Distinct Roles of Residual Xylan and Lignin in Limiting Enzymatic Hydrolysis of Organosolv Pretreated Woody Biomass Maobing Tu Forest Products Laboratory Auburn University 1
Tu Research Group: Carbohydrate-derived biofuels and bioproducts Woody biomass Pretreatment Pretreated biomass Enzymatic hydrolysis Hemicellulose Hydrolysate Microbial and Catalytic conversion Sugars Fermentation Co-products (Lactic acid, Acrylic acid, Butanol, Xylonic acid) Biofuels 2
Outline Background and Goals Effects of residual xylan/lignin on enzymatic hydrolysis Effects of pectinase/xylanase supplementation on enzymatic hydrolysis Ethyl xyloside production in SSF process Conclusion 3
Background Biomass Pretreatment Solid Phase Rich in Cellulose Liquid Phase Rich in Hemicellulose Enzymatic Hydrolysis Fermentation Fermentation Biofuels Enzymatic hydrolysis limiting factors Residual xylan and lignin Other factors (DP, crystallinity, pore size and accessible surface area) End product inhibition 4
Goals Goals Distinguish the different roles of residual xylan and lignin in enzymatic hydrolysis of pretreated woody biomass Improve enzymatic hydrolysis of woody biomass Hypotheses Residual xylan affects Initial hydrolysis rate Residual lignin affects Final hydrolysis yield 5
Methods Biomass Loblolly pine and sweetgum Organosolv Pretreatment (17 C, 1.1% H 2 SO 4 (w/w), 65% ethanol (v/v)) Chemical Composition Analysis (NREL) Langmuir Adsorption Isotherm Liquid Fraction SEM Solid Fraction Enzymatic Hydrolysis SSF 6
Methods Langmuir Adsorption Isotherm KC max 1 KC R K max Ʈ: The surface concentration of adsorbed cellulase enzymes; Ʈ max : The surface concentration of protein at full coverage; K: Langmuir constant; C: The free protein concentration in the bulk solution; R: The distribution coefficient 7
Results and discussion Table 1. Chemical composition of untreated biomass and pretreated biomass Compositions Untreated loblolly pine (%) Untreated sweetgum (%) OPLP substrate (%) OPSG substrate Acetone extractives 1.64±.8.99±.9 8.53±.19 8.47±.21 Acid-insoluble lignin 28.48±.4 23.56±.28 18.38±.17 8.16±.3 Acid-soluble lignin.26±.3 2.24±.2.23±.6.87±.3 Glucan 41.33±.49 41.19±.73 63.32±1.24 69.8±1.78 Xylan 6.34±.13 16.18±.5 2.93±.79 9.74±.53 Galactan 2.16±.9 1.87±.32 NA NA Arabinan 1.3±.11.83±.11.9±.21 NA Mannan 12.17±.39 3.33±.49 4.5±.59 3.1±.28 Total 93.69 9.2 98.79.5 OPLP: organosolv pretreated loblolly pine; OPSG: organosolv pretreated sweetgum. (%) 8
SEM images of OPLP (left) and OPSG (right) Lignin droplets 9
Glucose yield (%) Effect of enzyme loading on the hydrolysis of glucan in OPLP and OPSG 24 48 72 75 5 25 OPLP + 2 FPU OPLP + 1 FPU OPSG + 2 FPU OPSG + 1 FPU 75 5 25 1. Initial glucan hydrolysis rate (g L -1 h -1 ): OPLP >OPSG 1. vs.7 (1 FPU) 1.5 vs 1.2 (2 FPU) 2. Final glucan hydrolysis yield: OPSG>OPLP 89% vs 6% (1 FPU) % vs 81% (2 FPU) 3. Crossing point at 17 h 24 48 72 Time (h) OPLP: organosolv pretreated loblolly pine; OPSG: organosolv pretreated sweetgum. 1
