Comparative Study on Enzymatic Digestibility of Upland and Lowland Switchgrass Varieties Processed by Leading Pretreatment Technologies

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1 Comparative Study on Enzymatic Digestibility of Upland and Lowland Switchgrass Varieties Processed by Leading Pretreatment Technologies Youngmi Kim, Nathan S. Mosier, Michael R. Ladisch Purdue University V. Ramesh Pallapolu and Y. Y. Lee Auburn University Bonnie Hames Ceres Steven R. Thomas DOE - Golden Field Office Rebecca Garlock, Venkatesh Balan, and Bruce E. Dale Michigan State University Bryon S. Donohoe, Todd B. Vinzant, and Richard T. Elander NREL Matthew Falls, Rocio Sierra, and Mark T. Holtzapple Texas A&M University Jian Shi, Mirvat A. Ebrik, Tim Redmond, Bin Yang, and Charles E. Wyman University of California Riverside Ryan E. Warner Genencor, A Danisco Division 2009 AIChE Annual Meeting, Nashville, Nov 08-13, 2009

2 Acknowledgments Material in this work supported by US Department of Energy Office of the Biomass Program, Contract DE-FG36-04GO14017 For gifts of enzymes Genencor, a Danisco Division Switchgrass Ceres CAFI Team Collaborators LORRE Staff Linda Liu, Rick Hendrickson

3 Switchgrass A plentiful, warm-season perennial grass Low fertility requirement Tolerant of poor soils High yield (6-8 ton/acre) Photos courtesy of Department of Agronomy, Purdue University

4 Switchgrass Variety Morphological type Lowland type: Adapted to poor drainage Tall, coarse, thicker stems, Grows faster and higher yield potential than upland Alamo, Kanlow Upland type: Short, fine stems, tolerant to cold temperature and drought Shawnee, Dacotah, Trailblazer, Cave-in-Rock, Caddo Physiological type (based on latitude-of-origin) Southern ecotype: Higher biomass productivity, higher risk of winterkill Northern ecotype: Earlier flowering, shorter, lower yield, lower cell wall concentration, better winter survival than southern ecotypes Cassida et al., Crop Sci. (2005)

5 Factors Affecting Biomass Yields and Composition Morphological type Latitude-of-origin Harvest Season Lowland Upland Southern Northern Fall Biomass Yield > > > Moisture level > > > Cellulose > > < Lignin > > < Soluble Carbohydrates Storage Polysaccharides unknown unknown > unknown unknown > Ash < < > N < < > After- Winter Other factors, such as N fertilizer, precipitation, storage method, are not considered in this study. Cassida et al., Crop Sci. (2005); Adler et al., Crop Sci. (2006); Wiselogel et al., Bioresource Tech. (1996)

6 CAFI3-Allotted Switchgrass Alamo 1 Alamo 2 Shawnee Dacotah Latitude-of-Origin 29 N 38 N 46 N Ecotype Southern Lowland Northern Upland Morphology Thick stems Thin stems Harvest Location Ardmore, OK 34 N (Elev. 870 ft) Stillwater, OK 36 N (Elev. 960 ft) Pierre, SD 44 (Elev ft) Plant date June, 2005 June, 2007 June, 2005 December, 1999 Harvest date Post processing and Storage December, 2006 November, 2007 December, 2006 May, 2008 (Plot was allowed to stand over the winter) Small square bales stored in a building Dried at 50 C to less than 1 Moisture Milled using a Knife mill or hammer mill to 2-6 mm size

7 Objectives Compare compositions of CAFI3-allotted switchgrass Apply different pretreatment technologies and compare enzymatic digestibility

8 12% % Water-Extractable Free Sugars in CAFI3 Switchgrass % Water-Extractable Free Sugars in Raw Switchgrass 1 8% 6% 4% 2% 9.6% 6.9% 8.2% 0.8% Alamo 1 Alamo 2 Shawnee Dacotah

9 Cell Wall Composition of CAFI3 Switchgrass (Samples were hot-water washed by x10 v/w DI water to remove water-soluble free sugars) 10 % Dry Mass of Pre-Washed Switchgrass % % 3.6% 4.3% 4.6% 4.6% 3.3% % 1.2% 4.7% 20.8% 20.9% 21.5% 22.8% 59.7% 60.5% 58.9% 61.4% Acetyl Ash Protein Lignin Structural Carbohydrates Alamo 1 Alamo 2 Shawnee Dacotah

10 Effect of Ecotype and Harvest Season on Composition Lowland vs Upland, Late-Fall Harvest vs Spring Harvest 4 35% Cellulose 3 25% 2 15% 1 5% Ecotype: Lowland (A) Upland (S) Upland (D) Harvest: Late-fall Late-fall Spring Latitude-of-origin ( o N) Lignin Water Extractable Sugars Protein Ash

