No abstract available. / Thesis (M.Sc.Eng.)-University of KwaZulu-Natal, Durban, 2009.

Thesis (PhD. (Microbiology)) --University of Limpopo, 2013 / The current study investigated four fungal species namely Aspergillus niger FGSC A733, Aspergillus versicolor EF23, Penicillium citrinum AZ01 and Trichoderma harzianum NCGR 0509 for their abilities to produce cellulases and xylanases in submerged and solid state fermentations. Five different substrates (carboxymethyl cellulose, xylan, common thatch grass, wheat bran and Jatropha curcas seed cake) were examined for their potential use as low cost feedstock for fermentation by the fungal species. Aspergillus niger FGSC A733 produced the highest titres of cellulase and xylanase in solid state fermentations using wheat bran as a substrate. However, because of the need to lower the cost of enzyme production, Jatropha seed cake a relatively underutilised oilseed cake was used. Supplementation of the Jatropha seedcake with 10% common thatch grass (Hyperrhenia sp) resulted in a fivefold increase in the levels of xylanase produced. Cellulase production was not affected by this supplementation. Addition of ammonium chloride increased production of xylanase while cellulase production was not affected nitrogen supplementation. Maximum xylanase was produced on Jatropha seed cake at 25 °C after 96 hours while cellulase was maximally produced at 40 °C after 96 hours of solid state fermentations. Peak production of xylanase was obtained at an initial pH of 3 whilst cellulase was maximally produced at an initial pH of 5. The crude xylanase was most active at pH 5 and cellulase at pH 4. The optimum temperature for cellulase activity was 65 °C and that of xylanase was 50 °C. Under optimized conditions, 6087 U/g and 3974 U/g of xylanase and cellulase per gram of substrate used were obtained respectively. The diversity of cellulases was investigated so as to determine the most appropriate enzyme mixture for saccharification of the common thatch grass. Proteins from the four species under investigation were partially purified by affinity chromatography on swollen Avicel. The proteins were analysed using sodium dodecyl sulphate-polyacrylamide gel electrophoresis SDS-PAGE and zymography. Potential cellulase bands from SDS-PAGE were sequenced by mass spectrometry. The basic logical alignment tool (BLAST) and Clustal W were used for matching and identifying the sequences with closely related ones in the databases. The identified proteins from Penicillium citrinum AZ01 and Aspergillus versicolor EF23 were found to closely resemble a catalytic domain of cellobiohydrolase from Trichoderma sp. The three proteins obtained from Aspergillus niger showed resemblance to 1,4-beta glucan cellobiohydrolase A precursor from Aspergillus niger FGSC A733 was also found to have cellobiase and endoglucanase activity was determined using cellobiase and carboxymethyl cellulose as substrates. Cellulase and xylanase zymograms of proteins from A. niger FGSC A733 demonstrated six active bands ranging from 20 kDa to 43 kDa for cellulase and a 31 kDa active band for xylanase. The cellulase produced by Aspergillus niger FGSC A733 on Jatropha seed cake under optimised conditions was used for saccharification of 2% (w/v) common thatch grass (CTG) in combination with Celluclast™. Celluclast™ and Aspergillus niger cellulase were mixed at different ratios and the amount of glucose produced over time was monitored using high performance liquid chromatography (HPLC). A ratio of 2 volumes Celluclast™ to one volume Aspergillus niger cellulase was chosen for the saccharification process. The main enzymes in the mixture were identified using peptide mass fingerprinting as endoglucanases from the Celluclast™ and cellobiase from the Aspergillus niger cellulase. Concentration of the Celluclast™ tenfold times (164 FPU) improved the yield of glucose by 42.8 and 37.8% in acid and alkali pre-treated CTG, respectively. Concentrating Aspergillus niger cellulase (13.2 FPU) decreased the production of glucose by 4.8% in acid pre-treated CTG while in alkali pre-treated CTG, a 5% increase in glucose production was observed. Increasing the substrate loading of acid pre-treated CTG from 2% to 10% (w/v) resulted in a two and a half times increase in glucose production while an increase of 1.5 g/l glucose was obtained from 7% (w/v) alkali pre-treated CTG. Addition of xylanases from Aspergillus niger to the Celluclast™-Aspergillus niger cellulase mixture decreased glucose production by 16.3% on acid pre-treated CTG while there was an increase of 18.3% glucose in alkali pre-treated CTG. Addition of enzyme preparations from Aspergillus versicolor EF23, Penicillium citrium AZ01 and Trichoderma harzianum NCGR 0509 to the Celluclast™-Aspergillus niger cellulase mixture resulted in lower glucose production both in acid and alkali pre-treated CTG. Addition of Pentopan™ improved glucose production by 8 and 25% on 10% acid and 7.5% alkali loading of pre-treated CTG respectively. The optimal conditions for the production of the glucose rich hydrolysate in 10% (w/v) acid and 7% (w/v) alkali pre-treated CTG was found to be the use of Celluclast™-Aspergillus niger cellulase-Pentopan™ mixture (164 FPU Celluclast™ and 13 FPU Aspergillus niger cellulase, 7178 IU) Pentopan™ at 50 °C for 32 hours. The fermentability of the glucose in glucose-rich CTG hydrolysates to ethanol using Saccharomyces cerevisae WBSA 1386 and Candida shehatae CSIR Y-0492 was investigated. The highest yield of ethanol produced by S. cerevisae WBSA 1386 was 9.8 g/l in the alkali pre-treated CTG hydrolysate and 8.7 g/l in acid pre-treated CTG. C. shehatae CSIR Y-0492 produced 9 g/l of ethanol in alkali pre-treated CTG within 48 hours while acid pre-treated CTG hydrolysate produced 8.8 g/l of ethanol within 24 hours of the fermentation process. Addition of the nutrient supplement boosted the ethanol yield in the acid pre-treated hydrolysates. The consumption of glucose during fermentation by S. cerevisae WBSA 1386 and C. shehatae CSIR Y-0492 on average was 97%. The C. shehatae CSIR Y-0492 was expected to produce much higher ethanol yield than the Saccharomyces because of its ability to utilize xylose for ethanol production. This however was not observed in this investigation. The conclusion of this study is that it is possible to produce bioethanol from Hyperrhenia spp. (CTG) using a combination of fungal enzymes for the production of fermentable sugars.

