Department of Microbiology

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Biodegradation of winery wastewater
(Stellenbosch : University of Stellenbosch, 2003-04) Malandra, Lida; Bloom, M.; Wolfaardt, Gideon M.; University of Stellenbosch. Faculty of Science. Dept. of Microbiology.
ENGLISH ABSTRACT: Large volumes of wastewater are generated annually during the grape harvest season from various processing and cleaning operations at wineries, distilleries and other wine-related industries. South African regulatory bodies dictate that wastewater should have a pH of 5.5 to 7.5 and a chemical oxygen demand (COD) lower than 75 mg/L. However, winery wastewater has a typical pH of 4 to 5 and a COD varying between 2 000 and 12 000 mg/L. Urban wineries channel the wastewater to local sewage treatment facilities and are often heavily fined for exceeding governmental requirements. Rural wineries usually have little or no treatment operations for their wastewater and it is often irrigated onto crops, which may result in environmental pollution and contamination of underground water resources. Various criteria are important in choosing a wastewater treatment system, such as an ecofriendly process that is flexible to withstand various concentration loads and characteristics, requiring low capital and operating costs, minimal personal attention and do not require too much land. In this study, a large variation in COD, pH and chemical composition of the winery wastewater was observed that could be related to varying factors such as the harvest load, operational procedures and grape variety. Wastewater from destemming and pressing operations contained higher concentrations of glucose, fructose and malic acid, which originated from the grape berries. The fermentable sugars (glucose and fructose) contributed to almost half of the COD with a smaller contribution from ethanol and acetic acid. The low pH can be ascribed to relative high concentrations of organic acids in the wastewater. The efficacy of biological treatment systems depends strongly on the ability of microorganisms to form biofilm communities that are able to degrade the organic compounds in the wastewater. Preliminary identification of microorganisms that naturally occur in winery wastewater indicated the presence of various bacterial and yeast species that could be effective in the biological treatment of the wastewater. When evaluated as pure cultures under aerobic conditions, some of the yeast isolates effectively reduced the COD of a synthetic wastewater, whereas the bacterial isolates were ineffective. The most effective yeast isolates were identified as Pichia rhodanensis, Kloeckera apiculata, Candida krusei and Saccharomyces cerevisiae. Our search for cost-effective biological treatment systems led to the evaluation of a Rotating Biological Contactor (RBC) for the treatment of winery wastewater. The RBC was evaluated on a laboratory scale with 10% (v/v) diluted grape juice and inoculated with a mixed microbial community isolated from winery wastewater. The results showed a reduction in the COD that improved with an extended retention time. Evaluation of the RBC on-site at a local winery during the harvest season resulted on average in a 41% decrease in COD and an increase of 0,75 pH units. RFLP analysis of the biofilm communities within the RBC confirmed a population shift in both the bacterial and fungal species during the evaluation period. The most dominant yeast isolates were identified with 18S rDNA sequencing as Saccharomyces cerevisiae, Candida intermedia, Hanseniaspora uvarum and Pichia membranifaciens. All these species are naturally associated with grapes and/or water and with the exception of Hanseniaspora uvarum, they are able to form either simple or elaborate pseudohyphae.
Characterisation of L-malic acid metabolism in strains of Saccharomyces and the development of a commercial wine yeast strain with an efficient malo-ethanolic pathway
(Stellenbosch : Stellenbosch University, 2002-12) Volschenk, Heinrich; Van Vuuren, H. J. J.; Bloom, M.; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
