USDA /IFAFS Project Status Report
(Auburn University, April 2003)
Pretreatment with Aqueous Ammonia at Room Temperature
During this project period, we have investigated on a pretreatment method
drastically different from the conventional ARP process. The method is quite simple.
Corn stover is soaked in 30% aqueous ammonia and left at room temperature for
extended period (1-30 days). It is done without agitation under atmospheric pressure in a
closed vessel. The purpose of this work was to see if this method could alleviate two
main problems of the conventional ARP. The first problem is the high-energy input (high
temperature and relatively high liquid throughput). The second problem is that about half
of hemicellulose (4060%) is removed during the ARP process along with lignin. The
ARP effluent therefore has to be reprocessed for maximum utilization of hemicellulose
sugars, which is a significant cost factor. The room temperature treatment method has a
potential to resolve these problems because of no heat input during the treatment phase
and limited interaction of ammonia with hemicellulose at room temperature. In our
previous work, we have also found that high degree of delignification is not a necessary
condition to achieve high enzymatic digestibility.
Experimental Procedure
Ground corn stover (9-30 mesh) was treated with 29.5 wt% of aqueous ammonia
in capped glass lab bottles at room temperature for 1-30 days. The solid-to-liquid ratio
was kept at 1:12 for 1–10 days bottles and at 1:15 for 20–30 days bottles. After the
soaking, the solids were separated by filtering, washed with cold water, and subjected to
enzymatic digestibility test as described in Table 1.
Table 1. Treatment conditions
Items
Temperature
Reactor
Treatment time
Ammonia concentration
Solid liquid ratio (S:L)
Conditions
Room temperature (22–24ºC)
Tight seal vessel (Pyrex glass laboratory
bottle)
1-30 days
29.5 wt%
1:8 to 1:15
Enzymatic hydrolysis conditions: (same as previous reports)
1. Cellulase enzyme (Spezyme CP, Lot 301-00348-257, activity: 31.2 FPU) :
15 FPU/g of glucan loading
2. -Glucosidase supplementation (Novozyme 188, 40 IU/g of glucan)
3. Conditions: 0.05M citrate buffer (pH 4.8), 50C, 150 rpm.
4. -Cellulose (Sigma, Lot No. 11K0246)
Summary of Results
Composition: Figure 1 summarizes the time progression of composition data upon
ammonia soaking. The major change of composition is that of lignin. Lignin removal by
soaking process was in 35–67% range. Delignification over 10-day period is 59.1%. It
has steadily increased to 67.2% after 30 days. The glucan content is well preserved
showing no significant changes up until 20 days. Approximately 10% of total initial
xylan was removed in 4 days. Xylan removal increased slightly with soaking time
reaching 13% after 30 days of soaking. This is in sharp contrast to 40–60% dissolution of
xylan in the conventional ARP.
Enzymatic Digestibility: The digestibility test results are summarized in Figure 2. The
digestibilities of the samples treated for 8–30 days are 89–94% with 60 FPU/g of glucan
of enzyme loading. With 15 FPU/g of glucan, the digestibilities were somewhat lower
being in the range of 82–87%. The 10-day sample shows 83.8% of digestibility with 15
FPU/g of glucan. In direct comparison with the ARP samples, the digestibility of the
room temperature soaking was lower, but only by 2-4%. Delignification and digestibility
generally increase with the soaking time. However, increases after 10 days were rather
insignificant.
Effect of L/S Ratio: Tests with varying L/S ratio (Fig. 3) indicate that lignin removal and
enzymatic digestibility increase slightly when the L/S ratio was increased from 8 to 15
(54→59% for delignification, 82→84% for digestibility @ 15 FPU/g of glucan). Xylan
removal increased only by 1%.
Concluding Remarks
Room temperature soaking with aqueous ammonia appears to be a feasible
pretreatment method applicable for corn stover. In direct comparison with the ARP, the
process is simple, less energy intensive, and retains higher fraction of xylan. The
enzymatic digestibility of this method is only slightly lower than the ARP. On the
downside, the processing time is long and requires higher amount of ammonia and total
liquid per unit mass of feedstock. The latter would increase the cost of ammonia recovery.
Actual consumption of ammonia, however, is expected to be lower than the ARP due to
reduced acetate generation. It is of our utmost interest to see how the process economics
of this method measures up.
The literature information implies that the major occurrence of alkaline treatment of
lignocellulosic biomass at room temperature is swelling of cellulose. What is unusual in
our finding is that treatment over extended period causes not only swelling but also a
significant delignification, both factors contributing to improved digestibility.
40
36.1
36.3 36.7
Composition [%]
35
36.4
36.4
35.5
36.3
36
34.8
30
Glucan
Xylan
25
20.4
20.1
20
19.3
18.8
18.4
18.2
17.8
18.1
17.8
15
10
0
5
10
15
20
25
30
Reaction time [d]
20
Lignin
17.2
Composition [%]
15
11.12
10
9.83
8.82
7.52
7.88
5.71
7.02
5
5.64
0
0
5
10
15
20
25
30
Reaction time [d]
.Figure 1. Variation of solid composition with soaking time.
Note. 1–8 days; solid:liquid ratio=1:12, 10–30 days; solid-liquid ratio=1:15, reaction temperature=22–24ºC
(room temperature), 29.5 wt% ammonia.
All sugar and lignin content based on the oven-dry untreated biomass.
100
100
8-30 day-60 FPU
90
80
Enzymatic digestibility [%]
Enzymatic digestibility [%]
90
4 day-60 FPU
70
1 day-60 FPU
60
50
40
30
Untreated-60 FPU
20
80
70
40
30
20
0
0
48
72
96
1 day-15 FPU
50
10
24
4 day-15 FPU
60
10
0
8-30 day-15 FPU
Untreated-15 FPU
0
24
72
96
Time [h]
Time [h]
Untreated-60 FPU
1 d- 60 FPU
8 d-60 FPU
20 d-60 FPU
48
α-Cellulose-60 FPU
4 d-60 FPU
10 d-60 FPU
30 d-60 FPU
Untreated-15 FPU
1 d-15 FPU
8 d-15 FPU
20 d-15 FPU
α-Cellulose-15 FPU
4 d-15 FPU
10 d-15 FPU
30 d-15 FPU
Figure 2. Effect of soaking time on enzymatic hydrolysis.
Note. 1–8 days; solid:liquid ratio=1:12, 10–30 days; solid:liquid=1:15, reaction; reaction temperature=22–24ºC (room temperature); 29.5 wt% ammonia; solid
and liquid ratio is based on wt.
50
85
Digestibility at 72 h
40
83.4
83.8
83.0
81.9
80
Glucan
Composition [%]
35
36.5
36.9
35.7
36.0
30
75
25
Xylan
20
15
19.1
19.1
18.7
10
5
70
18.1
65
K-Lignin
7.9
7.6
7.6
15 FPU Digestibility at 72 h [%]
45
7.0
0
60
1:8
1:10
1:12
1:15
Solid : Liquid [-]
Glucan
Xylan
Lignin
72 h digestibility
Figure 3. Effect of solid to liquid ratio on solid composition and digestibility.
Note. Reaction time= 10 days (240 h); reaction temperature=22–24ºC (room temperature); 29.5 wt%
ammonia; solid-liquid ratio is based on wt.
All sugar and lignin content based on the oven-dry untreated biomass.
Enzymatic digestibility; 15 FPU/g of glucan, digestibility at 72 h.