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

Thesis--University of Helsinki, 1991. / Includes bibliographical references.

Thesis--University of Helsinki, 1991. / Includes bibliographical references.

Orientador: Heloiza Ferreira Alves-Prado / Banca: Fabiana da Fonseca Zanoelo / Banca: Ana Maria Cassiolato / Banca: Paulo César Ceresini / Banca: Gustavo Orlando Bonilla Rodriguez / Resumo: O presente trabalho configura-se no estudo da produção de enzimas degradadoras de parede celular de plantas (EDPCP), produzida por Rhizoctonia solani AG-1 IA obtido de culturas de arroz, braquiária e soja. As enzimas inicialmente estudadas foram celulases, β-glucosidase, xilanase, pectinases, amilase, lípases e proteases excretadas extracelularmente para o meio de cultivo. Foram avaliados 87 isolados de Rhizoctonia solani AG-1 IA, e adotado o cultivo em estado sólido utilizando farelo de trigo como fonte de carbono inicial para indução da produção extracelular das enzimas e avaliar o potencial dos isolados. O estudo do efeito da fonte de carbono foi realizado com cinco isolados de cada tipo de cultura, os quais foram cultivados em farelo de trigo, braquiária triturada, palha de soja, palha de milho, sabugo de milho e palha de arroz. Os melhores resultados para atividade xilanase foi do isolado PA_B1F6 obtido de culturas de braquiária quando cultivado em farelo de trigo (15,82 U mL-1 ), para CMCase e Avicelase os melhores resultados foram obtidos com os isolados MA_217 e TO_064 obtidos de culturas de soja quando cultivado em palha de milho (3,24 U mL-1 ) e palha de soja (2,91 U mL-1 ), respectivamente, e para β-glucosidase os melhores resultados foram obtidos com o isolado TO_022 (2,75 U mL-1 ) obtido de cultura de soja cultivado em braquiária. A atividade de amilase foi superior no cultivo em farelo de trigo para o isolado ROB4D7 (142,88 U mL-1 ) obtido da cultura de braquiária e a pectinase do isolado PA_B1F6 (17,69 U mL-1 ) obtido da cultura de braquiária quando cultivado em substrato braquiária. O isolado MT_S085 obtido da cultura de soja apresentou melhor atividade de protease (1154,52 UAP mL-1 ) no cultivo com farelo de trigo e melhor atividade lípase (30,57 U mL-1 ) quando cultivado em substrato de braquiária. Os cinco isolados de cada tipo de cultura foram... / Abstract: The Present work covers the study of the production of degradative enzymes from plant cell wall (EDPCP) produced by Rhizoctonia solani AG-1 IA isolated from rice, signalgrass and soybean. The enzymes studied initially were cellulases, β-glucosidase, xylanase, pectinase, amylase, lipase and proteases extracellularly secreted into the culture medium. It was evaluated 87 isolates of Rhizoctonia solani AG-1 IA and adopted cultivation in the solid state using wheat bran as the initial carbon source for inducing the production of extracellular enzymes and assess the potential isolates. The study of the effect of carbon source was performed with five isolates of each type of culture which were cultured on wheat bran, comminuted signalgrass, soybean straw, corn stover, corn cobs and rice straw. The best result results for xylanase activity was the isolated PA_B1F6 from cultures of signalgrass grown in wheat bran (15.82 U mL- 1 ) to Avicelase and CMCase and the best results were obtained for the isolated of soybean MA_217 and TO_064 when grown corn straw (3.24 U ml-1 ) and soybean straw (2.91 U ml-1 ), respectively. For β-glucosidase the best results were obtained for the isolated of soybean TO_022 (2.75 U ml-1 ) grown in signalgrass. The amylase activity was higher in culture with wheat bran for isolated ROB4D7 from the signalgrass culture (142.88 U mL-1 ). The best pectinase activity was by the PA_B1F6 isolated signalgrass culture (17.69 U mL-1 ) when grown in signalgrass as substrate. The MT_S085 isolated from soybean showed better protease activity (UAP 1154.52 ml-1 ) in culture with wheat bran. For this same isolated, but when grown in signalgrass substrate was observed the best lipase activity (30.57 U mL-1 ). The five isolates of each type were grown in culture substrates that indicated better xylanase activity to evaluate the activity profile over time. Isolates obtained from ... / Doutor

