Xylitol production from lignocellulosic hydrolysates

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1 Xylitol production from lignocellulosic hydrolysates Young-Jae Jeon a, Hyoun-Sung Shin b and Peter L. Rogers a a : School of Biotechnology and Biomolecular Sciences The University of New South Wales b : LPBio Co. Ltd, Seoul, Korea address:

2 Overview Current trends in biofuel production Needs for biorefinery/higher value products Why xylitol as a higher value product? Isolation of xylitol assimilation deficient mutant Its characterisation by fermentation studies Xylitol production from lignocellulosic hydrolysate Conclusion

3 Biofuel production in global boom Bioethanol in global boom (over decade) -Energy security -Environmental concerns associated with climate chage Million Litres Source from World Fuel Ethanol; Analysis and Outlook,Christoph Berg and F. O. Lich(21)

4 Current trends in bioethanol production Current technology for bioethanol -Relies on food based substrate (sugarcane, corn, and cassava) -Food vs energy competition Sustainable technology for bioethanol production -Using renewable lignocelluosic biomass

5 Biochemical process for 2 nd generation bioethanol Biomass recalcitrance High energy input Enzyme production cost 1~2 days Higher production cost compared to gasoline or diesel

6 Biorefinery concept Glycerol Xylose Glycerol Xylose Cell biomass Xylitol

7 Biodiesel production and glycerol price 3 3 Biodiesel production (million gallons) Crude glycerol production (million lbs) Crude glycerol price (cents/lbs) Year Year Biodiesel production (million gallons) crdue glycerol (8%) production (million lbs) crdue glycerol (8%) price (cents/lbs) Source adapted from Yazdani and Gonzalez (27) Current Opinion in Biotechnology

8 Biodiesel and glycerol Biodiesel Phase Glycerol Phase

9 Suitability test of raw materials for 2 nd generation bioethanol process 3 Sugar concentrations (g/l) Glucose Xylose Arabinose Wheat straw Sugarcane bagasse Sorghum straw Arundo donax Sugarcane tops Oil mallee Pine Eucalyptus Raw materials Jeon et al. (21, Letters in Applied Microbiology, 1: )

10 Why xylitol? Natural sweetener Anti-cariogenic properties Benefits used in chewing gums and toothpaste No side effects compared to artificial sweeteners viz, Saccharin, Aspartame etc. Non-nutritive Benefits as an alternative sweetener for diabetics Potential for higher value product association with ethanol

11 Current xylitol production Chemical process, hydrogenation of xylose derived from hemicellulose with an active Ni catalyst Major expensive cost associated with xylose purification Temperature (13~1 o C) and pressure (2~2 bar) Current market capacity 2, ~4, tonne per annum (Werpy et al., 24 and Granström et al., 27) Alternative process under being developed -Using mutant yeast strains and/or controlling environmental conditions such as dissolved oxygen Characteristics of current biological process -Xylitol yield: 7 ~8 %; Controlled oxygen; Low productivity

12 Chemical mutagenesis to enhance xylitol production by xylitol assimilation deficiency Chemical mutagenesis using NTG

13 Screening of xylitol assimilation deficient mutant of Candida tropicalis SS2

14 Growth on glucose C. tropicalis SS2 3 3 Glucose, ethanol and biomass (g/l) Glucose, ethanol and biomass (g/l) C. tropicalis IFO Time (h) Time (h) Fermentation profiles on the semi-synthetic medium containing g/l glucose. Symbols:, glucose;, ethanol;, biomass

15 Growth on xylose Time (h) 3 Glucose, xylose, and biomass (g/l) Time (h) Fermentation profiles on the semi-synthetic medium containing g/l xylose. Symbols:, glucose;, xylose;, ethanol;, xylitol;, biomass Xylitol (g/l) C. tropicalis SS2 Xylitol (g/l) Glucose, xylose, ethanol and biomass (g/l) C. tropicalis IFO618

