Cellulose hydrolysis in subcritical water

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Enzymatic digestibility of Cellulose enhances after pretreatment for few seconds in subcritical water.

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Cellulose hydrolysis in subcritical water

  1. 1. Enhancement of Enzymatic Digestibility of Microcrystalline Cellulose by Treatment in Subcritical Water Sandeep Kumar, Rajesh Gupta, Y.Y. Lee, and Ram B. Gupta* gupta@auburn.edu Department of Chemical Engineering, Auburn University, Auburn, AL
  2. 2. Outline Introduction Lignocellulosic biomass Subcritical water ? Objective Effect of subcritical water treatment Experimental study Subcritical water treatment in continuous flow reactor Enzymatic digestibility Results Conclusion 2
  3. 3. Introduction : National biofuel action plan New US Renewable Fuels Standard Energy security and renewable fuel 40 35 30 Billion gallons 25 20 15 10 5 0 2008 2009 2010 201 201 1 2 201 201 201 201 201 201 201 2020 2021 2022 3 4 5 6 7 8 9 Co rn starch based Cellulo sic A ny o ther bio fuel 3 Frank D. Haagensen, Novozymes NA, Inc., Presentation in Auburn University, March 5th, 2008
  4. 4. Corn Stover Bagasse Switchgrass Wood chips Lignocellulosic Biomass Other (Extractives, Ash etc) 5 -15% Lignin 15 - 25% Cellulose 38 - 50% Hemicellulose 23 - 32% 4 http://www.nrel.gov
  5. 5. Ethanol from lignocellulosics Lignocellulosic Enzymatic biomass Pretreatment hydrolysis Fermentation Ethanol Pretreatment to improve cellulose accessibility Pretreatment enhances •Rate of production of monomeric sugars •Yield of monomeric sugars 5
  6. 6. Pretreatment methods Physical Physio-chemical Chemical 1. Mechanical Comminution 1. Steam explosion 1. Acid / alkali 2. SO2 / CO2 Catalyzed Steam 2. Irradiation explosion 2. Organosolv 3. Ammonia fiber explosion 3. Subcritical / hot compressed water Critical point of water Subcritical water properties Tc= 374 oC, Decreased Increased Pc= 22.1 MPa, Density Ionization constant ρ c= 0.375 g cm-3 Dielectric constant Diffusivity Viscosity Water is a non-toxic, environmentally benign and inexpensive 6
  7. 7. Objective Effect of temperature and residence time on cellulose structure in a subcritical water treatment process Changes in enzymatic reactivity after subcritical water treatment Factors affecting enzymatic reactivity of cellulose 7
  8. 8. Cellulose hydrothermal reaction pathway Non reducing end Reducing end Water-soluble products (n = 2 to 8) (Oligomers, Cellobiose) Hydrolysis products Glucose Degradation products (Glycoaldehyde, Anhydroglucose, HMF, Furfural, Organic acid etc) 8 . Bobleter, O., 1994. (Prog. Polym. Sci., )19, 797–841.
  9. 9. Enzymatic hydrolysis of cellulose by cellulase enzyme Amorphous domain (Substrate for Endo-glucanase) Reducing end Cellulose Reducing ends (Substrate for Exo-glucanase) Cellobiose β-Glucosidase Glucose 9
  10. 10. Factors effecting enzymatic reactivity Crystallinity of cellulose Degree of polymerization Accessibility Polymorph of cellulose Six known polymorphs Cellulose; I, II, III1, IIIII, IVI, and IVII 10
  11. 11. Analytical techniques Solids characterization Liquid products Degree of polymerization Total organic carbon (TOC) by viscosimetry High pressure liquid X-ray diffraction (XRD) chromatography (HPLC) Scanning electron microscope(SEM) Fourier transform infra-red (FTIR) Differential scanning calorimetry (DSC) 11