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.

Thesis (MSc)--Stellenbosch University, 2001. / ENGLISH ABSTRACT: The use of fungal enzymes as ruminant feed digestibility enhancers was investigated. Currently, ruminants may not digest 38 to 80 % of fibrous forages' content. A renewed interest in the potential of feed enzymes for ruminants was prompted by the high costs of livestock production, together with the availability of newer enzyme preparations. Direct application of enzyme preparations can improve in vitro dry matter (DM) and neutral detergent fibre (NDF) degradation, indicating that direct-fed fibrolytic enzymes may be effective in enhancing in vivo digestion of forages. Two commercial enzyme products, Fibrozyme and Celluclast, and fungal extracellular enzyme extracts from Aureobasidium pullulans, Trichoderma reesei, Aspergillus aculeatus, and Thermomyces lanuginosus were evaluated for enhancing in vitro feed digestibility. Fibrozyme addition to both wheat straw and lucerne hay did not improve their in vitro digestibilities, even after a two hour pre-incubation period. The four fungal enzyme extracts did not enhance wheat straw's digestibility, but marginal increases were evident for lucerne hay. Celluclast addition resulted in marginal increases in the digestibility of both oat hay and oat silage, with no enhanced effect on lucerne hay and NaOH-treated wheat straw. No relationship could be found between the level of enzyme activity and the degree of feed digestion in the in vitro assay. Enzyme hydrolysis with Celluclast, in the absence of rumen fluid, gave more conclusive results. All the feed samples tested showed a positive response to Celluclast addition, even the less digestible feeds, namely sugarcane bagasse and wheat straw. In vitro results show that the assays were unsuccessful, because almost all of the experiments conducted showed inconclusive results. Alternative feed evaluation assays, which include the in vivo, in sacco and in situ methods of analysis, as well as gas production measurement and in vitro analysis with the DAISyII system, should be evaluated. A more detailed study of feed digestibility should be motivated by determining which feeds are hydrolysable, their chemical composition, i.e. how accessible the feeds are, and also evaluation of feed mixtures. The enzyme supplements also need to be evaluated for optimum temperature and pH, as well as the compilation of enzyme cocktails. / AFRIKAANSE OPSOMMING: Die gebruik van swamensieme om die verteerbaarheid van herkouervoere te verhoog, is ondersoek. Tussen 38 en 80 % van veselagtige voere se inhoud is tans onverteerbaar. 'n Hernieude belangstelling in die potensiaal van voerensieme vir herkouers word deur die hoë koste van veeproduksie, asook die beskikbaarheid van nuwe ensiempreparate gedryf Direkte byvoeging van ensiempreparate kan die in vitro droëmateriaal (DM) en neutrale onoplosbare vesel (NOV) vertering verbeter, wat daarop dui dat fibrolitiese ensieme wat direk gevoer word, effektief mag wees tydens die in vivo vertering van voer. Twee kommersiële ensiemprodukte, Fibrozyme en Celluclast, en die vier ekstrasellulêre ensieme van vier swamme, naamlik Aureobasidium pullulans, Trichoderma reesei, Aspergillus aculeatus, en Thermomyces lanuginosus is vir hul vermoë om die in vitro verteerbaarheid van voere te verbeter getoets. Byvoeging van Fibrozyme by beide koringstrooi en lusernhooi het geen verbetering in hulonderskeie in vitro verteerbaarheid tot gevolg gehad nie, selfs nie eens na 'n twee uur vooraf inkubasieperiode nie. Koringstrooi se verteerbaarheid is nie verbeter deur die byvoeging van die vier swam-ensiempreparate nie, maar 'n minimale verbetering is wel waargeneem in die verteerbaarheid van lusernhooi. Byvoeging van Celluclast het 'n minimale verbetering in beide hawerhooi en hawerkuilvoer se verteerbaarheid tot gevolg gehad, maar geen effek op lusernhooi of NaOH-behandelde koringstrooi se verteerbaarheid nie. Geen verwantskap is tussen die vlak van ensiemaktiwiteit en die mate van vertering tydens die in vitro toets gevind nie. Ensiematiese afbraak met Celluclast, in die afwesigheid van rumenvloeistof, het meer konkrete resultate gelewer. Al die voermonsters het 'n positiewe respons op die byvoeging van Celluclast getoon, selfs ook die minder verteerbare voere, nl. suikerrietbagasse en koringstrooi. In die wyer konteks was die resulate van die in vitro verteringstoetse egter onbeduidend as gevolg van groot variasie in die metings. Alternatiewe voerontledingstoetse, wat moontlik beter resultate mag lewer, sluit in in vivo, in sacco en in situ analises, asook die meting van gasproduksie en in vitro analise met die DAISyII sisteem. 'n Meer uitgebreide studie van voerverteerbaarheid wat die bepaling van die afbraak van voere, hul chemiese samestelling, met ander woorde toeganklikheid van voere, en die ondersoek van voermengsels behels, behoort aandag te geniet. Die ensiemmengsels behoort ook ten opsigte van samestelling, optimum temperatuur en pH ondersoek teword.

