1. Biomass Refining CAFI Auburn University Soaking in Aqueous Ammonia (SAA) for Pretreatment of Corn Stover Tae Hyun Kim and Y. Y. Lee Department of Chemical Engineering Auburn University AIChE 2004 Annual Meeting Austin, TX. Nov, 2004

2. Biomass Refining CAFI Auburn University Research objective  Overall objective To develop a economically viable and environmentally benign process for the pretreatment of biomass.  Factors of interests Energy and capital costs Enzymatic digestibility Fermentability

3. Biomass Refining CAFI Auburn University Corn stover composition Note. NREL supplied corn stover to CAFI members (source: BioMass AgriProducts, Harlan IA). Corn stover was washed and dried in small commercial operation, knife milled to pass ¼ inch round screen 36.1% 21.4% 17.2% 7.1%

4. Biomass Refining CAFI Auburn University Ammonia as a pretreatment reagent Highly selective and effective in delignifying the biomass Easy to recover and reuse because of high volatility. One of the most widely used commodity chemicals (one-fourth the cost of sulfuric acid). Non-polluting and non-corrosive chemical.

5. Biomass Refining CAFI Auburn University ARP (Ammonia Recycle Percolation) A pretreatment method based on aqueous ammonia, which uses a flow-through column reactor (percolation). Lignin is believed to be a major hindrance to enzymatic hydrolysis. Efficient delignification; it gives a high and adjustable degree of delignification (70-85%). It has been proven to be effective for pretreatment of biomass; digestibility is 90% for glucan and 78% for xylan with 15 FPU/g-glucan.

6. Biomass Refining CAFI Auburn University Corn stover ARP laboratory reactor and reaction conditions Data Acquisition System Oven & Reactor Receiver Tank NH 3 Feeding N 2 Back Pressure Aq. NH 3 50 ml of 15 wt% Aqueous NH 3 (1 reactor void volume) Reactor (15 g of Corn Stover) Reactor Volume:70.9 cm 3 Reactor Void Volume: 45.0 cm 3 Flow through column reactor Flow rate; 5 ml/min Reaction temp; 170°C

7. Biomass Refining CAFI Auburn University Problems in ARP 1. High capital cost (High pressure equipment). 2. High-energy cost (high temperature). 3. Half of xylan is removed (xylan recovery is difficult in the downstream processing) SAA (soaking in aqueous ammonia) at room temperature was proposed.

8. Biomass Refining CAFI Auburn University Features of SAA at room temperature Reaction conditions Room temperature/ Atmospheric pressure 30 wt.% of aqueous ammonia Solid-to-liquid ratio: 1:8 Treatment time: 10 days Features Batch Process / no agitation Mild reaction conditions Low equipment cost Lignin is removed/Xylan is retained Simple downstream process SAA apparatus Corn stover

9. Biomass Refining CAFI Auburn University Effect of soaking time on compositional change in SAA at room temperature- treated corn stover Note. S:L ratio=1:12, reaction temp.=22–24ºC (R.T.), 29.5 wt.% NH 3. All sugar content based on the oven-dry untreated biomass Glucan Xylan Lignin

10. Biomass Refining CAFI Auburn University Digestibility vs. soaking time Note. S:L ratio=1:12, 1-60 days treat.; reaction temp.=R.T., 29.5 wt.% NH 3. 4 days 10~60 days Untreated 86% with 10 days-treated corn stover 1 days

11. Biomass Refining CAFI Auburn University Problems in SAA at room temperature 1. Longer reaction time (about 10 days). -> Large capital cost (huge reactor) 2. High liquid input (S:L=1:8~1:10). -> Large operating cost SAA at moderate temperature was proposed.

12. Biomass Refining CAFI Auburn University Features of SAA at moderate temperature Reaction conditions Temperature: 60 °C 15 wt.% of Aqueous Ammonia S:L Ratio: 1:6 Treatment Time: 12 hours Atmospheric pressure Features Batch process /no agitation Lignin is removed/Xylan is retained Simple downstream process

13. Biomass Refining CAFI Auburn University Effect of temperature, S/L Ratio, and reaction time on Lignin removal 15 wt.% of NH 3 ; 24 h reaction time; 1:10 of solid-to-liquid ratio. 15 wt.% of NH 3 ; 60°C of reaction temperature 24 h 12 h 6 h Solid-to-liquid ratio And Reaction time Reaction temperature

14. Biomass Refining CAFI Auburn University Xylan content in SAA-treated corn stover 24 h 12 h 6 h 15 wt.% of NH 3 ; 24 h reaction time; 1:10 of solid-to-liquid ratio. 15 wt.% of NH 3 ; 60°C of reaction temperature Solid-to-liquid ratio And Reaction time Reaction temperature

15. Biomass Refining CAFI Auburn University Selectivity of delignification over xylan removal upon various temperature and ammonia concentration

16. Biomass Refining CAFI Auburn University Enzymatic Digestibility of Glucan and Xylan in SAA-treated Corn Stover 85% at 72 h 78% at 72 h  - Cellulose Untreated Treatment conditions: 60°C, 15 wt.% NH 3, 1:6 of S/L, and 12 h

17. Biomass Refining CAFI Auburn University Simultaneous saccharification and fermentation (SSF) oMicroorganism : S. cerevisiae, D 5 A oSubstrate : SAA-treated corn stover oEnzyme loadings: 15 FPU of Spezyme CP + 30 CBU of Novozyme 188 per gram-glucan oYP medium (1% of Yeast extract, 2% of Peptone) oAnaerobic condition

18. Biomass Refining CAFI Auburn University SSF of “ SAA-treated ” corn stover using D 5 A yeast Ethanol yield, SAA-treated corn stover Ethanol yield, a-Cellulose Glucose, SAA- treated C.S. Glucose,  - cellulose 73% of ethanol yield is much lower than 85% of the glucan digestibility.

