the time of fermentation and manpower, to increasing the efficiency of conversion of substrate in conidia.

O método empregado na produção de fungos entomopatogênicos teve poucas alterações em mais de 40 anos de utilização no Brasil. A produção adotada nas biofábricas em larga escala é basicamente através da fermentação sólida em arroz com elevado uso de mão de obra e longo tempo de produção. Assim, o presente estudo consistiu em desenvolver estratégias que possam ser implementadas nas diferentes etapas do sistema de produção de Metarhizium anisopliae ESALQ1037, como adição ao arroz de materiais inertes (bagaço de cana-de-açúcar, maravalha, esponja vegetal e palha de arroz) e outros substratos (milheto, flocos de arroz e sagu) para separação dos grãos de arroz e aumento da aeração, uso de blastosporos como inóculo em sistemas bifásicos tendo como substrato sólido o arroz ou mistura de farelos (trigo, milho, aveia, arroz, soja e algodão) ou suportes de crescimento e esporulação (bagaço de cana-de-açúcar, vermiculita, tecido de lã, palha de arroz e maravalha). Foi testado o uso da esterilização por fervura em substituição à autoclavagem e recuperação de conídios por extração em água como substituto ao peneiramento, além de armazenamento em condições de alta e baixa temperatura e umidade: 97%UR e 22ºC; 97%UR e 4ºC; 0%UR e 22ºC; 0%UR e 4ºC. O meio líquido M11 (a base de 45g L-1 glicose e 36g L-1 extrato de levedura) foi o mais adequado para produção de blastosporos (3,0 x 108 blastosporos/mL) e biomassa. A inoculação de blastosporos em arroz resultou em produção maior e mais rápida de conídios (4,51 x 108 conídios/g) em 7 dias de crescimento fúngico. O farelo de trigo inoculado com blastosporos produzidos em meio M11 resultou em maior produção de conídios (1,2 x 109 conídios/g) quando comparado aos farelos e arroz inoculados com suspensão de conídios (máximo de 7,8 x 108 conídios/g). O bagaço de cana-de-açúcar autoclavado seco foi o suporte que resultou em maior produção do fungo quando associado ao meio líquido. A adição ao arroz de bagaço de cana-de-açúcar umedecido, maravalha e esponja vegetal não umedecidas promoveu incremento na produção de conídios (> 3,3 x 109 conídios/g) quando comparado ao arroz somente (1,00 x 109 conídios/g). A exposição do arroz à fervura por 10 e 15 minutos resultou em produção de conídios semelhante à produção em arroz autoclavado, sem a presença de contaminantes. Farelo de trigo foi o substrato mais promissor para produção do fungo especialmente quando misturado ao farelo de arroz (1,75 x 109 conídios/g) na fermentação sólida. Conídios extraídos do arroz por peneiramento ou lavagem em água e recuperados com ou sem a adição de argilas, terra de diatomácea e caulim apresentaram viabilidade superior a 92% após armazenamento por 60 dias a 22°C e 97% UR. A 22°C e 0% UR, somente os conídios extraídos do arroz por peneiramento e os extraídos em água e recuperados com a adição de argila preta e caulim apresentaram viabilidade superior a 92% após armazenamento por 60 dias. Estratégias testadas neste estudo para produção e extração de conídios de M. anisopliae são alternativas viáveis para serem incorporadas na produção em larga escala e reduzir o tempo de fermentação e mão de obra, aumentando a eficiência de conversão de substrato em conídios. / The method used in the production of entomopathogenic fungi had few changes in more than 40 years of use in Brasil. The production of large-scale biofactories is basically through solid fermentation in rice with high labor and long production time. Thus, the present study consisted in developing strategies that could be implemented in the different stages of the production system of Metarhizium anisopliae ESALQ1037, as addition to rice of inert materials (sugarcane bagasse, shavings, vegetable sponge, rice straw) and other substrates (millet, rice flakes and sago) for separation of rice grains and increased aeration, use of blastospores as inoculum in biphasic systems having as a solid substrate the rice or mixture of bran (wheat, corn, oats, rice, soybean and cotton) or growth and sporulation supports (sugarcane bagasse, vermiculite, wool, rice straw and shavings). The use of boiling sterilization to replace autoclaving and recovery of conidia by water extraction as a substitute for sieving was tested, as well as storage under high and low temperature and humidity conditions: 97% RH and 22 ºC; 97% RH and 4°C; 0% RH and 22°C; 0% RH and 4°C. The