1. 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

2. Outline Introduction Overall Objectives Optimization Experiments
Storage Experiments
Conclusions

3. 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)

4. Introduction Pretreatment Pretreatment / Incubation
Hydrolysis / Fermentation

5. Overall Objectives Can we do it? How well does LMAA work?
Will LMAA help storage?
How much will LMAA cost?

6. Optimization Experiments

7. 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

8. 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

9. 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)

10. 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.

11. Experimental Modifications
Input
Output
Original setup
Modified setup
Ammonia loading:
0.1 g/g biomass g/g biomass
Optimization of LMAA conditions
Increase glucan digestibility
Ammonia diffusion
Hydrolysis conditions

12. 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

13. 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

14. 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%

15. 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%

16. 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%

17. 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

18. Impact of LMAA on Long-Term Storage

19. 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:

20. 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

21. 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

22. 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

23. Results - Visible Mold Growth

24. 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

25. Results - Mass Loss Mass loss (g) in sealed bags
Relationship between mass changes (wet basis) and time in sealed containers.

26. 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

27. Results - Glucan
No significant reduction was observed between open and sealed conditions

28. Results - Xylan
No significant reduction was observed between open and sealed conditions.
Higher ammonia loading tended to retain more xylan

29. 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

30. Impacts 3 MS Students 1 PhD Student 2013-2014 2015
5 conference papers at ASABE
2015
2 conference papers at ASABE

31. Thank you! Any questions?

32. Optimization Conditions
Suggested optimize conditions (Incubation conditions):
Time: 79hr; Temperature: 67°C
Ammoniation:
0.1 g NH3/g biomass

33. Regression analysis of the mass loss of the seal containers.
TRT 1
Y= X X
0.9952
TRT 2
Y= X X
0.9998
TRT 3
Y= X X
0.9992
TRT 4
Y= X X
0.9922
TRT 5
Y=-9*10-5X X
0.9939
TRT 6
Y=-4*10-5X X
0.999
TRT 7
Y= X X
0.9784
TRT 8
Y= X X
0.9683
TRT 9
Y= X X+0.09
0.9943

34. 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

35. 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

36. Results --- Ash General trend
Higher M.C. led to higher ash content
With increasing time, the difference in ash content tended to decrease.

37. Results --- Lignin
Anhydrous ammonia has the potential to remove lignin.