Construction And Fermentation Of Flocculating Saccharomyces Cerevisiae For Ethanol Production

The ability of yeast cells to flocculate is of considerable importance for the fuel ethanol industry, as it provides an effective, environment-friendly and simple way to separate yeast cells from the final culture fluid. The aim of this study is to constuct a recombinant yeast using the flocculent gene FLO1 by the genetic modification method. The research includes the following: First, the recombinant plasmid pYX-FLO was constructed using the expression vector pYX212 and gene FLO1 and then was transformed into S. cerevisiae W303-1A. The transformant yeast W303-1A F4 was obtained. Secondly, the recombinant plasmid pYX-FLO-kan was constructed using the previous vector pYX-FLO and kanMX cassette and then was transformed into the industrial yeast Angel yeast and ZWA46, respectively. The industrial flocculating yeast Ay-F12 and ZWA46-F2 were obtained. Thirdly, The repeated-batch fermentation with cell recycled and continuous fermentation were performed with the recombinant yeast ZWA46-F2. Lastly, the cell wall protein Flo1p encoded by the gene FLO1 was studied. The main results were described as follows:1. The flocculation ability of the transformant W303-1A F4 reached 70 % and was stable during 14 serial batch cultivations. The flocculation onset of the strain is in the early stationary growth phase, not coincident with the glucose depletion in the cultural medium. And the flocculation ability of the transformant showed no difference with the initial pH ranging from 2.5 to 7.0. The above results made it possible that the industrial flocculating yeast could be obtained using flocculation gene FLO1 controlled by promotor TPI.2. The flocculation ability of the industrial flocculating Ay-F12 reached 80 %. The flocculation onset of the strain is in the early stationary growth phase, not coincident with the glucose depletion in the cultural medium. And the flocculation ability of the transformant showed no difference with the initial pH ranging from 3.5 to 5.5. The maximum ethanol concentration of 8.1 % (v/v) with an ethanol yield on glucose of 84.5 % of the theoretic value was achieved. Furthermore the ethanol concentration of the transformant strain Ay-F12 and its other properties such as the biomass, residual glucose concentration and change of pH were similar to those of the wild-type strain.3. The flocculation ability of the transformant Ay-F12 showed no difference with the temperature ranging from 30℃to 50℃. And the ethanol concentration reached 6.7 % at 40℃, which combined the good property of ehanol production and the thermotolerance.4. The flocculation ability of the industrial flocculating yeast ZWA46-F2 reached more than 90 % and was stable during 20 serial batch cultivations. The flocculation onset of the strain is in the early stationary growth phase, not coincident with the glucose depletion in the cultural medium. And the flocculation ability of the transformant showed no difference with the initial pH ranging from 2.5 to 6.0.5. The batch fermentation was performed in the 5 L fermentor with the industrial recombinant ZWA46-F2. The maximum ethanol concentration of 8.6 % (v/v) with an ethanol yield on glucose of 89.8% of the theoretic value was achieved. Furthermore the ethanol concentration of the transformant strain ZWA46-F2 and its other properties were similar to those of the wild-type strain ZWA46.6. The repeated-batch fermentation was performed with the industrial recombinant ZWA46-F2. In the 6th batch, the final ethanol concentration was 8.5 % for 30 h, which corresponded to a volumetric productivity of 2.27 g/l h. And the productivity was improved by 78.7 %, compared with that of 1.27 g/l h in the first batch fermentation. The ethanol concentration reached 9.0 % at the end of the 11th and 12th batches, which corresponded to the yield of 0.5 g/g. As the volumetic productivity declined 1.26 g/l h in the 22th round, which was lower than the ordinary batch fermentation, 22 rounds of repeated-batch fermentation were accessible.7. The single-stage continuous fermentation was performed with the industrial recombinant ZWA46-F2. The volumetric productivity reached 3.82 g/l h. And its productivity of the single-stage fermentation coupled with repeated-batch fermentation was 5.82 g/l h.8. Comparison of the four fermentations, such as batch fermentation, repeated-batch fermentation, single-stage continuous fermentation and continuous fermentation coupled with repeated-batch fermentation, showed that the productivity of single-stage continuous fermentation was 3 folds as that of the ordinary batch fermentation, and the productivity of single-stage continuous fermentation coupled with repeated-batch fermentation was 2.6 folds as that of the single-stage continuous fermentation. The results proved that the continuous fermentation with the recombinant ZWA46-F2 was prior to the batch fermentation.9. The cell wall protein Flo1p of the recombinant W303-1A F4 was purified. The result of western blot showed that its MW was about 200 kDa. And Flo1p belongs to Flo1 type charactorized by the inhibition of sugars. Study of the N-terminal part of the protein Flo1p showed that its active region was from amino acid 196 to 209, enabling the protein to selectively bind the mannose sugars, and the region from amino acid 210 to 240 could not bind sugars but had some negative effect on the active region.