M11 liquid medium (the base of 45g L-1 glucose and 36g L-1 yeast extract) was the most suitable for the production of blastospores (3.0 x 108 blastospores / mL) and biomass. The inoculation of blastospores in rice resulted in larger and faster production of conidia (4.51 x 108 conidia / g) in 7 days of fungal growth. The wheat bran inoculated with blastospores produced in M11 medium resulted in higher conidia production (1.2 x 109 conidia / g) when compared to the bran and rice inoculated with conidial suspension (maximum of 7.8 x 108 conidia / g). The dried autoclaved sugarcane bagasse was the carrier that resulted in higher fungus production when associated with the liquid medium. The addition of moistened sugarcane bagasse and non-moistened shavings and vegetable sponges promoted increase in the production of conidia (> 3.3 x 109 conidia / g) when compared to rice only (1.0 x 109 conidia / g). Exposure of the boil rice for 10 and 15 minutes resulted in conidial production similar to the production in autoclaved rice without the presence of contaminants. Wheat bran was the most promising substrate for the fungus production especially when mixed with rice bran (1.75 x 109 conidia / g) in solid fermentation. Conidia extracted from rice by sieving or washing in water and recovered with or without the addition of clays, diatomaceous earth and kaolin showed viability higher than 92% after storage for 60 days at 22 °C and 97% RH. At 22 °C and 0% RH, only the conidia extracted from the rice by sieving and those extracted in water and recovered with the addition of black clays and kaolin had viability higher than 92% after storage for 60 days. Strategies tested in this study for the production and extraction of conidia of M. anisopliae are viable alternatives to be incorporated in the large scale production and to reduce the time of fermentation and manpower, to increasing the efficiency of conversion of substrate in conidia.

Call number: LD2668 .T4 1979 F47 / Master of Science

Call number: LD2668 .T4 1985 C84 / Master of Science

Doctor of Philosophy / Department of Animal Sciences and Industry / James S. Drouillard / Beta-adrenergic agonists, which are synthetic catecholamines, increase rate of gain, improve feed efficiency, and decrease carcass fat, when fed to cattle before slaughter. However, little attention has been given to the potential effects of beta-adrenergic agonists on the rumen ecosystem. Natural catecholamines, such as norepinephrine, epinephrine, and dopamine, have been observed to stimulate bacterial growth. The objectives of this research were to determine if ractopamine hydrochloride (RAC) a synthetic catecholamine has direct effects on growth and fermentation products of ruminal bacteria, and to determine the effects of protein source on ruminal fermentation and proteolysis when cattle are fed RAC. The effects of varying concentrations of RAC on ruminal fermentation were evaluated in vitro. Ractopamine hydrochloride had a quadratic effect on in vitro gas production (P < 0.05). Total VFA production was not changed with RAC (P > 0.50). Different concentrations of RAC were evaluated in vitro with different nitrogen sources to determine effects of nitrogen degradability on response to RAC. There was an interaction between RAC and nitrogen substrate (P < 0.01), with more degradable forms of nitrogen eliciting greater changes in in vitro dry matter disappearance (IVDMD) with RAC supplementation. Significant main effects also were detected for RAC, substrate, and hour (P < 0.001). In vitro analysis of proteolysis revealed that RAC lowered ammonia and amino acid concentrations (P < 0.001). In vivo ruminal ammonia concentrations also were lower when RAC was fed in combination with dry-rolled corn, but not when fed in conjunction with steam-flaked corn (grain processing × RAC, P < 0.01). Addition of RAC, steam-flaked corn, and distiller’s grains (DG) all resulted in lower ruminal ammonia concentrations (P < 0.01). Amino acid concentrations were decreased when RAC was added to diets with DG but were unchanged in diets without added DG (DG × RAC, P < 0.05). Results from these studies suggest that RAC affects fermentation by ruminal microflora. However, there were no differences in growth or fermentative end products of pure bacterial cultures with the addition of RAC (P > 0.10). Overall beta-adrenergic agonists alter ruminal fermentation, which could have important implications for diet formulation.

