1. Improve Xylose Utilization 1.The significance of improving xylose utilization: The commercialization of second-generation bioethanol has not been realized due to several factors, including poor biomass utilization and high production cost. It is generally accepted that the most important parameters in reducing the production cost are the ethanol yield and the ethanol concentration in the fermentation broth. Ethanol is recovered by distillation, which becomes economically feasible when the concentration exceeds 40 g/L (Zacchi and Axelsson, 1989 ). This concentration therefore serves as benchmark when different process alternatives are evaluated. Hence, almost complete conversion of both hexoses and pentoses must be achieved in SSF at a high solid content.

2. The probable main reason for this is that xylose and glucose compete for the same transport systems, and the affinity for xylose is approximately two orders of magnitude lower than for glucose. Therefore, the glucose concentration must be low to obtain efficient xylose uptake. However, glucose in fact enhances xylose utilization at low but nonzero concentrations. This can be attributed to induction of suitable xylose transporter systems, improved cofactor generation and induction of glycolytic enzymes. A low but nonzero glucose concentration– giving a high xylose to glucose ratio – is therefore desirable in a process aiming at co - fermentation of glucose and xylose. 2.Why xylose utilization is so difficult ?

3. 3.How to improve xylose utilization: Method 1: A Fed-Batch SSCF Instead of a batch SSCF process, one may instead use a fed-batch SSCF process. In this way the following advantages are gained : 1) The viscosity of the medium can be maintained low due to a gradual feeding of new material to the reactor, in which the viscosity decreases due to enzymatic degradation 2) the effect of toxicity of the hydrolyzate can be decreased as a result of both adaptation of the yeast and gradual biological detoxification ; 3)there may be a beneficial effect on the xylose uptake from a changed concentration ratio of xylose to glucose in the medium. --------Olofsson K, Rudolf A, Lidén G: Designing simultaneous saccharification and fermentation for improved xylose conversion by a recombinant strain of Saccharomyces cerevisiae. J Biotechnol 2008, 134: 112-120.

4. ------Olofsson K, Rudolf A, Lidén G: Designing simultaneous saccharification and fermentation for improved xylose conversion by a recombinant strain of Saccharomyces cerevisiae. J Biotechnol 2008, 134: 112-120. Material : wheat straw Pretreatment method : acid treatment Microorganism: S. cerevisiae TMB3400 Method: Fed-batch SSCF with 4% WIS initially, 4 additions during 12 h to a final WIS-content of 7%. Fed-batch SSCF with 4% WIS initially, 8 additions during 24 h to a final WIS- content of 7%. Fed-batch SSCF with 6% WIS initially, 4 additions during 12 h to a final WIS- content of 9%. Fed-batch SSCF with 6% WIS initially, 8 additions during 24 h to a final WIS- content of 9%.

5. Results :

6. Method 2: Enzyme feeding --------- Olofsson K, Wiman M, Lidén G: Controlled feeding of cellulases improves conversion of xylose in simultaneous saccharification and co- fermentation for bioethanol production. J Biotechnol 2010,145 :168-175 Material : spruce Pretreatment method : acid treatment Microorganism: S. cerevisiae TMB3400

7. Method and Results:

8. Method 3: Both enzyme and substrate feeding Material : wheat straw Pretreatment method : acid treatment Microorganism: S. cerevisiae TMB3400 Method: --------Olofsson K, Palmqvist B, Liden G: Improving simultaneous saccharification and co- fermentation of pretreated wheat straw using both enzyme and substrate feeding. Biotechnol Biofuels 2010,3:17

9. Results :

10. Method 4:Prefermentation ----Bertilsson M, Olofsson K, Lidén G: Prefermentation improves xylose utilization in simultaneous saccharification and co-fermentation of pretreated spruce. Biotechnol Biofuels 2009,2:8. Prefermentation: the concept of prefermentation, that is, the fermentation of initially available free glucose in the liquid before addition of enzymes, was tested. The idea was to reduce the competitive inhibition on xylose uptake by hexoses in this way. Material : Wood chips from spruce Pretreatment method : steam-pretreated Microorganism: S. cerevisiae TMB3400

11. Results:

12. Method 5: Integration of first-generation and second- generation processes --Borbála Erdei, Balázs Frankó,Mats Galbe and Guido Zacchi: Separate hydrolysis and co-fermentation for improved xylose utilization in integrated ethanol production from wheat meal and wheat straw. Biotechnology for Biofuels 2012, 5:12 Material : wheat straw and wheat meal Pretreatment method : steam pretreated Microorganism: S. cerevisiae TMB3400

13. Method:

14. Results:

15. Method 6: Two-step SSCF ----- Jin, M.J., Lau, M.W., Balan, V., Dale, B.E., 2010. Two-step SSCF to convert AFEX-treated switchgrass to ethanol using commercial enzymes and Saccharomyces cerevisiae 424A(LNH-ST). Bioresour. Technol. 101, 8171–8178 Material : switchgrass Pretreatment method : AFEX-treated Microorganism: S. cerevisiae 424A(LNH-ST)

16. Method: For two-step SSCF(Fig. 1B), xylanase and pectinase were added first to perform pre- hydrolysis for 8 h. Yeast cells and a small percentage of Spezyme CP and Novozyme 188 were then added to the hydrolyzate to carry out SSCF (step 1) for 60 h. After 60 h, the rest of Spezyme CP and Novozyme 188 enzymes were fed to re-lease glucose from glucan, which was subsequently fermented to ethanol by the yeast (SSCF step 2).

17. Results:

18. Method 7: extending the pre-hydrolysis time ---- Mingjie Jin, Christa Gunawan, Venkatesh Balan, Ming W. Lau, Bruce E. Dale:Simultaneous saccharification and co-fermentation (SSCF) of AFEX pretreated corn stover for ethanol production using commercial enzymes and Saccharomyce cerevisiae 424A(LNH-ST). Bioresource Technology 110 (2012) 587–594 Material : corn stover Pretreatment method : AFEX-treated Microorganism: S. cerevisiae 424A(LNH-ST)

19. Method and Results :