Solubilization of lignin and hemicellulose during hydrothermal pretreatment

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1 Solubilization of lignin and hemicellulose during hydrothermal pretreatment Heather L. McKenzie 1, Nancy L. Engle 2, Joshua F. Emory 2, Marcus B. Foston 3, Arthur Ragauskas 3, Bruce A. Tomkins 2, Timothy Tschaplinski 2, Gary J. Van Berkel 2, Charles E. Wyman 1 1 Department of Chemical & Environmental Engineering, Center for Environmental Research and Technology, University of California, Riverside, BioEnergy Science Center, Riverside, CA 2 Environmental Sciences Division and Chemical Sciences Division, Oak Ridge National Laboratory, BioEnergy Science Center, Oak Ridge, TN 3 School of Chemistry and Biochemistry, Georgia Institute of Technology, BioEnergy Science Center, Atlanta, GA October 25, 2011

2 Acknowledgments ArborGen LLC Brookhaven National Laboratory Verenium Corporation Georgia Institute of Technology Mascoma Corporation National Renewable Energy Laboratory Oak Ridge National Laboratory Dartmouth College Samuel Roberts Noble Foundation University of Georgia University of Tennessee Washington State University North Carolina State University Virginia Polytechnic Institute and State University Cornell University University of California-Riverside University of Minnesota The BioEnergy Science Center is a U.S. Department of Energy Bioenergy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science. 2

3 Outline Introduction and objectives Materials and methods Results and discussion Influence of flow rate Release of lignin and phenols from P. trichocarpa and isolated lignin Changes in molecular weight and composition of lignin Release of xylan and xylooligomers from P. trichocarpa and Birchwood xylan Summary 3

4 Introduction and objectives 4

5 Introduction Ethanol from cellulosic biomass is one option for renewable, low GHG emission transportation energy. Cellulosic biomass Process boundaries Pretreatment Cellulose (primarily glucan) Hemicellulose (primarily xylan) Lignin Biological steps: Enzyme production Hydrolysis Fermentation Process heat, electricity Utilities Lignin, etc Ethanol recovery Residue processing Exported electricity H-lignin S-lignin G-lignin Process effluents Release of hemicellulose and lignin during pretreatment is poorly understood. Chemical structures from Fengel and Wegener (1984). 5 Block diagram courtesy of Dr. Wyman. Overcome natural recalcitrance of biomass Make enzymes. Breakdown polymers glucan and xylan to sugars, glucose and xylose. Ferment all sugars Fuel ethanol

6 Objectives Perform hydrothermal flowthrough pretreatment in order to: compare the release of lignin and lignin derivatives from poplar and isolated lignin. compare the release of xylose and xylooligomers from poplar and birchwood xylan. These comparisons will provide indications of the role of lignin-carbohydrate interactions. 6

7 Materials and methods 7

8 Materials Substrates: - Populus trichocarpa (Pt). - Lignin isolated from Pt by J. Seokwon and Dr. S. Cao (IL). - Birchwood xylan (BX). Left: Fluidized sand bath (Techne, Princeton, NJ). Middle: Custom 10 ml batch reactor. Right: Custom flowthrough reactor system. 8

9 Methods: Pretreatment Solids were loaded and sealed into reactor. Pretreatment was conducted with 180 o C water at conditions summarized below. Liquid samples were collected continuously during run. Residual solids were collected by filtration and massed at the end of a run. Material Mass per run (g) Flow rate (ml/min) Temperature ( o C) Time (min) Sampling Period (min) Pt BX IL

10 Methods: Analytical procedures Measurement Procedure Performed by Sugar monomer and K-lignin content of hydrolysate and the residual solids. M n and M w of lignin Functional groups in lignin Phenols in liquid hydrolysate Xylooligomers in liquid hydrolysate Strong acid hydrolysis with HPLC Gel permeation chromatography (GPC) Heteronuclear single quantum coherence (HSQC) Gas chromatography-mass spectrometry (GCMS) Ultra-high pressure liquid chromatography (UPLC) H. McKenzie, UCR Dr. Foston, GIT Dr. Foston, GIT N. Engle and Dr. Tschaplinski, ORNL. Drs. Emory, Tomkins, and Van Berkel, ORNL 10