Xylose yield (%) Effect of enzyme loading on the hydrolysis of xylan in OPLP and OPSG 24 48 72 1. Xylan hydrolysis (rate): OPSG>OPLP 75 5 75 5.14 vs.3.21 vs.5 2. Xylan hydrolysis (yield): OPSG>OPLP 87% vs 43% 25 OPLP + 2 FPU OPLP + 1 FPU OPSG + 2 FPU OPSG + 1 FPU 25 % vs 66% 24 48 72 Time (h) OPLP: organosolv pretreated loblolly pine; OPSG: organosolv pretreated sweetgum. 11
Results and discussion Initial glucan hydrolysis rate: OPLP (1. g L -1 H -1 )>OPSG (.7 g L -1 H -1 ) 1 FPU OPLP (1.5 g L -1 H -1 )>OPSG (1.2 g L -1 H -1 ) 2 FPU Final glucan hydrolysis yield: OPSG (89%) > OPLP (6%) 1 FPU OPSG (%)>OPLP (81%) 2 FPU Xylan hydrolysis (initial rate and final yield): OPSG>OPLP Question: Why the initial hydrolysis rate (glucan) was higher in OPLP than that in OPSG? 12
Adsorbed cellulases on substrates (mg/g) Cellulase adsorption isotherms on OPLP and OPSG..5 1. 1.5 2. 4 4 3 3 2 2 1 1 Cellulase adsorption on OPSG Cellulase adsorption on OPLP..5 1. 1.5 2. Free cellulases in solution (mg/ml) Cellulases Ʈ max (mg/g) K(mL/mg) R(L/g) Cellulases on OPLP 35.9 3.11.11 Cellulases on OPSG 6.19 1.31.8 Celluclast on EPLP* 87.69 3.48.31 Celluclast on SELP* 11.5 1.45.15 13
Initial hydrolysis rate (g/l/h) Correlation between distribution coefficient and initial hydrolysis rate 2.4 2.2 2. 1.8 1.6 1.4 1.2 1..5.1.15.2.25.3.35 Distribution coefficient R (L/g) R 2 =.9, initial hydrolysis rate is well correlated to the Distribution coefficient (R) 14
Glucose yield (%) Effect of pectinase on enzymatic hydrolysis of OPLP and OPSG 24 48 72 75 5 25 OPLP + Pectinase OPLP OPSG + Pectinase OPSG 75 5 25 Pectinase addition (3.6 mg/g glucan) 1.Initial rate (g L -1 h -1 ) OPLP >OPSG 1. vs.7 (1 FPU) 1. vs.8 (+Pectinase) 2. Final yield 6% to 67% in OPLP 89% to 99.9% in OPSG 24 48 72 Time (hr) 15
Xylose yield (%) Effect of pectinase on enzymatic hydrolysis of OPLP and OPSG 24 48 72 75 5 75 5 1. Xylose yield increased 43% to 48% in OPLP, 87% to 99.9% in OPSG 25 OPLP + Pectinase OPLP OPSG + Pectinase OPSG 25 24 48 72 Time (h) 16
Glucose yield (%) Effect of xylanase on enzymatic hydrolysis of OPLP and OPSG 24 48 72 75 5 25 OPLP 1 FPU + Xylanase OPLP 1 FPU OPSG 1 FPU + Xylanase OPSG 1 FPU 75 5 25 Xylanase addition (2.7 mg/g glucan) 1.Initial rate (g L -1 h -1 ): OPLP >OPSG 1. vs.7 (1 FPU) 1. vs 1. (+Xylanase) 2. Final yield 6% to 67% in OPLP 89% to 99.9% in OPSG 24 48 72 Time (hr) 17
Xylose yield (%) Effect of xylanase on enzymatic hydrolysis of OPLP and OPSG 24 48 72 75 5 75 5 1. Xylose yield increased 43% to 52% in OPLP 87% to % in OPSG 25 OPLP 1 FPU + Xylanase OPLP 1 FPU OPSG 1 FPU + Xylanase OPSG 1 FPU 25 24 48 72 Time (hr) 18
Results and discussion Pectinase supplementation (3.6 mg/g glucan) Initial hydrolysis rate (OPSG) did not change much (.7 vs.8) Initial hydrolysis rate (OPLP) kept the same (1.) Final hydrolysis yield increased in both substrates From 6% to 67% in OPLP; from 89% to 99.9% in OPSG Xylanase supplementation (2.7 mg/g glucan) Initial hydrolysis rate (OPSG) increased by 5% (.7 vs 1.) Initial hydrolysis rate (OPLP) kept the same (1.) Final hydrolysis yield increased in both substrates From 6% to 67% in OPLP; from 89% to 99.9% in OPSG 19
