Alcohol fermentation of lactose was investigated using a recombinant flocculating Saccharomyces cerevisiae, expressing the LAC4 (coding for beta-galactosidase) and LAC12 (coding for lactose permease) genes of Kluyveromyces marxianus. Data on yeast fermentation and growth on a medium containing lactose as the sole carbon source are presented. In the range of studied lactose concentrations, total lactose consumption was observed with a conversion yield of ethanol close to the expected theoretical value. For the continuously operating bioreactor, an ethanol productivity of 11 g L(-1) h(-1) (corresponding to a feed lactose concentration of 50 g L(-1) and a dilution rate of 0.55 h(-1)) was obtained, which is 7 times larger than the continuous conventional systems. The system stability was confirmed by keeping it in operation for 6 months.

A review on the main aspects associated with yeast flocculation and its application in biotechnological processes is presented. This subject is addressed following three main aspects -the basics of yeast flocculation, the development of "new" flocculating yeast strains and bioreactor development. In what concerns the basics of yeast flocculation, the state of the art on the most relevant aspects of mechanism, physiology and genetics of yeast flocculation is reported. The construction of flocculating yeast strains includes not only the recombinant constitutive flocculent brewer's yeast, but also recombinant flocculent yeast for lactose metabolisation and ethanol production. Furthermore, recent work on the heterologous β-galactosidase production using a recombinant flocculent Saccharomyces cerevisiae is considered. As bioreactors using flocculating yeast cells have particular properties, mainly associated with a high solid phase hold-up, a section dedicated to its operation is presented. Aspects such as bioreactor productivity and culture stability as well as bioreactor hydrodynamics and mass transfer properties of flocculating cell cultures are considered. Finally, the paper concludes describing some of the applications of high cell density flocculation bioreactors and discussing potential new uses of these systems.

Alcoholic fermentation of cheese whey permeate was investigated using a recombinant flocculating Saccharomyces cerevisiae, expressing the LAC4 (coding for beta-galactosidase) and LAC12 (coding for lactose permease) genes of Kluyveromyces marxianus enabling for lactose metabolization. Data on yeast fermentation and growth on cheese whey permeate from a Portuguese dairy industry is presented. For cheese whey permeate having a lactose concentration of 50 gL(-1), total lactose consumption was observed with a conversion yield of ethanol close to the expected theoretical value. Using a continuously operating 5.5-L bioreactor, ethanol productivity near 10 g L(-1) h(-1) (corresponding to 0.45 h(-1) dilution rate) was obtained, which raises new perspectives for the economic feasibility of whey alcoholic fermentation. The use of 2-times concentrated cheese whey permeate, corresponding to 100 gL(-1) of lactose concentration, was also considered allowing for obtaining a fermentation product with 5% (w/v) alcohol.

Background: Consolidated bioprocessing, which combines saccharolytic and fermentative abilities in a single microorganism, is receiving increased attention to decrease environmental and economic costs in lignocellulosic biorefineries. Nevertheless, the economic viability of lignocellulosic ethanol is also dependent of an efficient utilization of the hemicellulosic fraction, which contains xylose as a major component in concentrations that can reach up to 40% of the total biomass in hardwoods and agricultural residues. This major bottleneck is mainly due to the necessity of chemical/enzymatic treatments to hydrolyze hemicellulose into fermentable sugars and to the fact that xylose is not readily consumed by Saccharomyces cerevisiae-the most used organism for large-scale ethanol production. In this work, industrial S. cerevisiae strains, presenting robust traits such as thermotolerance and improved resistance to inhibitors, were evaluated as hosts for the cell-surface display of hemicellulolytic enzymes and optimized xylose assimilation, aiming at the development of whole-cell biocatalysts for consolidated bioprocessing of corn cobderived hemicellulose. Results: These modifications allowed the direct production of ethanol from non-detoxified hemicellulosic liquor obtained by hydrothermal pretreatment of corn cob, reaching an ethanol titer of 11.1 g/L corresponding to a yield of 0.328 g/g of potential xylose and glucose, without the need for external hydrolytic catalysts. Also, consolidated bioprocessing of pretreated corn cob was found to be more efficient for hemicellulosic ethanol production than simultaneous saccharification and fermentation with addition of commercial hemicellulases. Conclusions: These results show the potential of industrial S. cerevisiae strains for the design of whole-cell biocatalysts and paves the way for the development of more efficient consolidated bioprocesses for lignocellulosic biomass valorization, further decreasing environmental and economic costs.