ENGLISH ABSTRACT: L-Malic and tartaric acid are the most prominent organic acids in wine and playa crucial role in winemaking processes and wine quality, including the organoleptic quality and the physical, biochemical and microbial stability of wine. The production of premium wines depends on the oenologist's skill to accurately adjust wine acidity to obtain the optimum balance with other wine components to produce wine with optimum colour and flavour. Strains of Saccharomyces, in general, rarely degrade L-malic acid completely in grape must during alcoholic fermentation, with relatively minor modifications in total acidity during vinification. The degree of L-malic acid degradation, however, varies from strain to strain. Some strains of Saccharomyces are known to be able to degrade a higher percentage of L-malic acid, but the underlying reason for this phenomenon is unknown. The underlying mechanisms of this phenomenon have been partially revealed during preliminary transcriptional regulation research during this study. In contrast, S. pombe cells can effectively degrade up to 29 gil L-malic acid via the malo-ethanolic pathway that converts L-malic acid to pyruvate and CO2, and ultimately to ethanol under fermentative conditions. A number of reasons for the weak degradation of L-malic acid in Saccharomyces cerevisiae have been postulated. Firstly, S. cerevisiae lacks the machinery for the active transport of L-malic acid found in S. pombe and relies on rate-limiting simple diffusion for the uptake of extracellular L-malic acid. Secondly, the malic enzyme of S. cerevisiae has a significantly lower substrate affinity for L-malic acid (Km = 50 mM) than that of S. pombe (Km = 3.2 mM), which contributes to the weaker degradation of L-malic acid in S. cerevisiae. Lastly, the mitochondrial location of the malic enzyme of S. cerevisiae, in contrast to the cytosolic S. pombe malic enzyme, suggests that the S. cerevisiae malic enzyme is inherently subject to the regulatory effects of fermentative metabolism. The malate permease gene tmael) and the malic enzyme gene (mae2) of S. pombe was therefore cloned and co-expressed in single or multi-copy under regulation of the constitutive S. cerevisiae 3-phosphoglycerate kinase (PGK1) promoter and terminator sequences in a laboratory strain of S. cerevisiae. This introduced a strong malo-ethanolic phenotype in S. cerevisiae where L-malic acid was rapidly and efficiently degraded in synthetic and Chardonnay grape must with the concurrent production of higher levels of ethanol. Functional expression of the malo-ethanolic pathway genes of S. pombe in a laboratory strain of S. cerevisiae paved the way for the genetic modification of industrial wine yeast strains of Saccharomyces for commercial winemaking. A prerequisite for becoming an inherited component of yeast is the stable integration of the malo-ethanolic genes into the genome of industrial wine yeast strains. Genetic engineering of wine yeasts strains of Saccharomyces is, however, complicated by the homothallic, multiple ploidy and prototrophic nature of industrial strains of Saccharomyces. Transformation and integration of heterologous genes into industrial strains of Saccharomyces require the use of dominant selectable markers, i.e. antibiotic or toxic compound resistance markers. Integration of these markers into the yeast genome is, however, not acceptable for commercial application due to the absence of long-term risk assessment and consumer resistance. A unique strategy for the integration of the S. pombe mae} and mae2 expression cassettes without the incorporation of any non-yeast derived DNA sequences was. The malo-ethanolic cassette, containing the S. cerevisiae PGK} promoter and terminator regions together with the S. pombe mae] and mae2 open reading frames, was integrated into the VRA3 locus of an industrial strain of Saccharomyces bayanus EC 1118 during co-transformation with a phleomycin-resistance plasmid, pUT332. After initial screening for phleomycin resistance, S. bayanus EC1118 transformants were cured of the phleomycin-resistance plasmid, resulting in the loss of non-yeast derived DNA sequences. After correct integration of the mae] and mae2 expression cassettes was verified, small-scale vinification in synthetic and Chardonnay grape must with stable transformants resulted in rapid and complete degradation of L-malic acid during the early stages of alcoholic fermentation. Integration and expression of the malo-ethanolic genes in S. bayanus ECll18 had no adverse effect on the fermentation ability of the yeast, while sensory evaluation and chemical analysis of the Chardonnay wines indicated an improvement in wine flavour compared to the control wines, without the production of any off-flavours.
Characterisation of the malate transporter and malic enzyme from Candida utilis
(Stellenbosch : University of Stellenbosch, 2011-10) Saayman, Maryna; Bloom, M.; Van Zyl, Willem Heber; University of Stellenbosch. Faculty of Science. Dept. of Microbiology.