Submitted in fulfilment of the requirements for the degree of Master of Engineering, Durban University of Technology, Durban, South Africa, 2015. / Fermentation processes are vital for the production of numerous bioproducts. Fermentation being the mass culture of micro – organisms for the production of some desired product, is an extensive field, with immense prospects for study and improvement. Enzyme production is of significance as these proteins are biological catalysts, finding niches in numerous industries, xylanase for example is utilized in the pulp and paper, animal feed, biofuel and food production processes. During enzyme production, a critical step is biomass separation, whereby the valuable product, the enzyme, is removed from the broth or micro – biological culture before it is denatured. This is typically achieved via centrifugation. The aim of this study was to develop and evaluate a submerged membrane fermenter system with the specific outcome of increasing the rate of production of xylanase, from the thermophilic fungal species Thermomyces lanuginous DSM 5826. Preliminary shake flask experiments were performed to determine the optimal production conditions, followed by partial characterization of the enzyme. A bioreactor was then fabricated to include a flat sheet membrane module, with outlets for permeate and broth withdrawal and inlets for feed and sterile air input. Experiments were conducted to determine the optimal dilution rate for maximum volumetric productivity. Results from the shake flask experiments indicated that the best conditions for xylanase production, yielding xylanase activity of 5118.60 ± 42.76 U.mL-1 was using nutrient medium containing beechwood xylan (1.5 % w/v), yeast extract (1.5 % w/v), potassium dihydrogen phosphate (0.5 % w/v), adjusted to a pH of 6.5 and inoculated with 1.0 mL of spore solution, rotating in a shaking incubator set to 150 rpm at 50 °C. Apart from analysis of the effect of the carbon source on xylanase activity, coarse corn cobs were used in the shake flask experiments as a cost saving initiative. The pH optima was determined to be 6.5 while the temperature optima of the enzyme was 70 °C. SDS PAGE analysis revealed that the molecular weight of the enzyme was between 25 and 35 kDa and qualitative analysis via a zymogram revealed clear zones of hydrolysis on a xylan infused agarose gel. During short run membrane fermenter experiments the percentage increase in enzyme activity between the batch operation (610.58 ± 34.54 U.mL-1) and semi – continuous operation (981.73 ± 55.54 U.mL-1) with beechwood xylan nutrient replenishment was 60.78 %. The maximum volumetric productivity achieved with beechwood supplementation after 192 hours in semi – continuous operation (5.32 ± 0.30 U.mL-1.hr-1) was 2.1 times greater than that of batch operation (2.54 ± 0.14 U.mL-1.hr-1) which equates to an increase of 110.28 % in productivity measured at its peak. The increase in total activity between batch (610 576.92 U) and beechwood xylan medium supplemented semi – continuous mode (1 184 937.50 U) resulted in a 94.07 % increase. During long run experimental periods, the increase in production of xylanase between the batch (873.26 ± 61.78 U.mL-1) and the xylan medium membrane system (1522.41 ± 107.65 U.mL-1) was determined to be 74.34 % while an overall average increase in productivity between the batch and xylan fed membrane system was 43.25%. The total enzyme activity with in membrane mode with beechwood xylan nutrient medium feed was 160 % greater than the batch process offering a 2.6 – fold increase. Experiments where de – ionized water was alternated with beechwood xylan nutrient medium had no significant impact on the productivity or enzyme activity. The optimal dilution rate for maximum volumetric productivity as determined to be 0.0033 hr-1. The results are indicative of the potential viability of such a design, yielding the desired outcome of a membrane integrated system to significantly increase the production of enzymes during fermentation.

