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Yongming Zhu 1 , Tae Hyun Kim 2 , Y. Y. Lee 1 , Rongfu Chen 3 and Richard T. Elander 4

Yongming Zhu 1 , Tae Hyun Kim 2 , Y. Y. Lee 1 , Rongfu Chen 3 and Richard T. Elander 4 1 Department of Chemical Engineering, Auburn University, AL 36849; 2 Eastern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, Wyndmoor, PA 19038;

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Yongming Zhu 1 , Tae Hyun Kim 2 , Y. Y. Lee 1 , Rongfu Chen 3 and Richard T. Elander 4

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  1. Yongming Zhu1, Tae Hyun Kim2, Y. Y. Lee1, Rongfu Chen3 and Richard T. Elander4 1Department of Chemical Engineering, Auburn University, AL 36849; 2Eastern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, Wyndmoor, PA 19038; 3SNC Lavalin GDS, Inc. Houston, TX 77096; 4National Bioenergy Center, National Renewable Energy Laboratory, Golden, CO 80401 Enzymatic Production of Xylooligosaccharides from Corn Stover and Corn Cobs Treated with Aqueous Ammonia 27th Symposium on Biotechnology for Fuels and Chemicals Denver, CO. May, 2005

  2. Abstract Xylooligosaccharides (XOS) in purified form are currently marketed as a high-value food additive in Asian countries. We have investigated on a novel method of producing food-grade XOS from corn cobs and stover, which is simpler than the conventional methods. The process starts with SAA (soaking in aqueous ammonia) treatment of a feedstock which results in clean and xylan-rich substrates. The pretreated substrates are then subjected to selective enzymatic hydrolysis using endo-xylanases for production of mostly XOS and small amount of xylose. Refining of xylooligosaccharides was accomplished by carbon adsorption followed by ethanol elution. By this method, all of monomeric sugars and impurities were removed and the XOS were collected in relatively high yield. Xylanolytic hydrolysis of the SAA treated corn stover maintained high digestibility of the remaining cellulose, above 80% with 10 FPU/g-glucan. As a feedstock for XOS production, corn cobs are better than corn stover because of high xylan content and high density. The high packing density of corn cobs reduces water use in both SAA treatment and enzymatic digestion, which eventually leads to high XOS concentration.

  3. Introduction • Xylooligosaccharides (XOs): a high-value food additive known to selectively promote the proliferation of bifidobacteria (beneficial microorganism in the intestine). • Desired DP: 2-7 • Naturally present in bamboo shoots, fruits, vegetables, etc. • Commercial-scale production is based on hydrolysis of hemicellulose in lignocellulosic materials (LCM)

  4. Chemicals for fractionation (alkalis, acids or oxidizing agents) Cellulose, lignin and/or lignin degradation products CHEMICAL PROCESSING Xylan-containing LCM Enzymes HYDROLYTIC DEGRADATION XO Isolated xylan or soluble xylan fragments Conventional process for xylooligosaccharides production Xylan Purification – A complex and costly step (Vazquez et al., 2001, Trends in Food Science & Technology, 11, 387-393)

  5. Introduction (continued) • SAA (soaking in aqueous ammonia) is a pretreatment method that retains all of glucan and most of xylan yet achieves high delignification. The enzymatic digestibility is high for both glucan and xylan of the treated corn stover. • The SAA treated corn stover can therefore be used as a substrate for enzymatic production of xylooligosaccharides, avoiding the costly purification step in the conventional method

  6. Objectives • To examine the feasibility of producing low-DP xylooligosaccharides by enzymatic hydrolysis of xylan in SAA treated corn stover and corn cobs using endo-xylanase enzyme • To investigate the fundamentals of enzymatic hydrolysis of xylan in corn stover pretreated by aqueous ammonia.

