Low-Moisture Anhydrous Ammonia (LMAA) Pretreatment of Corn Stover Minliang Yang Ming-Hsun Cheng Xiong Cao Kurt A. Rosentrater Department of Agricultural and Biosystems Engineering Iowa State University
Outline Introduction Overall Objectives Optimization Experiments Storage Experiments Conclusions
Pretreatment is required for efficient hydrolysis. Introduction Procedure of ethanol production Pretreatment is required for efficient hydrolysis. Hydrolysis Glucose (Fermentable) Cellulose (Glucan, β-1,4-glucose)
Introduction Pretreatment Pretreatment / Incubation Hydrolysis / Fermentation
Overall Objectives Can we do it? How well does LMAA work? Will LMAA help storage? How much will LMAA cost?
Optimization Experiments
Batch Reactors Sealed batch reactor (690mL): Optimized Conditions: 0.1 g ammonia/g biomass, 80°C, 84h Results: 89% of the maximum theoretical ethanol yield 24.9 g/L ethanol production in fermentation Scaled-up reactor (3L): 4 x larger Conditions: 0.1 g ammonia/g biomass, 75°C, 72h Results: 71.6% of the maximum ethanol yield $3.86 /gal of bioethanol
Phase One – Results Glucan Digestibility of treated vs. untreated corn stover *Note: Conditions for untreated treatment: 50% moisture content and medium size; Conditions for optimal treatment: 72h, 75°C, 50% moisture content and medium size. Compared with untreated corn stover, LMAA pretreatment method greatly improved enzymatic digestibility (% improvement=119) 8
Phase Two – Results Glucan Digestibility of treated vs. untreated corn stover 71.6 ° ° This is the highest digestibility from the treated group, which was 71.6% compared with the untreated corn stover, which was 26.6%. As can be seen from the picture, pretreated corn stover was much higher than unpretreated corn stover, which validated the effect of anhydrous ammonia in pretreatment. *Note: Conditions for untreated treatment: 50% moisture content and medium size; Conditions for optimal treatment: 72h, 75°C, 50% moisture content and medium size. Compared with untreated corn stover, LMAA pretreatment method greatly improved enzymatic digestibility (% improvement=213)
Phase Two – Results 71.6 60.3 58.0 * Note: Conditions for 144h treatment: 144h, 120C, 80% moisture content and large size; Conditions for 24h treatment: 24h, 120C, 80% moisture content and large size. Longer incubation time may improve digestibility, but not very obvious. The left one is the time effect on enzymatic digestibility, it shows that longer incubation time may improve digestibility, but not very obvious. The right one is the temperature effect on enzymatic digestibility, it shows that lower incubation temperature may improve digestibility compared with higher one. *Note: Conditions for 20C treatment: 24h, 20C, 80% moisture content and small size; Conditions for 120C treatment: 24h, 120C, 80% moisture content and small size. Lower incubation temperature may improve digestibility compared with higher.
Experimental Modifications Input Output Original setup Modified setup Ammonia loading: 0.1 g/g biomass 0.18 g/g biomass Optimization of LMAA conditions Increase glucan digestibility Ammonia diffusion Hydrolysis conditions
Ammoniation & Pretreatment Modifications Pretreatment Conditions Ammonia pressure Original setup: 0.1 g NH3/g biomass (db) Increase NH3 loading: 0.18 gNH3/ g biomass (db) Biomass moisture content 50% (DM) Ammoniation time Original setup: 30 min Increase to 80 min Incubation Temperature: 75 oC Time: 72 to 144h
Enzymatic Hydrolysis Modifications NREL LAP (NREL, Enzymatic saccharification of lignocellulosic biomass. Laboratory Analytical Procedure (LAP). 2008) Enzyme loading Original loading: 15 FPU Cellulase (GC220) 30 CBU β-glucosidase (Novozyme 188) Increase loading: 15 FPU Cellulase (GC220) 60 CBU β-glucosidase (Novozyme 188) Hydrolysis Temperature: 75 oC Time: 72 & 144 hr RPM: 68
Phase 3 – Results Last year’s conditions Results 0.1 g NH3 / g biomass (db) 30 min ammoniation 75 oC and 72 hr incubation 15 FPU cellulose 30 CBU β-glucosidase Results Particle size smaller than 1mm Digestibility: 46.8% Particle size smaller than 0.5mm Digestibility: 49.5%
Phase 3 – Results Results New ammoniation and hydrolysis conditions 0.1 g NH3 / g biomass (db) 60 min ammoniation 75 oC and 72 hr incubation 15 FPU cellulose 60 CBU β-glucosidase Results No NH3 remained increased digestibility Digestibility increased at least 20%