BACKGROUND:Sweet sorghum is a domesticated grass containing a sugar-rich juice that can be readily utilized for ethanol production. Most of the sugar is stored inside the cells of the stalk tissue and can be difficult to release, a necessary step before conventional fermentation. While this crop holds much promise as an arid land sugar source for biofuel production, a number of challenges must be overcome. One lies in the inherent labile nature of the sugars in the stalks leading to a short usable storage time. Also, collection of sugars from the sweet sorghum stalks is usually accomplished by mechanical squeezing, but generally does not collect all of the available sugars.RESULTS:In this paper, we present two methods that address these challenges for utilization of sweet sorghum for biofuel production. The first method demonstrates a means to store sweet sorghum stalks in the field under semi-arid conditions. The second provides an efficient water extraction method that can collect as much of the available sugar as feasible. Operating parameters investigated include temperature, stalk size, and solid-liquid ratio that impact both the rate of sugar release and the maximal amount recovered with a goal of low water use. The most desirable conditions include 30degreesC, 0.6 ratio of solid to liquid (w/w), which collects 90 % of the available sugar. Variations in extraction methods did not alter the efficiency of the eventual ethanol fermentation.CONCLUSIONS:The water extraction method has the potential to be used for sugar extraction from both fresh sweet sorghum stalks and dried ones. When combined with current sugar extraction methods, the overall ethanol production efficiency would increase compared to current field practices.

Thesis (MSc)--University of Stellenbosch, 2001. / ENGLISH ABSTRACT: The importance of indigenous fruit wines is not well researched and documented. There is a need to develop and exploit these valuable food resources through improved production practices, storage, preservation and utilization technologies. The maruia fruit is beneficial in many ways, it can be used for making juice, jam, beer or can be eaten as a whole fruit. The highly nutritive nature of the fruit, its distinctive tropical flavor, its wild occurrence and demand by the local and international communities for the by-products of the fruit necessitated efforts to optimize the technological processes for the production of the possible by-products. This study focuses on the fermentation technology of the maruia fruit. The effect of enzymes prior to the fermentation process and post-fermentation was evaluated. For pre-fermentation processes we focused on the ability of commercial enzymes to increase juice yield, improve the clarification and filterability. For pre- and post-fermentation applications, aroma release was considered. The results indicated a significant increase in the yield depending on the enzyme used. An increase of at least 2% was recorded and a maximum of 12% yield increase was observed. The enzymes also had a phenomenal effect on the release of bound monoterpenes and hence enhancing the flavor of the juice. The panel of judges confirmed the results from the gas chromatography analyses by noting an increase in flavor intensity in the enzyme treated juice. The possibility of selecting a yeast strain that performs best during the fermentation of maruia pulp was also looked at. This study aimed at selecting a strain that produces wine and distillate with the typical maruia flavor complex. We showed the effect of the different yeast strains, in the wines and distillates, on the principal volatile compounds. We then correlated the performance of the different strains as perceived by the panel to the various volatile compounds. The effect of fermentation temperature on the performance of the different yeast strains was also considered. Fermenting the maruia pulp at different temperatures resulted in the production of wines and distillates with different volatile profiles for the different yeast strains. The wines and distillates fermented at a low temperature of 15°C were preferred to the wines and distillates fermented at 30°C. However, not all strains performed well at 15°C, strains like NT116 performed better at 30°C. The different commercial strains produced wines and distillates with significantly different flavor profiles. These differences in the flavor profiles were reflected in the sensory evaluation where, depending on the interaction of the volatile compounds some wines and distillates were preferred to others. The effect of the different commercial enzymes and yeast strains should thereof be further evaluated and optimized on a larger scale. This would