Summary of Progress (Auburn University)
(April 2, 2002)
Revision of report on Mar 2.
1. Enzymatic digestibilities of ARP and dilute-acid treated
corn stover
Enzymatic digestibilities were determined for ARP treated samples (previously
reported) and dilute acid treated samples using 2 different levels of enzyme loading (10,
and 60 FPU/g glucan). Untreated -cellulose was used as the reference.
Pretreatment conditions:

ARP pretreatment conditions: 15wt% of ammonia, 170C, 90min, 5mL/min of
flow rate, 325psig N2 back pressure.

Dilute acid pretreatment conditions: 0.07wt% of sulfuric acid, 180C, 30min,
5mL/min of flow rate 325psig, bed shrinking flow through reactor.
Table 1-1. Composition of solid after pretreatment
Composition
% S.R.*
Lignin
Glucan
Xylan
Untreated
ARP
Dilute acid
100
18.53
40.19
21.71
53.63
2.83
36.09
9.24
54.15
12.49
37.61
1.31
*S.R. : solid remaining after treatment
The enzymatic digestibility profiles are shown in Fig. 1-1 ~ 1-3.
Fig. 1-1: ARP treated corn stover
Fig. 1-2: Dilute-acid treated corn stover
Fig. 1-3: Comparison of the two at low enzyme loadings (10 FPU/g glucan)
Pretreated
60 FPU
10 FPU
α-Cellulose 60FPU
Untreated 60FPU
Figure 1-1. Enzymatic digestibility of corn stover treated by ARP a
a
Pretreatment conditions: 15wt% of ammonia, 170C, 60min, 5mL/min of flow rate 325psig.
Enzymatic hydrolysis conditions: cellulase enzyme (Spezyme CP, Lot 301-00348-257, activity:
31.2 FPU), 60FPU/g glucan for untreated and -cellulose, -glucosidase supplementation
(Sigma, Cat No. G-0395, excess amount, ~30 IU/g glucan), pH 4.8, 50C, 150 rpm. -cellulose
(Sigma, Lot No. 11K0246)
α-Cellulose 60 FPU
60 FPU
10 FPU
Pretreated by dilute acid
Untreated 60FPU
Figure 1-2. Enzymatic digestibility of corn stover pretreated by dilute
acid a
a
Pretreatment conditions: 0.07wt% of sulfuric acid, 180C, 30min, 5.0mL/min of flow rate
325psig N2, bed shrinking flow through reactor. Enzymatic hydrolysis conditions: cellulase
enzyme (Spezyme CP, Lot 301-00348-257, activity: 31.2 FPU), 60FPU/g glucan for untreated
and -cellulose, -glucosidase supplementation (Sigma, Cat No. G-0395, excess amount, ~30
IU/g glucan), pH 4.8, 50C, 150 rpm. -cellulose (Sigma, Lot No. 11K0246)
ARP 10 FPU
FPU
α-Cellulose 60 FPU
Acid 10 FPU
FPU
Untreated 60 FPU
Figure 1-3. Comparison of Enzymatic digestibilities of ARP and diluteacid treated corn stover with low enzyme loadings
2. XRD for ARP treated corn stover
The XRD profiles were measured for ARP treated samples using the facilities of
Material Science Department, Auburn. The diffraction patterns for treated and untreated
corn stover and -cellulose are shown in Fig.2-1. The XRDs were measured under wet
condition for all samples. The water content retained in the sample is characterized by its
scattering which has a maximum at 2=28. The crystallinity index (C.I.) was calculated
with the diffraction intensities, I002 at 002 peak position (222.5) and I18 at 2=18
(amorphous), as a ratio of [(I002-I18)/I002]100 ( Segal et al., 1959).
Table 2-1 and Fig.2-2 show the Crystallinity Index, digestibility, and the solid
composition for corn stover pretreated by ARP with different durations.