11 Results and discussion 11

12 Influence of flow rate Pt was pretreated at 180 o C for 10 minutes with water at 20 ml/min (triplicate) and 25 ml/min. Glucan and xylan yields and percent lignin removal are plotted as a function of time. Reducing the flowrate from 25 ml/min to 20 ml/min did not affect final yields. Glucan and Xylan Yield (wt%) Xylan Yield 25 ml/min 20 ml/min Lignin Removal 20 ml/min 25 ml/min Glucan Yield 25 ml/min 20 ml/min Pretreatment Time (min)

13 Lignin removal from P. trichocarpa and Isolated lignin More lignin is removed from IL during flowthrough pretreatment than during batch pretreatment. This indicates that lignin fragments are removed from the reactor before they can return to the solid phase. The flowthrough reactor improves understanding of product evolution as a function of time. Fraction Solubilized (wt%) Material Q(ml/min) T( o C) t(min) IL 20 ml/min 180 o C 12 min Pt IL IL IL Batch 180 o C 12 min IL

14 Lignin removal from P. trichocarpa and Isolated lignin 50 Fraction Solubilized (wt%) Pt ml/min o C min log(r o )= Material Q(ml/min) T( o C) t(min) Pt IL 20 ml/min 180 o C 12 min log(r o )=3.43 IL At approximately equal severity, more lignin is removed from Pt than from IL. IL IL

15 Release of phenols per gram of raw lignin Cumulative release of phenol metabolites (g/g raw lignin) from P. trichocarpa Total m p /m raw = t R 2 = hydroxybenzoic acid Time (t, min) coniferyl alcohol sinapyl alcohol hydroquinone Cumulative release of phenol metabolites (g/g raw lignin) Greater release of monomers from Pt. This may be due to extractives in raw Pt GCMS performed by: N. Engle and Dr. Tschaplinski, 15 ORNL. from Isolated lignin syringaldehyde vanillin sinapyl alcohol hydroquinone Total m p /m raw = t R 2 = hydroxybenzoic acid coniferyl alcohol Time (t, min)

16 Release of phenols per gram of raw lignin Cumulative release of phenol metabolites (g/g raw lignin) from P. trichocarpa Total m p /m raw = t R 2 =0.97 m p /m raw = t 4-hydroxybenzoic acid Time (t, min) coniferyl alcohol sinapyl alcohol hydroquinone IL s rate of monomer production from per gram of raw lignin is 2/3 of that of Pt. Cumulative release of phenol metabolites (g/g raw lignin) from Isolated lignin syringaldehyde vanillin sinapyl alcohol hydroquinone Total m p /m raw = t m p /m raw = t R 2 = hydroxybenzoic acid coniferyl alcohol Time (t, min) 16

17 P. Trichocarpa vs. Isolated lignin Given: greater lignin removal from Pt than IL, greater phenol production from Pt than IL, and greater rate of phenol production from Pt than IL, it seems that lignin-carbohydrate interactions promote the release of lignin. 17

18 Average molecular weight of lignin 3000 Number Averaged Molecular Weight (M n, g/mol) Q(ml/min) T( o C) t(min) Number averaged molecular weight Raw Flow Batch Raw Weight Averaged Molecular Weight (M w, g/mol) Q(ml/min) T( o C) t(min) Weight averaged molecular weight Raw Flow Batch Raw The decrease in M n and M w indicates that IL undergoes depolymerization reactions. GPC by Dr. 18 Foston, GIT

19 Bonds in isolated lignin Raw lignin- aliphatic region Methoxy B β 50 Methoxy group A β-o-4 ether B γ A γ 60 A α C α A β Carbohydrates C(ppm) C B β-5/α-o-4 phenyl-coumararan B β H H(ppm) HSQC by Dr. 19M. Foston, GIT. C spirodienone (Samuel et al., 2010)

20 Bonds in isolated lignin Methoxy B β 50 Raw Methoxy ml/min A α B γ A γ C (ppm) A α A γ B γ C (ppm) 180 o C 12 min B β C α A β Carbohydrates 80 B β C α A β H (ppm) 1 H (ppm) Methoxy ml/min Methoxy B β 50 Batch A α A γ B γ C (ppm) 180 o C 192 min A α A γ B γ C (ppm) 180 o C 12 min 80 C α A β 80 B β H (ppm) 1 H (ppm) 20

