USDA/IFAFS Project Status Report
Michigan State University
August 23, 2002
Areas of Investigation:
Measuring the effect of the batch system of Ammonia Fiber Explosion (AFEX)
pretreatment on:
Enzymatic digestion of glucan and xylan of corn stover after pretreatment
Ethanol production from AFEX treated corn stover versus untreated stover
Material and Methods:
Pretreatment
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Corn stover ( provided by NREL “new batch”)
Liquid Ammonia from AGA
Ammonia Fiber Explosion (AFEX) system (figure 1)
Ammonia loading range: 0.77 to 1.3 : 1 (kg NH3: kg dry biomass)
Biomass moisture content: 20-60% (dry weight basis)
Temperature range: 60-90 C
Pressure range: 150-250 psi
Saccharification (SAC), Hydrolysis of Lignocellulosic Biomass
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Hydrolysis was conducted according to the standard method published by NREL
(LAP-008)
Cellulase enzyme (provided by NREL, CAS 9012-548, activity: 28 FPU/ml)
Cellulase load : 25 FPU/gram of glucan
Glucan load : 0.5 gram per flask
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Simultaneous Saccharification and Fermentation (SSF)
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SSF was conducted according to the standard method published by NREL (LAP-008)
Cellulase enzyme (provided by NREL, CAS 9012-548, activity: 28 FPU/ml)
Cellulase load : 15 FPU/gram of glucan
Glucan Load: 1.5 gram per flask
Yeast: Saccharomyces cerevisiae D5A provided by NREL
The sugars were analyzed in a BioRad High Performance Liquid Chromatograph using an
Aminex HPX87P column (HPLC Carbohydrates Analysis Column) at 85ºC and a BioRad
Deashing Cartridge as a guard column. The mobile phase used was degassed HPLC
water at a flow rate of 0.6 ml/min. The injection volume used was 20 L and run time 20
minutes.
The ethanol analysis was performed in a Perkin Elmer Autosystem Gas Chromatograph.
The carrier gas was Helium at a flow rate of 5 mL/min. The column used was Altech
Econo-Cap (15m x 0.53 mm x 1.2 m). The oven temperature was 80ºC while the
injector temperature was 175ºC and the detector was 250ºC. The run time for each
injection was 5 minutes. The results for sugars and ethanol are presented below.
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Figure 2: Sugar Analysis from SAC at 168 hrs.
Figure 3: Glucose Yield from SAC at 168 hrs
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Cellobiose Concentration in SAC
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Untreated
4.5
0.77:1, 73ºC, 20 %
Concentration (g/L)
4
0.77:1, 83ºC, 60%
1.3:1, 61ºC, 60%
3.5
a-cellulose
3
2.5
2
1.5
1
0.5
0
0
20
40
60
80
100
Time (hrs)
120
140
160
180
Figure 4: Time profile of cellobiose concentration during SAC
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Concentration (g/L)
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7
6
5
4
Untreated
3
0.77:1, 73ºC, 20 %
2
0.77:1, 83ºC, 60%
1
1.3:1, 61ºC, 60%
a-cellulose
0
0
20
40
60
80
100
120
140
160
180
Tim e (hrs)
Figure 5: Time profile of glucose concentration during SAC
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Xylose Concentration
5
4.5
Concentration (g/L)
4
3.5
3
Untreated
0.77:1, 73ºC, 20 %
2.5
0.77:1, 83ºC, 60%
2
1.3:1, 61ºC, 60%
a-cellulose
1.5
1
0.5
0
0
20
40
60
80
100
Time (hrs)
120
140
160
180
Figure 6: Time profile of Xylose Concentration during SAC
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0.2
1:1, 58C, 60%
Untreated
0.18
0.16
Glucose Concentration g/l
0.14
0.12
0.1
0.08
0.06
0.04
0.02
0
0
20
40
60
80
100
120
140
160
180
Time, hr
Figure 7: Glucose produced from AFEX treated and untreated corn stover during SSF
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1:1, 58C, 60%
Untreated
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Ethanol Concentration , mg/l
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5
4
3
2
1
0
0
20
40
60
80
100
120
140
160
180
Time, hr
Figure 8: Ethanol produced from AFEX treated and untreated corn stover in SSF
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Results:
Figure 1 shows the experimental setup of the AFEX pretreatment in a 300 ml reactor.
Figure 2 shows the results from hydrolysis of AFEX treated corn stover at various
conditions. This figure illustrates that all treated samples produced much more glucose
and xylose as compared to untreated corn stover.
Figure 3 illustrates the glucose yield of the AFEX treated samples at various conditions.
As this figure shows the untreated corn stover glucose yield is about 40% while that of
the AFEX treated samples ranges from 50 to 83 %.
The best three samples (those which produced more glucose and xylose) among the
AFEX treated samples were chosen for further analysis and are presented in figures 4 to
6. Figure 4 presents the time profile of cellobiose concentration during SAC.
Figure 5 shows the time profile of glucose concentration during SAC. As this figure
demonstrates the AFEX treated samples produced almost double the amount of glucose
as compared to the untreated corn stover.
Figure 6 shows the time profile of xylose concentration during SAC. As this figure
demonstrates the AFEX treated samples produced almost triple the amount of xylose as
compared to the untreated corn stover.
Figure 7 and 8 show the time profile of ethanol and glucose during SSF of treated and
untreated samples. Ethanol increases the first 48 hours and then remains constant while
in the untreated sample decreases slightly and then stays relatively constant (Figure 8).
The production of ethanol from treated sample was up to 114% different (higher) than
from untreated. A drastic increase in glucose concentration at 96 hours with no increase
in ethanol production may imply that the ethanol concentration at this point is high
enough to inhibit the yeast fermentation.
Ongoing and Future Work:
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Measurement of the treated and untreated samples crystallinity in order to
correlate with digestibility of cellulose by both enzymatic hydrolysis and SSF.
Measure the impact of pretreatment conditions like temperature, moisture content
and ammonia loading in the digestibility, crystallinity and bond rearrangement of
corn stover.
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