Bioresource Technology 97 (2006) 583591

Eect of structural features on enzyme digestibility of corn stover

Sehoon Kim, Mark T. Holtzapple *

Department of Chemical Engineering, Texas A&M University, College Station, TX 77843-3122, USA

Received 28 March 2005; accepted 29 March 2005

Available online 14 June 2005

Abstract

Corn stover was pretreated with excess calcium hydroxide (0.5 g Ca(OH)2/g raw biomass) in non-oxidative and oxidative con-
ditions at 25, 35, 45, and 55
C. The enzymatic digestibility of lime-treated corn stover was aected by the change of structural fea-
tures (acetylation, lignication, and crystallization) resulting from the treatment.
Extensive delignication required oxidative treatment and additional consumption of lime (up to 0.17 g Ca(OH)2/g biomass).
Deacetylation reached a plateau within 1 week and there were no signicant dierences between non-oxidative and oxidative con-
ditions at 55
C; both conditions removed approximately 90% of the acetyl groups in 1 week at all temperatures studied.
Delignication highly depended on temperature and the presence of oxygen. Lignin and hemicellulose were selectively removed
(or solubilized), but cellulose was not aected by lime pretreatment in mild temperatures (2555
C), even though corn stover was
contacted with alkali for a long time, 16 weeks.
The degree of crystallinity slightly increased from 43% to 60% with delignication because amorphous components (lignin, hemi-
cellulose) were removed. However, the increased crystallinity did not negatively aect the 3-d sugar yield of enzymatic hydrolysis.
Oxidative lime pretreatment lowered the acetyl and lignin contents to obtain high digestibility, regardless of crystallinity.
The non-linear models for 3-d hydrolysis yields of glucan (Yg), xylan (Yx), and holocellulose (Ygx) were empirically established as
a function of the residual lignin (L) for the corn stover pretreated with lime and air.
 2005 Published by Elsevier Ltd.

Keywords: Corn stover; Lime pretreatment; Structural features; Acetyl; Lignin; Crystallinity

1. Introduction tion is sucient to obtain high digestibility regardless

The enzymatic hydrolysis of lime-treated biomass is
aected by structural features resulting from the treat-
ment. Key structural features that aect digestibility
are the extent of acetylation, lignication, and
crystallization.
Lime pretreatment removes amorphous substances
(e.g., lignin and hemicellulose), which increases the crys-
tallinity index. Chang and Holtzapple (2000) reported
correlations between enzymatic digestibility and three
structural factors: lignin content, crystallinity, and acetyl
content. They concluded that (1) extensive delignica-
*
Corresponding author. Tel.: +1 409 845 9708; fax: +1 409 845 6446.
E-mail address: m-holtzapple@tamu.edu (M.T. Holtzapple).
of acetyl content and crystallinity; (2) delignication
and deacetylation remove parallel barriers to enzymatic
hydrolysis; and (3) crystallinity signicantly aects initial
hydrolysis rates but has less eect on ultimate sugar
yields. These results indicate that an eective lignocellu-
lose treatment process should remove all the acetyl
groups and reduce the lignin content to about 10% in
the treated biomass. Further lignin reduction incurs an
extra cost; therefore, it is not justied for enzymatic
hydrolysis.
Lee and Fan (1982) reported that the rate of
enzymatic hydrolysis depends on enzyme adsorp-
tion and the eectiveness of the adsorbed enzymes,
instead of the diusive mass transfer of enzyme. Lignin
removal increases enzyme eectiveness by eliminating

0960-8524/$ - see front matter  2005 Published by Elsevier Ltd.

doi:10.1016/j.biortech.2005.03.040
584 S. Kim, M.T. Holtzapple / Bioresource Technology 97 (2006) 583591

non-productive adsorption site and by increasing access
to holocellulose (cellulose + hemicellulose).
In hardwoods, the O-acetyl groups are combined
with the xylose units, whereas in the softwoods, they
are combined with the mannose and glucose units of
glucomannans (Whistler and Jeans, 1943). Acetylation
sites are maximally 2 positions per anhydroxylose unit.
For natural xylan, the degree of acetylation is approxi-
mately 1. Deacetylation in alkaline solution increases
moisture content (swelling) (Mitchell et al., 1990).
Kong et al. (1992) reported that alkalis remove acetyl
groups from hemicellulose (mainly xylan) thereby reduc-
ing the steric hindrance of hydrolytic enzymes and
greatly enhancing carbohydrate digestibility. The re-
moval of acetyl groups from xylan is not mainly aected
by swelling, because there are no cation eects among
several dierent types of alkalis. They concluded that
the sugar yield in enzymatic hydrolysis is directly associ-
ated with acetyl group content, and not with the swelling
feature.
The acetyl groups of biomass can be cleaved by
hydrothermal treatment (autohydrolysis; P170
C in
water), because the hydronium ions from water autoion-
ization removes acetyl groups to give acetic acid in the
reaction medium (Garrote et al., 2002).
The degree of crystallinity of lignocellulosic biomass
has been considered an important factor in resisting
enzymatic hydrolysis (Chang and Holtzapple, 2000;
Puri, 1984; Rivers and Emert, 1989). It has been re-
ported that the particle size of biomass (excluding big
chunks) has no eect on enzymatic conversions of corn
stover (Kaar and Holtzapple, 2000), switchgrass (Chang
and Holtzapple, 2000), and bagasse (Sinitsyn et al.,
1991).
In this study, the enzymatic digestibility of untreated
and lime-treated corn stovers was correlated with three
structural features: acetylation, lignication, and
crystallinity.
2. Methods
2.1. Lime pretreatment
Corn stover was supplied from NREL (National
Renewable Energy Laboratory, Golden, CO) in two dif-
ferent batches (Source: BioMass AgriProducts, Harlan,
IA). The stover was already washed, dried, and milled
to pass 1/4-in. round screen before being delivered to
our laboratory.
Corn stover was pretreated with lime (calcium
hydroxide) in the presence of water. Packed-bed PVC
columns (D L = 1 in. 17 in.) were used for the lime-
pretreatment reaction at 25 (ambient temperature), 35,
45, and 55
C. Raw biomass (15.0 g dry weight of corn
stover), calcium hydroxide (7.5 g dry weight), and dis-
tilled water (150 mL) were transferred into a reactor
after thoroughly being mixed using a spatula. After
the pretreatment time elapsed (016 weeks), the reactors
were moved out of the system randomly and cooled
down to ambient temperature. Samples were then col-
lected for various analyses. Lime was used in excess to