AbstractA phenotypic screening of 12 industrial yeast strains and the well-studied laboratory strain CEN.PK113-7D at cultivation temperatures between 12 °C and 40 °C revealed significant differences in maximum growth rates and temperature tolerance. Two Saccharomyces cerevisiae strains, one performing best at sub-, and the other at supra-optimal temperatures, plus the laboratory strain, were selected for further physiological characterization in well-controlled bioreactors. The strains were grown in anaerobic chemostats, at a fixed specific growth rate of 0.03 h-1 and sequential batch cultures at 12, 30, and 39 °C. We observed significant differences in biomass and ethanol yields on glucose, biomass protein and storage carbohydrate contents, and biomass yields on ATP between strains and cultivation temperatures. Increased temperature tolerance coincided with higher energetic efficiency of cell growth, indicating that temperature intolerance is a result of energy wasting processes, such as increased turnover of cellular components (e.g. proteins) due to temperature induced damage.

A flocculating yeast Saccharomyces cerevisiae ura3 was transformed by the method based on treatment of intact cells with lithium acetate plus single-stranded carrier DNA using the shuttle vector pYAC4. The transformation efficiency was above 10 3 transformants per microgram of plasmid DNA which is similar to other described yeast transformation systems. Under selective pressure, the transformed cells were stable and maintained the flocculation ability. Thus, this simple transformation system can be used for gene expression studies in flocculating yeasts, overcoming disadvantages of conventional methods such as the spheroplast one. Several methods to transform DNA into yeast cells are currently available, such as transformation of spheroplasts made with lytic enzymes (HINNEN et al. 1978; BEGGS 1978), transformation of intact cells made competent either by lithium acetate (ITO et al. 1983, SCHIESTL and GIETZ 1989) or by electroporation (MANIVASAKAM and SHIESTL 1993). To our knowledge, there are no published methods for preparing pYAC clones using transformation protocols with the lithium acetate method (BURKE and OLSON 1991). Yeast flocculation, defined as the aggregation of yeast cells into flocs or clumps which sediment rapidly in culture medium, is one of the most important properties of yeast strains used in traditional processes like brewing and winemaking. It might also be useful in modern biotechnology for the production of heterologous proteins and it has already shown its value in the development of continuous fermentation processes (TEIXEIRA et al. 1990) having a strong influence on the process overall performance. On one hand, it allows for the operation in a continuous mode at high cell concentration, thus increasing system overall productivity. On the other hand, biomass concentration in the effluent is reduced, thus decreasing product separation/purification costs. The flocculent yeast strain used in this work is a Saccharomyces cerevisiae strain with an ura3 gene constructed by ultraviolet mutagenesis and tested for its resistance towards 5-fluoro-orotic acid. The use of pYAC4 plasmid-a yeast artificial chromosome shuttle vector containing the yeast URA3 gene as selectable marker-to transform intact Saccharo-myces cerevisiae cells using lithium acetate and single-stranded carrier DNA is reported (VENÂNCIO et al. 1995). Materials and methods Strains and media: The flocculent wild-type (WT) haploid strain of S. cerevisiae NCYC869 was mutagenized by ultraviolet radiation and ura3 mutants were isolated by positive selection using 5-fluoro-orotic acid as described previously (LIMA et al. 1995). An auxotrophic strain A3 (MATa FLO1 ura3) was used for transformation experiments. Escherichia coli HB101 (Bio-Rad) was used for all bacterial transformations and plasmid propagations. Yeast strains were cultured in complete YEPD medium (2% glucose, 2% peptone, 1% yeast extract) or minimal SD medium (2% glucose,

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