ENGLISH ABSTRACT: Yeast species differ remarkably in their ability to degrade extracellular dicarboxylic acids and to utilise them as their only source of carbon. The fission yeast Schizosaccharomyces pombe effectively degrades L-malate, but only in the presence of an assimilable carbon source. In contrast, the yeast Saccharomyces cerevisiae is unable to effectively degrade L-malate, which is ascribed to the slow uptake of L-malate by diffusion. In contrast, the yeast Candida utilis can utilise L-malate as the only source of carbon and energy, but this is subject to substrate induction and catabolite repression. Very little research has been done on a molecular level in C. utilis and only a few of its genes have been studied. In this study, we have shown that the yeast C. utilis effectively degraded extracellular L-malate and fumarate, but in the presence of glucose or other assimilable carbon sources, the transport and degradation of these dicarboxylic acids was repressed. The transport of both dicarboxylic acids was shown to be strongly inducible by either L-malate or fumarate and kinetic studies suggest that the same transporter protein transports the two dicarboxylic acids. In contrast, S. pombe effectively degraded extracellular L-malate, but not fumarate, only in the presence of glucose or other assimilable carbon sources. The S. pombe malate transporter was unable to transport fumarate, although fumarate inhibited the uptake of L-malate. In order to clone the C. utilis dicarboxylic acid transporter, a cDNA library from C. utilis was constructed using a number of strategies to ensure representativeness and high transformation frequencies. The cDNA library was transformed in a S. cerevisiae strain carrying a plasmid containing the S. pombe malic enzyme gene (mae2) to allow screening for a malate-degrading S. cerevisiae clone. However, no positive clones that would indicate the successful cloning of the C. utilis malate transporter were obtained. The C. utilis malic enzyme gene, CuME, was subsequently isolated from the cDNA library based on conserved sequence homologies with the genes of S. cerevisiae and S. pombe, and characterised on a molecular and biochemical level. Sequence analysis revealed an open reading frame of 1926 bp, encoding a 641 amino acid polypeptide with a predicted molecular weight of 70.2 kDa. The optimum temperature for the C. utilis malic enzyme was 52°C and the enzyme was stable at 50°C for 2 hours. The inferred amino acid sequence showed significant homology with the malic enzymes of S. pombe and S. cerevisiae. Expression of the CuME gene is subject to glucose repression and substrate induction, as was observed for the dicarboxylic acid transporter from C. utilis. The CuME gene was successfully coexpressed with the S. pombe malate permease gene (mae1), resulting in a recombinant strain of S. cerevisiae able to effectively degrade L-malate.
Evaluation and optimisation of fungal enzymes for microbial bioprocessing of rooibos tea
(Stellenbosch : Stellenbosch University, 2005-03) Pengilly, Mia; Bloom, M.; Van Zyl, Willem Heber; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
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%.
Evaluation of commercial enzymes for the bioprocessing of Rooibos tea
(Stellenbosch : Stellenbosch University, 2005-04) Coetzee, Gerhardt; Bloom, M.; Van Zyl, Willem Heber; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
ENGLISH ABSTRACT: The Rooibos tea plant (Aspalathus linearis) is indigenous to South Africa and occurs only in the Western Cape's Cedarberg region. Rooibos tea is produced from the leaves and fine stems of the plant. The tea is normally prepared by brewing the leaves and consuming the liquor. However, the Rooibos plant is not only used to prepare tea; the plant extracts are also used in various neutraceutical and pharmaceutical products, including health drinks, iced tea, soaps and moisturising creams. Although the tea plant contains native enzymes responsible for the colour and aroma development of Rooibos tea, the disruption and maceration of the plant material during processing is insufficient to allow these enzymes proper access to the substrates responsible for Rooibos tea's characteristics. The current processing of Rooibos tea is also time consuming and is done under uncontrolled conditions, leading to unnecessary loss in aroma and antioxidant content. The addition of enzymes could improve the maceration of the plant material, shorten the processing time and improve the extraction of aroma, colour and antioxidant components. During this study, 16 commercially available microbial enzymes were evaluated on three different Rooibos substrates for the improvement of aroma and colour development, as well as the extraction of soluble solids (SS) and total polyphenols (TP). Thirteen enzymes were evaluated on spent tea for the enhanced extraction of soluble solids and to determine the best candidates for further evaluation on fermented and green Rooibos tea. Seven of the enzymes improved the yield in SS from spent tea. Up to 232% improvement was obtained, depending on the type of enzyme and dosage applied. The best six enzyme preparations were further evaluated on fermented Rooibos tea. For Depol™ 670L at 20 ul/g tea, the laboratory treatment increased the yield in SS by 44%, while small-scale industrial simulations increased the SS by 26%. However, an increase in the yield in SS was usually accompanied by a decrease in the %TP/SS ratio, indicating that mainly inactive compounds were extracted. Based on the results with the commercial enzymes, twelve "synthetic" enzyme cocktails, consisting of different combinations of commercial enzymes were designed, of which three cocktails released increased amounts of SS without decreasing the %TP/SS ratio significantly. Thirteen enzymes were evaluated on dried and freshly cut green Rooibos tea, with three enzymes (Depol™ 670L, Pectinex Ultra SP-L and Depol™ 692L) increasing the yield in SS between 21% and 66%, and the TP content between 11% and 47%. Laccase was the best candidate in improving colour development from green tea, with the improvement being slightly better at 50°C than at 40°C. All the "synthetic" cocktails containing laccase improved the colour extract of all three substrates evaluated, but also significantly decreased the TP and antioxidant content. However, lower dosages of laccase resulted in colour development with little loss in the antioxidant content. Due to the promising results obtained with the treatments of Rooibos tea with laccases, it was decided to clone and express the laccase gene (lacA) of Pleurotus ostreatus into Aspergillus niger. The gene was successfully transformed into A. niger, but the expression of the recombinant gene was not effective.