  7. Enzymes Endoxylanase: X2753 (Sigma-Aldrich), ~ 2500 xylanase units per gram. Cellulase: Spezyme CP (Genecor) 31.2 filter paper units (FPU) per mL. Analysis HPLC with a Bio-Rad Aminex HPX-87P column and a refractive index detector. NREL standard analytical procedure. SAA Treatment 20 g of dry corn stover soaked in 200 g of 15% ammonia solution (L/S =10), 90oC, 24h, or 28 g dry corn cob particles in 75 g of 15% ammonia solution (L/S =2.8), 60oC, 48h 250 mL SS-316 tubular reactor, heating and temperature control in a GC oven Materials and Methods

  8. Composition of untreated and SAA treated corn stover (dry basis)

  9. Effects of pH and Temperature on Activity of Endo-Xylanase(Substrate: SAA treated corn stover) 4 5 6 7 40oC 50oC 60oC 70oC pH Temperature 0.04g X2753/ g solids, 50℃ and 96 h 0.04g X2753/ g solids, pH=5, 96 h. • Optimum pH 5 • Optimum temperature 50oC

  10. Effect of Enzyme and Solid Loadings on the Activity of Endo-Xylanase(Substrate: SAA treated corn stover) Inoperable T g-enzyme / g-solids g-solids:mL-liquid pH=5, 50℃ and 96 h 0.04g X2753/ g solids, pH=5, 96 h. • Efficiency of enzyme (Yield/g-enzyme) dramatically decreased with increasing enzyme loading • Appropriate solids concentration is less than 10%

  11. DP3 DP5+ Xylose DP2 DP4 Xylooligosaccharides from hydrolysis of SAA treated corn stover • The xylooligosaccharides are composed mainly of DP3 and DP5+. • Portion of the xylooligosaccharides of DP5+ exists in the form of heteropolymers (i.e. arabinoxylan), • Hydrolysis conditions: pH=5, 50 ℃, 72 h. Enzyme loading: 0.04g / g-solids;

  12. Xylan digestibility of untreated and SAA treated corn stover • Enzymatic hydrolysis conditions: 5% solids, 0.04 g X2753 endo-xylanase/g solids, pH 5, 50℃, 150 rpm, 72h.

  13. Product refining - carbon adsorption followed by ethanol elution • Xylose and xylobiose (DP2) were completely removed by DI water washing • Most of xylooligosaccharides were recovered by 30% ethanol elution • Recovery of solubilized xylan as xylooligosaccharides was more than 70%, the balance being xylose and xylobiose, which were removed in water washing

  14. Digestibility of Glucan in SAA-treated corn stover after xylanlytic digestion SAA treated corn sover SAA-treated corn stover after xylanlytic digestion α-cellulose. 1 wt% glucan loading, 10 FPU/g-glucan pH 4.8, 50℃, 150 rpm untreated corn stover

  15. Production of xylooligosaccharides from corn cobs • Corn cobs are a portion of corn stover • Contains higher xylan • 30% vs 20% in corn stover • Allows higher solids loading in SAA because of high bulk density • liquid to solid ratio 2.8 (w/w) vs 10 for corn stover Higher productivity and concentration

  16. Composition of treated corn cobs (15% aqueous ammonia, L/S=2.8, 60℃ and 48h) 92.5%

  17. Xylooligosaccharides production from SAA treated corn cobs • Enzymatic digestion conditions: 0.04 g X2753 endo-xylanase/g solids, pH=5, 50℃, 150 rpm, 72h.

  18. Conclusions (1) • Corn stover and corn cobs treated with aqueous ammonia are substrates feasible for enzymatic production of food-grade xylooligosaccharides. • The products from enzymatic hydrolysis using endoxylanase consist predominantly of xylooligosaccharides. • Carbon adsorption/ethanol elution is an efficient method for refining of xylooligosaccharides.

  19. Conclusions (2) • Removal of xylan from SAA treated corn stover slightly decreased the glucan digestibility, perhaps due to collapse of the cellulose structure. • For production of xylooligosaccharides, corn cobs are more suitable than corn stover because of higher xylan content and higher solid loadings in both pretreatment and enzymatic digestion, resulting in higher product concentration.

  20. Acknowledgement This research was supported by US-EPA: Technology for a Sustaining Environment- Grant No. EPA-RD-83164501.

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