Phase 3 – Results Results Ammoniation of corn stover with particle size smaller than 0.5 mm 0.18 g NH3/ g biomass (DM) 80 min ammoniation 75 oC incubation 72 and 144 hr incubation 15 FPU cellulose 60 CBU β-glucosidase Results 3 days incubation Digestibility: 85.19% 6 days incubation Digestibility: 85.57%
Cost Impacts Increased from $3.86/gal to $4.14/gal Unit cost of bioethanol based on optimized conditions: For the 85% digestibility results, we increased the ammonia loading from 0.1g ammonia / 1g biomass (db) to 0.18g ammonia /1g biomass (db), which increased 80% ammonia loading. Increased from $3.86/gal to $4.14/gal
Impact of LMAA on Long-Term Storage
Background Why worry about storage? Seasonal harvest Stover not used immediately after the harvest Among all base reagents, ammonia has been found to be very effective for preservation Source: http://www.ccur.iastate.edu/news/newsletters/2012/sep_oct/stoverproject.html
Objectives To evaluate the effect of the LMAA pretreatment on biomass quality, specifically, changes in carbohydrates, lignin, and ash content; To optimize the storage time, and monitor the growth of fungi; To compare the effects of sealed vs. open storage conditions Help guide scale-up
Methods Grind corn stover to 2mm Adjust moisture content to 20 wt.%, 40 wt.%, 60 wt.% Ammoniated corn stover with ammonia loadings of 0, 0.1, 0.2 g NH3/g DM biomass Transfer the ammoniated corn stover into various plastic containers and open bottles
Methods Place the pretreated corn stover for 6h, 1d, 5d, 12d, 30d, 60d, and 90d at ambient temperature (~21°C) Conduct composition analysis, mass loss and visible mold observation for the pretreated corn stover over time Mold determined using an OCCULAR test All experiments were run in duplicate Sealed bag Open bottle
Results - Visible Mold Growth
Results - Visible Mold Growth Sealed containers Mold observed in sealed bags after 16 days in Treatment 3 (60 wt.% M.C., no LMAA); 1 day later, Treatment 2 (40 wt.% M.C., no LMAA) also appeared; No other mold growth observed in 20% wt % , no LMAA until 90 days Open containers No mold growth observed for any treatments – due to moisture loss LMAA treatments resulted in no mold growth for all storage times or conditions
Results - Mass Loss Mass loss (g) in sealed bags Relationship between mass changes (wet basis) and time in sealed containers.
Results - Mass Loss Mass loss (g) in open bottles Under the same moisture content, the higher ammonia loading (TRT 8,9) resulted in higher mass loss 60 wt.% moisture content resulted in highest mass loss Highly dependent on ambient temperature and humidity 20 wt.% corn stover 40 wt.% corn stover 60 wt.% corn stover
Results - Glucan No significant reduction was observed between open and sealed conditions
Results - Xylan No significant reduction was observed between open and sealed conditions. Higher ammonia loading tended to retain more xylan
Conclusions LMAA pretreated corn stover could be well preserved up to 90 days without mold growth nor reduction in carbohydrates Lignin removal was more obvious in sealed bags Mass loss in 0.2- was higher than 0.1 g/g biomass
Impacts 3 MS Students 1 PhD Student 2013-2014 2015 5 conference papers at ASABE 2015 2 conference papers at ASABE
Thank you! Any questions?
Optimization Conditions Suggested optimize conditions (Incubation conditions): Time: 79hr; Temperature: 67°C Ammoniation: 0.1 g NH3/g biomass
Regression analysis of the mass loss of the seal containers. TRT 1 Y=-0.0002X2+0.0345X+0.0162 0.9952 TRT 2 Y=-0.0003X2+0.0723X-0.0029 0.9998 TRT 3 Y=-0.0002X2+0.0529X+0.0239 0.9992 TRT 4 Y=-0.0001X2+0.0376X+0.0861 0.9922 TRT 5 Y=-9*10-5X2+0.0545X+0.0584 0.9939 TRT 6 Y=-4*10-5X2+0.0413X+0.0325 0.999 TRT 7 Y=-0.0003X2+0.0469X+0.1411 0.9784 TRT 8 Y=-0.0006X2+0.0973X+0.4176 0.9683 TRT 9 Y=-0.0001X2+0.0488X+0.09 0.9943
Methods Treatment Ammonia Loading Moisture Content 1 20 wt.% 2 40 wt.% 20 wt.% 2 40 wt.% 3 60 wt.% 4 0.1 5 6 7 0.2 8 9
Results --- Mass Loss Mass loss (g) in open bottles Highly dependent on ambient temperature and humidity Under the same moisture content, mass loss remained almost the same amongst the ammonia loadings The higher the moisture content, the higher the mass loss 20 wt.% corn stover 40 wt.% corn stover 60 wt.% corn stover
Results --- Ash General trend Higher M.C. led to higher ash content With increasing time, the difference in ash content tended to decrease.
Results --- Lignin Anhydrous ammonia has the potential to remove lignin.