The expression of fungal enzymes in Saccharomyces cerevisiae for bio-ethanol production from raw cornstarch
(Stellenbosch : Stellenbosch University, 2011-03) Viktor, Marko Johann; Bloom, M.; Van Zyl, Willem Heber; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
ENGLISH ABSTRACT: Reliable energy resources could be considered as one of the cornerstones of the prosperity of the human race. The growing human population is constantly exerting more pressure on the world’s natural resources, which include natural fossil fuels that are non‐renewable. There are concerns regarding the use of fossil fuels due to its growing scarcity and its negative impact on the environment. There is thus a growing need in the world for energy sources that are renewable, more or less carbon neutral and therefore with a minimum environmental impact. Renewable energy is currently being harnessed from the wind, water and sun, but to a limited extent. These forms of natural resources are very attractive for the production of renewable energy, but these technologies are difficult to apply in the current transportation sector. Biofuels provide an alternative to the current use of liquid fossil fuels and it could be able to sustain the current fleet of automobiles worldwide in the intermediate to long term with minimal adjustment to the engines of these vehicles. Extensive research has been done on the production processes for biofuels. Previous processes included the use of high temperatures and acids that further increased the total production cost and thus making biofuels less attractive as an alternative energy source. Recent research has suggested a wide range of organic materials as substrate for the production of biofuels, which include lignin, hemi‐cellulose, cellulose and starch. Processes based on hemi‐cellulose, cellulose and lignin as substrate are still in its early research stages and commercial application of these processes will only occur over the medium‐ to long‐term. Starch is a very good alternative source for the production of biofuels, but there is a need for a microbial system for the conversion of starch to bio‐ethanol in a single step, referred to as Consolidated Bioprocessing (CBP). This would reduce the overall production cost of bio‐ethanol and thus making starch‐based substrates more attractive as an alternative energy source. The cost saving will be mainly due to the elimination of the pre‐treatment of raw starch at high temperatures and the addition of enzymes for the liquefaction and saccharification of starch to simple sugars. However, as there is no currently no known microbial organism known that can produce the required enzymes (i.e. amylases) as well as ferment the resulting sugars to ethanol, heterologous expression of these enzymes in a host strain able to ferment sugars could provide the best alternative system. In the first part of this study, 36 fungal strains known for the production of amylases were screened and compared for the highest extracellular enzyme activity on raw corn starch. The best two candidates, i.e. Aspergillus tubingensis (T8.4) and Mucor cincinelloides (1180), were then further evaluated to determine which organism has the highest efficiency when combined with a Saccharomyces cerevisiae laboratory strain. In fermentation experiments, A. tubingensis (T8.4) in combination with S. cerevisiae Y102 yeast strain resulted in the highest yield of ethanol. Literature on A. tubingensis is limited compared with other Aspergillii and it was previously accepted that A. tubingensis has the highest homology with Aspergillus niger. However, other reports – including the present study ‐ found that A. tubingensis is closer related to other Aspergillus spp. with regard to its amylolytic enzymes. The α‐amylase gene of A. tubingenis has a homology of 99.00% with that of Aspergillus kawachii whereas the glucoamylase gene has a homology of 99.26% with that of Aspergillus shirousami. In the second part of this study, two recombinant S. cerevisiae strains were constructed to express the wild type A. tubingensis α‐amylase (Atamy) and glucoamylase (Atglu), respectively. The combination of the two recombinant yeast strains was able to completely hydrolyse and also utilize raw corn starch for the production of bio‐ethanol, with a yield of 11.04 g/l of ethanol, which translates to 98% of the theoretical yield from starch with a 52% conversion of the total raw starch. This rate of conversion is lower than other reports which indicated up to 82% and 96% of the theoretical yield of ethanol from raw and soluble starch, respectively, by α‐ and glucoamylase. Furthermore, the combined expressed of the two genes was much more effective than when only one of the two genes were expressed, with a yield of 0.32 g/l ethanol for only Atamy and 2.52 g/l ethanol for Atglu. This proved that the combination of the A. tubingensis genes were best suited for the production of biofuels from raw starch. This also proved that the concept of constructing an amylolytic yeast strain capable of raw starch hydrolysis and fermentation was indeed feasible.