16 Growth on Glucose/Xylose Yp/s=.93 g g-1 Yp/s=.12 g g Time (h) 3 Glucose, xylose, ethanol and biomass (g/l) 3 Xylitol (g/l) Glucose, xylose, ethanol and biomass (g/l) Time (h) Fermentation profiles on the semi-synthetic medium containing g/l glucose and g/l xylose. Symbols:, glucose ;, xylose;, ethanol;, xylitol;, biomass 3 Xylitol (g/l) C. tropicalis SS2 C. tropicalis IFO618

17 Growth of SS2 on Glycerol and Xylose Yx/s=.94, Yp/s =.97 g g-1 6 Yx/s=.3, Yp/s =.97 g g Time (h) Xylitol and biomass (g l-1) Glycerol and xylose (g l-1) B Xylitol and biomass (gl-1) Gylcerol and xylose (gl-1) A 3 Time (h) Fermentation profiles of C. tropicalis SS2 for xylitol production on the semisynthetic medium containing glycerol and xylose as substrates. A. For medium containing 2 g l-1 glycerol and g l-1 xylose; B. B. 2 g l-1 glycerol and g l-1 xylose. Symbols:, glycerol;, xylose;, xylitol;, biomass

18 Xylitol Production of SS Time (h) Xylitol, ethanol and biomass (g/l) Glucose, xylose and glycerol (g/l) Yx/s=.23, Yp/s =.97 g g-1 6 Fermentation profiles on the semi-synthetic medium containing 1g/l glucose, 2 g/l glycerol and 11 g/l xylose. Symbols:, glucose;, xylose;, glycerol ;, xylitol;, biomass

19 Fed batch fermentation profile of SS2 using xylose and glycerol Yp/s=.93 g g-1, Productivity = 3.3 g l-1 h Xylitol and biomass (gl-1) Glucose, xylose, glycerol and biomass (g l-1) Time (h) Fed-batch fermentation profile under the environmental conditions at ph=4., 3o C, aeration rate=1vvm, and rpm Symbols:, glucose;, xylose;, glycerol ;, xylitol;, biomass.

20 Acid bagasse hydrolysate (1% substrate loading) Sugar extraction from sugarcane bagasse using 2% H 2SO4, 1% substrate loading and 1hr at 134 oc. Sugar compounds (g/l) Sugarcane Bagasse Glucose (g/l) Xylose (g/l) Arabinose (g/l)

21 Xylitol production using 1% sugar cane bagasse with glycerol feeding Xylitol and biomass (g/l) Glucose, xylose and glycerol (g/l) Glycerol injection Time (h) Fermentation profile of C. tropicalis SS2 in the medium containing acid -hydrolysate of sugarcane bagasse with glycerol injection Symbols:, glucose;, xylose;, glycerol;, ethanol;, xylitol;, biomass Yp/s=.9 g g-1

22 Schematic Process for concentrated xylose stream from Sugar cane bagasse Bagasse Acid hydrolysis Extraction Neutralization Filtration Ion Exchange Evaporation/Concentration Xylitol production

23 Fed batch fermentation profile of SS2 using sugarcane bagasse hydrolysate and glycerol Yp/s=.97, Productivity = 3. g l-1 h Xylitol and biomass (gl-1) Glucose, xylose, and glycerol (g l-1) Time (h) Fed-batch fermentation profile using sugarcane bagasse hydrolysate and glycerol under the environmental conditions at ph=4., 3o C, aeration rate=1vvm, and rpm Symbols:, glucose;, xylose;, glycerol ;, xylitol;, biomass.

24 Summary Efficient microbial xylitol production process developed via chemical mutagenesis Evaluation of glycerol as primary substrate for cell growth Evaluation of xylitol production process as a higher value product in bio-refinery using acid sugarcane bagasse hydrolysate Achieved xylitol product yield,.97 g g-1 Productivity 3.4 g l-1 h-1

25 Acknowledgment Prof. Peter L. Rogers Dr Simon Shin from LP Bio Ltd., Korea Financial support from NCRIS Biofuel Subprogram Lab colleagues from UNSW Biofuel Laboratory

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