11 Pretreatment Conditions Pretreatment Method Substrate Chemicals Loading (per g dry biomass) Temperature ( o C) Duration Moisture Level (per g dry biomass) AFEX A 1.5 g NH min 2 g H 2 O S, D 1.5 g NH min 2 g H 2 O DA A, S, D g H 2 SO min 19 g H 2 O LHW A, S, D 6.7 g H 2 O min 6.7 g H 2 O Lime A, S, D 1 g Ca(OH) hr None SAA A, S 0.15 g NH 4 OH hr 9 g H 2 O D 0.15 g NH 4 OH hr 9 g H 2 O SO 2 D 0.05 g SO min 9 g H 2 O

12 Fate of Solids During Pretreatment of Dacotah Switchgrass % Total Initial Dry Mass Lignin (acid insoluble) Arabinan Xylan Cellulose Untreated AFEX DA LHW Lime SAA SO2

13 Composition of AFEX Pretreated Solids of CAFI3 Switchgrass (Pretreatment conditions: 1.5 g NH 3, 2 g H 2 O:g dry solids 140 o C/20 min for Alamo, 150 o C/30 min for Shawnee and Dacotah) % Dry Weight Untreated AFEX Protein Acetyl Lignin Arabinan Xylan Cellulose 2

14 Composition of Dilute Acid Pretreated/Hot-Washed Solids of CAFI3 Switchgrass (Pretreatment conditions: g H 2 SO 4, 19 g H 2 O:g dry solids 160 o C/10 min) 10 Untreated DA Lignin Xylan 8 Cellulose % Dry Weight 6 4 2

15 Composition of LHW Pretreated/Hot-Washed Solids of CAFI3 Switchgrass (Pretreatment conditions: 6.7 g H 2 O: g dry solids, 200 C, 10 min) 10 Untreated LHW Acetyl Lignin Arabinan 8 Xylan Cellulose % Dry Weight 6 4 2

16 Composition of Lime Pretreated/Hot-Washed Solids of CAFI3 Switchgrass (Pretreatment conditions: 1 g Ca(OH) 2 : g dry solids, 120 o C/4 hr) 10 Untreated Lime Lignin Arabinan Xylan 8 Cellulose % Dry Weight 6 4 2

17 Composition of SAA Pretreated/Hot-Washed Solids of CAFI3 Switchgrass (Pretreatment conditions: 0.15 g NH 4 OH, 9 g H 2 O : g dry solids, 90 o C/24 hr for Alamo and Shawnee, 160 o C/1 hr for Dacotah) 10 8 Untreated SAA Acetyl Lignin Arabinan Xylan Cellulose % Dry Weight 6 4 2

18 Enzymatic Hydrolysis Conditions Substrate loading: DA, LHW, Lime, SAA, SO 2 - Pretreated and hot-washed solids at 1% glucan loading AFEX - Pretreated solids at 1% glucan loading Enzyme dose: Spezyme CP and Novo188 at 15 FPU and 30 CBU per g glucan in the prewashed, untreated switchgrass (~27 mg protein/g glucan) Hydrolysis conditions: 150 RPM, 50 0 C ph 4.8, 0.05 M citrate buffer Sampling: 1 hr (for initial rate), 24 hr, and at the end of hydrolysis (168 hr)

19 Glucose Yields after 1 hr Hydrolysis 10 8 Alamo Shawnee Dacotah % Glucose Yield AFEX DA LHW Lime SAA Yields calculated based on glucan in pretreated/hot washed solids for all pretreatments except for AFEX

20 Glucose Yields after 24 hr Hydrolysis 10 8 Alamo Shawnee Dacotah % Glucose Yield AFEX DA LHW Lime SAA Yields calculated based on glucan in pretreated/hot washed solids for all pretreatments except for AFEX

21 Glucose Yields after 168 hr Hydrolysis 10 8 Alamo Shawnee Dacotah % Glucose Yield AFEX DA LHW Lime SAA Yields calculated based on glucan in pretreated/hot washed solids for all pretreatments except for AFEX

22 Xylose Yields after 168 hr Hydrolysis 10 8 Alamo Shawnee Dacotah % Xylose Yield AFEX DA LHW Lime SAA Yields calculated based on glucan in pretreated/hot washed solids for all pretreatments except for AFEX

23 Effect of Supplementary Xylanase (Multifect Pectinase) on Sugar Yields LHW pretreated Dacotah, No hot-water washing after pretreatment 15 FPU Spezyme CP+ 200 OSX M. Pectinase/g glucan= 27 mg protein/g glucan With Pectinase 8 % Glucose Yield 6 4 No Pectinase % Xylose Yield Hydrolysis Time (hr) Hydrolysis Time (hr) Yields calculated based on glucan and xylan in untreated biomass

24 Summary Harvest season and possibly age of stand are more important factors than ecotype of switchgrass in terms of affecting switchgrass saccharifaction. Spring harvest contains more lignocellulose and less ash, protein and water extractable free-sugars than fall/late fall harvest. After pretreatment, compositional variability between switchgrass batches becomes less related to ecotype and/or harvest season. Enzymatic digestibility of Dacotah switchgrass, which is upland variety and was harvested in spring, was the lowest among the switchgrass samples, regardless of the pretreatment methods applied. The recalcitrant nature of upland cultivar and spring harvest of switchgrass may require more severe pretreatment conditions.