Identification and characterisation of a Cryptococcus laurentii Abo 510 Phytase
(Stellenbosch : Stellenbosch University, 2004-03) Van Staden, Jason; Bloom, M.; Van Zyl, Willem Heber; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
ENGLISH ABSTRACT: Phosphorus is vital for growth of all life forms and is a fundamental component of nucleic acids, ATP and several other biological compounds. Oilseeds and cereal grains, two major constituents of the diet of animals, contain phytic acid, which is the main storage form of phosphorus in plant cells. Monogastric animals, such as poultry and pigs, are not capable of utilising the bound phosphorus in phytic acid since they do not produce phytase, the essential hydrolysing enzyme. Microbial phytase is therefore added to the animal feed to enhance the availability of phosphorus and thus minimise phosphorus pollution and phosphorus supplementation in diets. For a phytase to be effective in the poultry and swine industry, it needs to be able to release phytic acid phosphorus in the digestive tract, it must be thermostable to resist feed processing and must be inexpensive to produce. One approach for developing an efficient phytase for the animal feed industry is by identifying new phytases from microorganisms, plants and animals. In this study, 11 strains of the genus Cryptococcus were screened for 'phytase activity. Initially, a differential agar plate screening method was employed to determine if any Cryptococcus species were able to express phytase, after which production was confirmed in different liquid media. Cryptococcus laurentii Abo 510 was identified as a strain with significant phytase activity. The C. laurentii Abo 510 strain showed clear zones on the differentialmedia agar plates and the production of phytase at high levels was observed when using wet cells grown in liquid media. The C. laurentii Abo 510 strain produced maximal phytase activity at a relatively high temperature (62°C) and in an acidic pH range (pH 5.0). This phytase also showed a broad substrate specificity that may assist in the release of other phosphate compounds captured in feedstuff. Although the phytase did not require any metal ions for its activity, several metal ions caused inhibition of the phytase activity. The enzyme was stable when exposed to 70°C for up to 180 minutes with only 40% loss in activity. Phosphorus addition to the culture media and enzyme assay solution at concentrations exceeding 500 f.!Minhibited the phytase activity completely. Different carbon sources in the culture media also influenced the phytase activity. The enzyme was determined to be a cell wall-associated phytase with little intracellularactivity.
Optimization of the conversion of lignocellulosic agricultural by-products to bioethanol using different enzyme cocktails and recombinant yeast strains
(Stellenbosch : Stellenbosch University, 2011-03) Mubazangi, Munyaradzi; Van Zyl, Willem Heber; Bloom, M.; Garcia-Aparicio, Maria P.; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
ENGLISH ABSTRACT: The need to mitigate the twin crises of peak oil and climate change has driven a headlong rush to biofuels. This study was aimed at the development of a process to efficiently convert steam explosion pretreated (STEX) sugarcane bagasse into ethanol by using combinations of commercial enzyme cocktails and recombinant Saccharomyces cerevisiae strains. Though enzymatic saccharification is promising in obtaining sugars from lignocellulosics, the low enzymatic accessibility of the cellulose and hemicellulose is a key impediment thus necessitating development of an effective pretreatment scheme and optimized enzyme mixtures with essential accessory activities. In this context, the effect of uncatalysed and SO2 catalysed STEX pretreatment of sugarcane bagasse on the composition of pretreated material, digestibility of the water insoluble solids (WIS) fraction and overall sugar recovery was investigated. STEX pretreatment with water impregnation was found to result in a higher glucose recovery (28.1 g/ 100 bagasse) and produced WIS with a higher enzymatic digestibility, thus was used in the optimization of saccharification and fermentation. Response surface methodology (RSM) based on the 33 factorial design was used to optimize the composition of the saccharolytic enzyme mixture so as to maximize glucose and xylose production from steam exploded bagasse. It was established that a combination of 20 FPU cellulase/ g WIS and 30 IU -glucosidases/ g WIS produced the highest desirability for glucose yield. Subsequently the optimal enzyme mixture was used to supplement enzyme activities of recombinant yeast strains co-expressing several cellulases and xylanases in simultaneous saccharification and fermentations SSFs. In the SSFs, ethanol yield was found to be inversely proportional to substrate concentration with the lowest ethanol yield of 70% being achieved in the SSF at a WIS concentration of 10% (w/v). The ultimate process would however be a one-step “consolidated” bio-processing (CBP) of lignocellulose to ethanol, where hydrolysis and fermentation of polysaccharides would be mediated by a single microorganism or microbial consortium without added saccharolytic enzymes. The cellulolytic yeast strains were able to autonomously multiply on sugarcane bagasse and concomitantly produce ethanol, though at very low titres (0.4 g/L). This study therefore confirms that saccharolytic enzymes exhibit synergism and that bagasse is a potential substrate for bioethanol production. Furthermore the concept of CBP was proven to be feasible.

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