greatly help prevent variation in quality of the fermented by-products of the maruia fruit. / AFRIKAANSE OPSOMMING: Die belang van inheemse vrugtewyne is nie goed nagevors en gedokumenteer nie. Daar is 'n behoefte om hierdie waardevolle voedselbronne te ontwikkel en te benut, deur verbeterde produksiepraktyke, storing, preservering en benuttingstegnologieë. Die maroelavrug is veelsydig op baie wyses, deurdat dit gebruik word vir die maak van sap, konfyt, bier, of as heel vrug geëet kan word. Die vrug is hoog in voedingswaarde, het In kenmerkende tropiese geur, kom wild voor, en is in aanvraag by plaaslike en internasionale gemeenskappe vir die by-produkte van die vrug. Dit maak dit essensieel om die tegnologiese prosesse vir die produksie van hierdie moontlike by-produkte te optimiseer. Hierdie studie fokus op die fermentasie-tegnologie van die maroelavrug. Die effek van ensieme voor en na die fermentasie-proses is geëvalueer. Vir prosesse wat voor fermentasie plaasvind, het ons gefokus op die vermoë van kommersiële ensieme om sapopbrengs te verhoog, asook om verheldering en filtrering te verbeter. Vir beide voor- en na-fermentasie toepassings is die vrystelling van aroma gemonitor. Die resultate dui op 'n betekenisvolle verhoging in die sapopbrengs, afhangende van die ensiem wat gebruik is. 'n Verhoging van ten minste 2% is opgeteken, en 'n maksimum van 12% opbrengsverhoging is waargeneem. Die ensieme het ook 'n geweldige effek op die vrystelling van gebonde monoterpene gehad, en dus die verhoging in die geur van die sap. Die proepaneel het die resultate bevestig van die gaschromatografie-analises, deur 'n verhoging in die geurintensiteit in die ensiembehandelde sap te bemerk. Daar is ook gekyk na die moontlikheid om 'n gisras te selekteer wat die beste presteer tydens die fermentasie van maroela-pulp. Hierdie studie het die doelstelling gehad om In gisras te selekteer wat wyn en distillaat produseer met In tipiese maroelageurkompleks. Ons het die effek van verskillende gisrasse aangedui in die wyne en distillate, op grond van van vlugtige komponente. Ons het dan die prestasie van die verskillende rasse, soos waargeneem deur die paneel, gekorrelleer met die verskeie vlugtige komponente. Die effek van fermentasie-temperatuur op die werkverrigting van die verskillende gisrasse is ook in ag geneem. Fermentasie van die maroela-pulp by verskillende temperature het gelei tot die produksie van wyne en distillate met verskillende vlugtige profiele vir die verskillende gisrasse. Die wyne en distillate wat by In laer temperatuur van 15°C gefermenteer is, is verkies bo die wyne en distillate wat by 30°C gefermenteer is. Alle rasse het egter nie baie goed presteer by 15°C nie, soos byvoorbeeld NT116 wat beter presteer het by 30°C. Die verskillende kommersiële rasse het wyne en distillate geproduseer met betekenisvol verskillende geurprofiele. Hierdie verskille in geurprofiele is gereflekteer in die sensoriese evaluering waar, afhangende van die interaksie van die vlugtige komponente, sommige wyne en distillate bo ander verkies is. Die effek van die verskillende kommersiële ensieme en gisrasse moet verkieslik verder op groter skaal geëvalueer en geoptimiseer word. Dit sal veral help om variasie in kwaliteit van die gefermenteerde by-produkte van die maroelavrug te voorkom.

Thesis(DScAgric.)--Stellenbosch University, 1932. / No Abstract Available

Thesis (MSc)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: Scotch whiskey is of two main types, namely Scotch malt whiskey, made from malted barley alone, or Scotch grain whiskey, made from cereals, such as wheat or maize, together with malted barley. In both processes, the enzymes from the barley are responsible for starch conversion and should always be derived entirely from the malted barley. No exogenous enzymes are allowed to be added to any mashing. The enzymes involved in the conversion process to fermentable sugars, are the aand p-amylases, limit dextrinase and p-glucosidase. Maize, on the other hand, contains no enzyme activity, therefore enzymes need to be added when producing whiskey from maize alone. In other whiskey-producing countries where maize is freely available and cheaper than barley, the use of exogenous enzymes are allowed in the mashing process and is crucial for the formation of fermentable sugars from complex carbohydrates. The cost of the enzymes, however, can push the production cost of whiskey to higher levels. Saccharomyces cerevisiae does not have any amylolytic activity, but is an excellent fermenter and produces favourable organoleptic notes, which makes it very suitable for producing potable spirit. Efforts have been made to genetically improve industrial strains, relying on classical genetic