  12. 12. Enzymatic Digestibility NREL Laboratory Analytical Procedure (LAP #. 009) Cellulase enzyme (brand name: Spezyme CP) Enzyme loadings Low enzyme loading (3.5 FPU/g of glucan), and High enzyme loading (60 FPU/g of glucan) pH 4.8 substrate buffer Temperature 50 °C, 140 rpm Samples collected after 1 hr and 24 hrs 12
  13. 13. Experimental set-up (subcritical) Cellulose, size 20μm Cellulose slurry input (reactor) = 2.7 wt% 13
  14. 14. Experimental conditions At constant pressure (27.6 MPa) in continuous flow Group I 200 - 275 °C and residence time(t), 3.7 to 6.2 s Group II 300 - 315 °C and residence time, 3.4 to 5.2 s Severity index (Ro) Overend, R.P., Chornet, E., 1987. (Philosophical Transactions of the Royal Society of London )A321, 523-536. 14
  15. 15. Results: Subcritical water treatment Conversion (%) with severity index (R0) 45 % Conversion 30 200 - 275 °C 300 - 315 °C 3.7 - 4.1 s 3.4 - 5.2 s 15 0 4 6 8 10 12 lnRo Cellulose remained chemically stable upto 275 °C (t < 6.2 s) 15
  16. 16. Effect on the crystallinity of cellulose after the treatment 85 83 Crystallinity (%) 81 79 77 75 0 3 6 9 12 15 lnRo Removal of amorphous region increases crystallinity 16 crystallinity for cellulose was determined using XRD pattern (Segal et al., 1959)
  17. 17. Enzymatic reactivity at low enzyme loading 1h 24 h 75 200-275°C % Digestibility 47.2 42.2 50 25 7.9 6.2 0 0 4.1 4.5 7.6 7.9 9.1 9.4 lnRo 300-315°C 68.1 % Digestibility 75 54.6 47.2 48.5 50 22.0 25 7.9 11.1 13.0 0 0 10.7 11.3 11.7 lnRo Digestibility increased for group II (300-315 C)samples only 17
  18. 18. Total hydrolyzable cellulose at high enzyme loading % Digestibility 200-275 C 100 75.0 74.2 75 50 25 0 0 4.1 4.5 7.6 7.9 9.1 9.4 1h 24 h lnRo 300-315°C 90.6 100 % Digestibility 75.0 75 60.1 45.0 50 25 0 0 10.7 11.3 11.7 lnRo Decrease in degree of polymerization ? 18 Transformation of cellulose structure ?
  19. 19. Effect of temperature on degree of polymerization 375 332 Residence time, 3.4 - 6.2 s Degree of polymerization 325 296 275 247 248 225 175 125 119 75 180 200 220 240 260 280 300 320 Temperature (°C) Sharp decline in degree of polymerization after 300 °C 19
  20. 20. XRD patterns of group II (300-315 °C) samples New Peak lnRo = 11.7 lnRo = 11.3 Intensity lnRo = 10.7 Untreated 10 12 14 16 18 20 22 24 26 28 Angle (2θ) Onset of cellulose II (Polymorph) peaks 20
  21. 21. SEM, FTIR, and DSC results Untreated 300 °C 1µm 1µm SEM image showing cracks and trenches in the treated sample FTIR and DSC analysis No significant changes in bonding arrangements No changes in thermal properties 21
  22. 22. Conclusions Subcritical water can be used as an effective pretreatment medium for biomass without degrading or changing properties of cellulose Cellulose maintained crystallinity untill it dissolved Cellulose conversion to water soluble products starts above 275 °C in continuous flow reactor (short residence time) Presence of cellulose II polymorph was confirmed in the cellulose treated at 300 - 315 °C, and degree of polymerization decreased substantially at 315 °C For highly crystalline cellulose (> 80%), enzymatic reactivity improved only for group II samples (300 - 315 °C) 22
  23. 23. Acknowledgements National Science Foundation (grant NSF-CBET-0828269) Alabama Center for Paper and Bioresource Engineering Rajeev Kumar (CE-CERT, University of California, Riverside) for help in DPv analyses. Thank you !! 23
  24. 24. Liquid product composition 302 °C, 5.2 s (lnRo = 11.3) Other compounds 28% Hydrolysis products Degradation 8% 64% Majority are the hydrolysis products in liquid 24

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