19. Biomass Refining CAFI Auburn University Xylose accumulation in SSF of “ SAA- treated ” corn stover using D 5 A Xylose Glucose

20. Biomass Refining CAFI Auburn University Xylose inhibition on enzyme activity in the cellulose hydrolysis 3 wt.% of glucan loading Lower ethanol yield (73%) than the digestibility (85%) with SAA treated sample is due to inhibition by xylose.

21. Biomass Refining CAFI Auburn University SSCF test using “ recombinant E.coli ATCC ® 55124 (KO11) ” oLB medium (0.5% of Yeast extract, 1% of Tryptone) oSubstrate : 12 h SAA at moderate temp.-treated corn stover (1:6 of S:L ratio). oEnzyme loadings: 15 FPU of Spezyme CP + 30 CBU of Novozyme 188 per gram-glucan oSAA-treated corn stover has about 52% glucan and 26% xylan. o3% w/v glucan loading: (30g glucan+15g xylan)/L

22. Biomass Refining CAFI Auburn University SSCF of “ SAA-Treated ” Corn Stover by Recombinant E.coli ATCC ® 55124 (KO11) Ethanol yield, SAA-treated corn stover Theoretical max. based on glucan Ethanol yield,  -cellulose Glucose, SAA-treated C.S. Xylose, SAA-treated C.S. 113% of theoretical maximum based on glucan only - Utilization of xylose contributes to increase of ethanol yield from 73% to 113%.

23. Biomass Refining CAFI Auburn University Sugar concentration in SSCF of “ SAA-treated ” corn stover by recombinant E.coli ATCC ® 55124 Xylose Glucose *Recombinant E.coli consumed the glucose and xylose simultaneously.

24. Biomass Refining CAFI Auburn University Process options based on aqueous ammonia ARP (ammonia recycle percolation) 2004 SAA at room temp. SAA at moderate temp.  170°C, 2.3 MPa - High capital & energy cost  Remove 50% of xylan 2003  Room temp. & Atm. pressure  Retain >85% of xylan in solid  30% ammonia, 1:8~10 of S:L  Pretreatment time : 10 days  60 °C & Atm. pressure  Retain >85% of xylan in solid  15% ammonia, 1:6 of S:L  Pretreatment time: 12 h To reduce the pretreatment time to <1 day

25. Biomass Refining CAFI Auburn University Biomass-to-ethanol process using SAA- SSCF Lignin & xylooligomer (10- 15%) Glucan + >85%Xylan SAA- SSCF (Biomass-to-Ethanol) Lignocellulo sic biomass Fermentation SSCF Pretreatment Enzymatic hydrolysis Lignocellulo sic biomass Fermentation SSCF Xylooligomer and lignin-rich hydrolysate Conditioning Lignin + Sugar degradation products Pretreatment Enzymatic hydrolysis Lignin Separation Pentose sugar Glucan Conventional SSCF (Biomass-to-Ethanol)

26. Biomass Refining CAFI Auburn University Acid-treated solid only Acid-treated solid +Hydrolysate SAA-treated Solid Hydrolysate GlucanXylan Total Sugar Loading Glucan Xylan 3.0 g 1.38 g -None- 4.38 g ARP-treated 3.0 g 0.78 g 4.78 g 0 g 1.0 g Acid-treated 3.0 g 0.12 g 5.69 g 0.37 g 2.2 g ARP-treated solid +Hydrolysate ARP-treated solid only Sugar Loadings in SSCF Reactor Toxicity of hydrolysates in SSCF: various treated solids + hydrolysates (soluble sugars)

27. Biomass Refining CAFI Auburn University Summary Soaking in Aqueous Ammonia (SAA) is a simple and technically feasible pretreatment method for corn stover. SAA retains 85% of xylan in the solid. SSF of SAA-treated corn stover faces significant inhibition due to xylose accumulation in the reactor. In SSCF of SAA-treated corn stover using recombinant E.coli (KO11), glucan and xylan are concurrently converted. Ethanol yield from SSCF is 113% of theoretical maximum on the basis of glucan alone, or 73% on the basis of combined glucan and xylan in untreated corn stover. Leaving hemicellulose in solids is a desirable pretreatment strategy.

28. Biomass Refining CAFI Auburn University Acknowledgements US Department of Agriculture Initiative for Future Agricultural and Food Systems Program, Contract 00-52104-9663 US Department of Energy Office of the Biomass Program, Contract DE-FG36-04GO14017 Genencor International Our team from Dartmouth College; Michigan State, Purdue, and Texas A&M Universities; the University of British Columbia; and the National Renewable Energy Laboratory

29. Biomass Refining CAFI Auburn University Questions? Corn stover Wood chip Bagasse Rice straw Sawdust