Thesis (PhD)--Stellenbosch University, 2008. / ENGLISH ABSTRACT: This study strives to improve two current industrial processes by making them more cost effective through the use of hydrolytic enzymes or microbial systems. The first process targeted is the industrial conversion of starch to ethanol. In the second process, hydrolytic enzymes are applied to the manufacturing of instant coffee. The engineering of microbial systems to convert starch to bio-ethanol in a one-step process may result in large cost reductions in current industrial processes. These reductions will be due to decreased heating energy requirements, as well as a decrease in money spent on the purchase of commercial enzymes for liquefaction and saccharification. In this study, a recombinant Saccharomyces cerevisiae strain was engineered to express the wild-type Aspergillus awamori glucoamylase (GA I) and α-amylase (AMYL III) as well as the Aspergillus oryzae glucoamylase (GLAA) as separately secreted polypeptides. The recombinant strain that secreted functional GA I and AMYL III was able to utilise raw corn starch as carbon source, and converted raw corn starch into bio-ethanol at a specific production rate of 0.037 grams per gram dry weight cells per hour. The ethanol yield of 0.40 gram ethanol per gram available sugar from starch translated to 71% of the theoretical maximum from starch as substrate. A promising raw starch converter was therefore generated. In the second part of this study, soluble solid yields were increased by hydrolysing spent coffee ground, which is the waste generated by the existing coffee process, with hydrolytic enzymes. Recombinant enzymes secreted from engineered Aspergillus strains (β-mannanase, β-endoglucanase 1, β-endo-glucanase 2, and β-xylanase 2), enzymes secreted from wild-type organisms (β-mannanases) and commercial enzyme cocktails displaying the necessary activities (β-mannanase, cellulase, and pectinase) were applied to coffee spent ground to hydrolyse the residual 42% mannan and 51% cellulose in the substrate. Hydrolysis experiments indicated that an enzyme cocktail containing mainly β-mannanase increased soluble solids extracted substantially, and a soluble solid yield of 23% was determined using the optimised enzyme extraction process. Soluble solid yield increases during the manufacturing of instant coffee will result in; (i) an increase in overall yield of instant coffee product, (ii) a decrease in amount of coffee beans important for the production of the product, and (iii) a reduction in the amount of waste product generated by the process. / AFRIKAANSE OPSOMMING: Hierdie studie poog om twee huidige industriële prosesse te verbeter deur die prosesse meer kosteeffektief met behulp van hidroltiese ensieme en mikrobiese sisteme te maak. Die eerste industrie wat geteiken word, is die omskakeling van rou stysel na etanol, en die tweede om hidrolities ensieme in die vervaardiging van kitskoffie te gebruik. Die skep van mikrobiese sisteme om rou-stysel in ’n ’een-stap’ proses om te skakel na bio-etanol sal groot koste besparing tot gevolg hê. Hierdie besparings sal te wyte wees aan die afname in verhittingsenergie wat tydens die omskakelingsproses benodig word, asook ’n afname in die koste verbonde aan die aankoop van duur kommersiële ensieme om die stysel na fermenteerbare suikers af te breek. In hierdie studie is ’n rekombinante Saccharomyces cerevisiae-gis gegenereer wat die glukoamilase (GA I) and α-amilase (AMYL III) van Aspergillus awamori, asook die glukoamilase van Aspergillus oryzae (GLAA) as aparte polipeptide uit te druk. Die rekombinante gis wat die funksionele GA I en AMYL III uitgeskei het, was in staat om op die rou-stysel as koolstofbron te groei, en het roustysel na bio-etanol teen ’n spesifieke tempo van 0.037 gram per gram droë gewig biomassa per uur omgeskakel. Die etanolopbrengs van 0.40 gram per gram beskikbare suiker vanaf stysel was gelykstaande aan 71% van die teoretiese maksimum vanaf stysel as substraat. ’n Belowende gis wat roustysel kan omskakel na bio-etnaol was dus geskep. In die tweede deel van hierdie studie is die opbrengs in oplosbare vastestowwe vermeerder deur die koffie-afval wat tydens die huidige industrieële proses genereer word, met hidrolitiese ensieme te behandel. Rekombinante ensieme afkomstig vanaf Aspergillus-rasse (β-mannanase, β-endoglukanase 1, β-endo-glukanase 2 en β-xilanase 2), ensieme deur wilde-tipe organismes uitgeskei (β-mannanase), asook kommersiële ensiempreparate wat die nodige ensiemaktiwiteite getoon het (β-mannanase, sellulase en pektinase) is gebruik om die oorblywende 42% mannaan en 51% sellulose in koffie-afval te hidroliseer. Hidrolise eksperimente het getoon dat ’n ensiempreparaat wat hoofsaaklik mannanase bevat, die oplosbare vastestofopbrengs grootliks kan verbeter, met ’n verhoogde opbrengs van 23% tydens geöptimiseerde ensiembehandelings. ’n Verhoogde opbrengs in oplosbare vastestowwe tydens die vervaardiging van kitskoffie sal die volgende tot gevolg hê: (i) ’n toename in totale opbrengs van kitskoffie produk, (ii) ’n afname in die hoeveelheid koffiebone wat vir die produksie ingevoer moet word, en (iii) ’n afname in die hoeveelheid afval wat tydens die vervaardigingsproses produseer word.