techniques followed by the selection of broad traits, such as ethanol tolerance, absence of off-flavours and carbohydrate/starch utilisation. No strain has thus far been selected for total starch degradation during the fermentation of whiskey mash. Over the last decade, considerable progress has been made in the development of genetically improved strains for the distilling, wine, brewing and baking industries. The expression of heterologous genes introduced a new dimension in approaches to the genetic improvement of industrial strains. It would therefore be cost-effective to use a yeast strain that can produce active and sufficient enzymes to ferment raw starch efficiently to alcohol without lowering the quality of the end product. No such strain has been developed to date, but the continuous improvement of starch-utilising strains has made this goal more achievable. Two a-amylase genes, namely LKA 1 and LKA2, were previously isolated from Lipomyces kanonenkoae. In this study, we selected 4 strains on the basis of criteria that are important for whiskey-specific strains. The selected strains were transformed with LKA 1, as well as with a combination of LKA 1 and LKA2 genes. The wine yeast VIN13 was included in the transformation of LKA1 and LKA2 because of its rapid fermentation rate. The genes were integrated into the genomes of the yeast strains and were stable after many generations. Assays showed that a significant increase in enzyme activity was induced in the whiskey strains, compared to the untransformed strains. The strains also showed good fermentation ability in whiskey fermentations, although optimum alcohol production was still not achieved. / AFRIKAANSE OPSOMMING: Skotse whiskey bestaan uit 2 tipes, nl. mout whiskey, gemaak slegs van mout d.w.s. gars wat die mout proses ondergaan het, en graan whiskey wat gemaak word van gewasse soos mielies of koring, waarby mout gevoeg word. Die ensieme afkomstig van die mout is verantwoordelik vir die omsetting van stysel na fermenteerbare suikers en geen eksogene ensieme mag by die gars- of graanmengsel gevoeg word nie. Die ensieme wat betrokke is by die omsetting van stysel, is die a- en ~- arnitases, limiet dekstrinase en ~-glukosidase. Mielies bevat geen ensiemaktiwiteit nie, dus moet ensieme by die proses gevoeg word indien slegs mielies vir die vervaardiging van whiskey gebruik word. In whiskey produserende lande waar mielies vryelik beskikbaar is en goedkoper is as gars, word eksogene ensieme by die graanmengsel gevoeg vir die vrystelling van fermenteerbare suikers vanaf komplekse koolhidrate. Die hoë koste van die ensieme kan egter die produksiekoste van whiskey verhoog. Saccharomyces cerevisiae besit geen amilolitiese aktiwiteit nie, maar is 'n uitstekende fermenteerder en produseer gewensde organoleptiese geure. Om hierdie redes is S. cerevisiae baie geskik vir die produksie van drinkbare etanol. Navorsingspogings om industriële rasse geneties m.b.v. klassieke genetiese metodes te verbeter, kom wydverspreid in die literatuur voor. Dit sluit in die seleksie van rasse met 'n verskeidenheid van eienskappe soos etanol toleransie, die afwesigheid van afgeur produksie en koolhidraat/stysel benutting. Geen ras is egter tot op hede geselekteer vir totale stysel afbraak gedurende fermentasie nie. Groot vordering is gedurende die laaste dekade gemaak in die ontwikkeling van genetiese verbeterde rasse vir die wyn- stokery- en brouers industrieë. Die uitdruk van heterogene gene in gisrasse gee 'n nuwe dimensie aan die genetiese verbetering van industriële rasse. Die gebruik van 'n gisras wat aktiewe en genoegsame ensieme produseer om rou stysel te fermenteer, sonder om die kwalitiet van die eindproduk nadelig te beïnvloed, kan die produksiekoste van whiskey aansienlik verminder. Geen gisras met hierdie eienskap is tot op hede ontwikkel nie, maar die voortdurende verbetering van rasse om stysel af te breek maak hierdie doel meer bereikbaar. Twee a-amilase gene, nl. LKA 1 en LKA2 is voorheen uit Lipomyces kononenkoae geïsoleer. In hierdie studie is 4 gisrasse geselekteer op grond van die kriteria wat nodig is vir whiskey giste. Die geselekteerde rasse is getransformeer met LKA 1 sowel as 'n kombinasie van LKA 1 en LKA2 gene. Die wyngis VIN13 is ingesluit by die transformasie met die LKA1 en LKA2 gene, omrede VIN13 bekend is as 'n vinnige fermenteerder. Die gene is geïntegreer in die genoom van die verskillende gisrasse en is stabiel na vele generasies. Die getransformeerde rasse het 'n betekenisvolle verhoging in ensiemaktiwiteit teenoor die nie-getransformeerde rasse getoon. AI die transformante het ook goeie fermentasie vermoë getoon in whiskey fermentasie proewe. Optimum alkoholproduksie is egter nie verkry nie.