21 Lignin functional groups Raw Lignin- Aromatic Region S 2,6 S 2,6 100 RO OH O 6 2 P 5 3 PB p-hydroxybenzoate unit G Guaiacyl unit PB 2,6 G 2 G 6 G C(ppm) C S Syringyl unit H H(ppm) HSQC by Dr. 21M. Foston, GIT. S Oxidized syringyl unit, C α =O (Samuel et al., 2010)

22 Lignin functional groups 100 Raw S 2,6 100 Run 4 S 2,6 G 2 S 2,6 110 G 2 S 2, ml/min G 5 G C (ppm) G 6 G C (ppm) 180 o C PB 2,6 130 PB 2, min H (ppm) H (ppm) G 2 S 2, Run 5 20 ml/min S 2,6 G 2 S 2, Run 6 Batch G 5 G C (ppm) 180 o C G 5 G C (ppm) 180 o C PB 2, min PB 2, min H (ppm) H (ppm) 22

23 Evidence of lignin reactions S 2,6 S 2,6 S 2,6 G 2 G 5 G 6 S 2, C (ppm) Raw G 2 G 6 G C (ppm) 20 ml/min 180 o C 12 min PB 2,6 130 PB 2, Number Averaged Molecular Weight (M n, g/mol) Q(ml/min) T( o C) t(min) log(r o ) 1 H (ppm) Raw H (ppm) Based on drop in functional group signal intensity, expect M n and M w to decrease substantially. The molecular weight drops but not as much as expected from HSQC spectra. 23

24 Evidence of lignin reactions The retention of molecular weight, poor solubility, and low signal to noise ratio in the spectra all suggest large polymers in pretreated lignin solids. These observations coupled with the low intensity of characteristic lignin bonds suggest formation of new, unknown bonds in the solids. 24

25 Release of xylan from P. trichocarpa and Birchwood xylan Pt and BX were pretreated by flowing water at 25 ml/min at 180 o C through the solids. After 10 minutes, 71% and 100% of the xylan from Pt and BX had been released to liquid phase, respectively. Normalized mass of released xylan (m x, l /m x, raw, g/g raw xylan) Xylan released from Birchwood xylan Xylan released from P. trichocarpa Pretreatment time (minutes) 25

26 Distribution of xylooligomers from P. trichocarpa and Birchwood xylan 100 Distribution of xylooligomers (wt% xylose) HP HP Sampling period (minutes) From poplar: DP=1 DP=2 DP=3 DP=4 DP=5 DP=6 DP>7 From birchwood xylan: DP=1 DP=2 DP=3 DP=4 DP=5 DP=6 DP>7 UPLC performed by: Drs. Emory, Tomkins, and Van Berkel, ORNL Oligomers with DP<6 account for a larger fraction of the xylooligomers released from poplar than from xylan. 26

27 P. Trichocarpa vs. Birchwood xylan Given: the reduction in xylan removal from Pt, and the high fraction of low DP oligomers in xylan released from Pt, it seems that the lignin-carbohydrate interactions limit the release of high DP xylooligomers. Consistent with the hypothesis that lignin-carbohydrate bonds reduce xylooligomer solubility (Yang and Wyman, 2008). 27

28 Summary 28

29 Summary In order to better understand the release of xylan and lignin from biomass, P. trichocarpa, isolated lignin, and birchwood xylan were subjected to batch and flowthrough pretreatment with water at 180 o C. The liquid hydrolysate and residual solids were analyzed with numerous techniques. 29

30 Summary, continued Comparison of lignin removal from Pt and IL: More lignin was removed from Pt. Greater phenol production from Pt. Greater rate of phenol production in Pt. Strong evidence of lignin reactions during pretreatment: Release of phenol monomers. Loss of characteristic bonds and functional groups. Retention of molecular weight. 30

31 Summary, continued Comparison of xylan removal from Pt and BX: Less xylan was removed from Pt. More xylooligomers with DP<6 were released from Pt. The substantial differences in lignin and xylan removal from the native biomass and model substrates suggest that lignin-carbohydrate interactions appear to enhance lignin removal while limiting the release of xylan, especially large xylooligomers. 31

32 Thank you. Questions? 32