En réponse aux préoccupations environnementales, les procédés industriels comme la production de bioéthanol de deuxième génération sont apparus. Basés sur la conversion enzymatique de la cellulose, ces processus font face à un problème majeur, la réticence de la biomasse lignocellulosique à l'hydrolyse. Afin de résoudre ce problème et celui lié aux coûts d'utilisation de cocktails de cellulases, les recherches se sont axées sur diverses méthodes permettant d'augmenter l'hydrolyse de la cellulose. Les champignons filamenteux sont connus pour être des dégradeurs naturels du bois et, par conséquent, sont utilisés dans de nombreuses applications biotechnologiques. Récemment, quelques études ont révélé l'importance d'enzymes fongiques telles que la CDH et les GH61 dans la dégradation oxydative de la lignocellulose. Les travaux réalisés au cours de cette thèse ont permis de démontrer l'importance de ces enzymes oxydatives dans les phénomènes de déconstruction de la lignocellulose. L'utilisation de ces enzymes oxydatives offre de réelles voies d'amélioration de la production de bioéthanol et de compréhension de la dégradation in vivo des lignocelluloses par les champignons. / In response to environmental concerns, industrial processes such as second generation bioethanol production have emerged. Based on enzymatic cellulose conversion, these processes are confronted with a major problem, the recalcitrance of lignocellulosic biomass. To solve the problem caused by substrate recalcitrance and high cost of cellulase cocktails, research has focused on various methods to enhance cellulose hydrolysis. Fungi are known to be natural degraders of wood and consequently are used in derived biotechnological applications. Recently, several studies have revealed the importance of fungal enzymes such as GH61 and CDH in the oxidative degradation of lignocellulose. During the work done on this thesis, we demonstrated implication of these oxidative enzymes in lignocellulose deconstruction to enhance hydrolysis performed by more classical cellulases. Utilization of oxidative enzymes offers a suitable way for bioethanol processing enhancement and comprehension of the in vivo lignocellulosic degradation by fungi.