-Cellulose
Untreated
Figure 2-1. XRD pattern of untreated, -Cellulose, ARP pretreated
samples
Digestibility
Cristallinity Index
Xylan remaining
Figure 2-2. Effect of reaction time on solid composition, crystallinity
index (CI) and enzymatic digestibility in ARP a
a
All sugar and lignin content based on the oven-dry untreated biomass. Pretreatment conditions: 15wt% of
ammonia, 170C, 5mL/min of flow rate, 325psig
Enzymatic hydrolysis conditions: 72h, 60FPU/g glucan, pH 4.8, 50C, 150 rpm.
Table 2-1. Effect of reaction time on solid composition, crystallinity
index (CI) and enzymatic digestibility in ARP a
Sample
Untreated
10min
20min
40min
60min
90min
-cellulose
a
S.R.
Lignin
Glucan
Xylan
C.I.
Digestibility
[%]
[%]
[%]
[%]
[ ]
[%]
100
18.53
40.19
21.71
59.2
61.40
58.53
56.41
55.18
53.63
100
5.95
3.61
3.30
2.98
2.83
0.0
39.42
38.58
38.51
37.80
36.09
91.36
12.76
10.97
10.54
9.68
9.24
4.02
71.7
74.4
72.3
74.6
76.0
79.8
60 FPU
21.22
10 FPU
N/A
60 FPU
93.9
10 FPU
83.9
60 FPU
96.0
10 FPU
88.3
60 FPU
95.1
10 FPU
89.2
60 FPU
94.4
10 FPU
88.3
60 FPU
99.6
10 FPU
92.5
60 FPU
93.4
10 FPU
71.7
All sugar and lignin content based on the oven-dry untreated biomass. Pretreatment conditions: 15wt% of
ammonia, 170C, 5mL/min of flow rate, 325psig, Enzymatic hydrolysis conditions: 72h, 60 and 10 FPU/g
glucan, pH 4.8, 50C, 150 rpm. Untreated corn stover was wetted to maintain a constant moisture.
Summary of Findings:
The ARP treated samples have low lignin and high xylan content. The dilute-acid treated
samples have high lignin and low xylan content.
Enzymatic digestibilities of ARP sample are 99.6% with 60 FPU and 92.5% with 10 FPU,
whereas those of dilute acid treated samples are 89.9% with 60 FPU and 82.8% with 10 FPU.
Lignin content in the biomass appears to be one of the critical factors controlling the enzymatic
digestibility.
The hydrolysis rate of ARP treated sample are so fast that the yield of 60FPU loading
reached to 97% in 6 hour and the yield of 10FPU loading reached to >90% in 24hr. That of acid
treated sample, however, are relatively fast (75% in 6 hour for 60FPU loading) but maximum
yield was stopped at 89.9% (60FPU) and 82.8% (10FPU). There is little difference in maximum
digestibilities for two treated samples when enzyme loadings above 60 FPU are applied. The
difference of hydrolysis rate becomes very significant.
The intensity of XRD as well as the Crystallinity Index of corn stover rises upon ARP
treatment, perhaps due to removal of amorphous carbohydrates in biomass. The CI increases
slightly with increase of pretreatment time in ARP.
Very high enzymatic digestibility, 83.9% (10 min ARP treated) and 88.3% (20min ARP
treated), at low enzyme loading (10FPU) was observed for ARP samples treated for very short
reaction time (10 and 20min).
Appendix.
cellulose at various enzyme loading
Hydrolysis condition :
1. Cellulase enzyme (Spezyme CP, Lot 301-00348-257, activity: 31.2 FPU) :
60FPU/g glucan loading
2. -glucosidase supplementation (Sigma, Cat No. G-0395, excess amount, ~30 IU/g glucan),
3. Conditions : 0.05M citrate buffer (pH 4.8), 50C, 150 rpm.
4. Substrate : -cellulose (Sigma, Lot No. 11K0246)
Table 1. Enzymatic digestibility of -cellulose
Enzyme loading
60FPU
40FPU
20FPU
10FPU
0hr
0
0
0
0
6hr
12hr
24hr
48hr
72hr
96hr
48.77
45.19
61.32
56.30
75.82
69.89
92.11
83.30
93.50
89.63
93.67
88.03
40.98
34.22
50.95
41.23
62.38
48.47
73.43
58.74
82.05
70.13
83.89
71.66
Figure 1. Enzymatic digestibility of -cellulose (from table 1)