Initial hydrolysis rate (g/l/h) Final hydrolysis yield (%) Correlation between residual xylan/lignin and enzymatic hydrolysis 2.4 2.2 EPLP 11 15 2. 1.8 95 OPSG EPHP EPLP 1.6 1.4 1.2 OPLP EPHP OPSG 9 85 8 75 OPLP 1. 5 1 Xylan (%) 8 1 12 14 16 18 2 Lignin (%) Organosolv pretreated lodgepole pine (EPLP), loblolly pine (OPLP), sweetgum (OPSG) and hybrid polar (EPHP). 2
Ethanol concentration (g/l) Xylose concentration (g/l) Effects of enzyme loading on SSF 24 48 72 96 2. 24 48 72 96 2. 1 1 8 8 1.5 1.5 6 4 2 OPLP 2 FPU OPLP 1 FPU OPSG 2 FPU OPSG 1 FPU 6 4 2 1..5. OPLP 2 FPU OPLP 1 FPU OPSG 2 FPU OPSG 1 FPU 1..5. 24 48 72 96 Time (hr) 1. Initial ethanol production rate (OPLP>OPSG):.5 vs.3 (1 FPU).8 vs.6 (2 FPU) 2. Final ethanol yield: OPSG>OPLP 8.8 vs 7.8 (1 FPU), 1.3 vs 9.8 (2 FPU) 24 48 72 96 Time (hr) 1. Decreased in OPSG after 48hr with 2 FPU loading? 21
Results and discussion Ethanol production Initial ethanol production rate: OPLP>OPSG Final ethanol yield: OPSG>OPLP Same trend as glucan hydrolysis but the crossing point changed (58h) Xylose release Decreased after 48hr with 2 FPU loading Potential enzyme-catalyzed reaction between xylose and ethanol 22
Formation of ethyl xylopyranoside in the process of SSF uv(x,) 1..75 Ethyl xylopyranoside Ethanol hr 2 hr 48 hr.5.25 Xylose glycerol 96 hr. 8. 9. 1. 11. 12. 13. 14. 15. 16. 17. 18. 19. 2. 21. min uv(x,) 1..9.8.7.6 Chromatograms in SSF with 2FPU/g glucan (a)softwood; (b) hardwood.5.4.3.2.1. 8. 9. 1. 11. 12. 13. 14. 15. 16. 17. 18. 19. 2. 21. min 23
% LC/MS analysis of potential reaction product between xylose and ethanol as is Sweet gum SSF 2FPU SSF96hr_92612_3 42 (1.712) Cm (39:12) 179.912 1: TOF MS ES+ 3.21e3 O enzyme O OH OH CH 3 CH 2 OH OH OCH 2 CH 3 H OH OH OH OH Xylopyranose Ethanol Ethyl xylopyranoside 179.273 H 2 O 18.247 178.5125 178.955 178.573 178.1452 178.4682 178.6145 178.984 179.1429 179.9775 179.588 179.1866 179.6135 179.8453 179 18 18.157 m/z 24
Conclusion Interactions between xylan/lignin and cellulase affect the enzymatic hydrolysis of pretreated biomass Residual xylan controls the initial hydrolysis rate Residual lignin controls the final hydrolysis yield Two-phase hydrolysis Xylanase supplementation increases initial hydrolysis rate by removing xylan Linear correlation between initial hydrolysis rate and distribution coefficient Potential enzyme-catalyzed production of ethyl xyloside 25
Acknowledgement AAES and OVPR at Auburn University Dr. Yonnie Wu (Chemistry and Biochemistry) Dr. Sushil Adhikari (Biosystems Engineering) Graduate students (Mi Li, Rui Xie, Lu Lu and Jing Li) 26
SEM images of enzymatic hydrolyzed OPLP and OPSG Enzymatic hydrolyzed biomass OPLP(left) and OPSG (right) ) after 72 hr with 1 FPU 27