R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 Reactors

Water
Jacket

Manifold
(N2 or Air)

Manifold Drain
(Water) Temperature Water
Frame sensor

Tank P
Temperature
Controller

Tray Float
Pump ATM CO2
N2 scrubber
o
C Heating Element or
Air

Fig. 1. Schematic diagram of the jacketed reactor system for lime pretreatment in the non-oxidative (N2 supply) and oxidative (air supply)
conditions.
 S. Kim, M.T. Holtzapple / Bioresource Technology 97 (2006) 583591 585

maintain the alkaline pH (P12.0) and to determine the 600

actual amounts of lime consumed during the pretreat- I002 Iam
ment. Nitrogen and air were purged into each reactor 500 CrI = 100
I002
to make non-oxidative and oxidative conditions, respec-
tively, after being preheated and saturated in the im- 400

Intensity (I)
mersed cylinder in the heating tank. Air was scrubbed
of carbon dioxide by passing it through a lime-water 300
slurry in a bottle. A schematic diagram of the whole
reactor system is shown in Fig. 1. 200
Iam I002
2.2. Determination of lime consumed 100
during the pretreatment
0
10 12 14 16 18 20 22 24 26
The amounts of lime unreacted after the pretreatment 2 (degree)
at each condition were determined by pH neutralization
with a standard solution of acid, 5-N HCl (ACS certi- Fig. 2. X-ray diraction pattern of raw corn stover.
ed). The amounts of lime consumed for pretreatment
were estimated by the subtraction of the amounts of The denition of crystallinity index is
lime unreacted from the initial amounts of lime for each I 002
I am
pretreatment. CrI  100 3
I 002

2.3. Estimation of delignication and deacetylation
Klason and acid-soluble lignin content in corn stover
were determined by NREL standard procedures nos. 03
and 04, respectively.
Delignication was dened by the weight fraction of
original lignin removed as follows:
Delignification 1
W L 1 washed-only (control) and the lime-treated, pH-neutral-
Here, WL is the fraction of residual lignin expressed as
follows:
L  YT
WL 2 (at least 6 times) with distilled water to remove water-
L0
where L and L0 are the Klason lignin contents of treated
biomass and raw biomass, respectively, and YT is the
pretreatment yield of the total solids determined after
the lime pretreatment. Acid-soluble lignin content was
not included in this study, because its variation was rel-
atively large and interfered with the accurate estimation
of delignication at lower lignin contents.
where I002 = intensity of the diraction from the 0 0 2
plane at 2h = 22.6
and Iam = intensity of the back-
ground scatter measured at 2h = 18.7
(Segal et al.,
1959), as shown in Fig. 2.
2.5. Enzymatic hydrolysis
Substrates for the enzyme reaction were the untreated
ized, and washed corn stovers. After lime pretreatment,
the treated samples were neutralized with 5-N HCl to
remove the unreacted lime and washed several times
soluble components. The substrate concentration was
10 g cellulose/L. Cellulase enzyme (Spezyme CP, Lot
1.0
0.8
g lignin removed
Delignification

g initial lignin

Acetyl content in untreated and treated corn stover 0.6

was determined as described in the previous report
(Kim and Holtzapple, 2005). Deacetylation can be esti-
0.4
mated the same way as delignication dened in Eqs. (1)
and (2).
0.2
2.4. Crystallinity
0.0
The crystallinities of untreated and treated corn 0.00 0.05 0.10 0.15 0.20
stovers were measured by the XRD Laboratory, Lime consumption
Department of Geophysics, Texas A&M University g Ca(OH)2 reacted
(College Station, TX) using a Rigaku Powder X-ray Dif- g raw biomass
fractometer (Rigaku Denki Co., Japan). The specimen Fig. 3. The fractional changes of lignin solubilized as a function of the
was scanned at 2
/min from 2h = 10
to 26
with a step fraction of lime consumed in the non-oxidative condition (m) and
size of 0.05
. oxidative condition (d).
586 S. Kim, M.T. Holtzapple / Bioresource Technology 97 (2006) 583591

301-00348-257) was kindly provided by NREL. Excess more delignication. In the lower range of lime con-
amounts of b-glucosidase (Novozyme 188, 250 CBU/g sumption (60.06 g lime reacted/g raw biomass), approx-
of activity) was added to completely convert cellobiose imately 50% of original lignin in raw biomass was
to glucose, i.e., 40 CBU/g cellulose. Cellulase was added removed easily. This phenomenon was independent of
at 15 FPU/g cellulose. Cellulase activity was periodically the presence of oxygen. However, removing the remain-
determined as lter paper units per mL, as described by ing fraction of the lignin required oxidative treatment
NREL standard procedure no. 06. Citrate buer with additional lime consumption.
(0.05 M, pH 4.8, in reaction mixture) and sodium azide
solution (0.03 (w/v)% in reaction mixture) were used to 3.2. Eect of lime pretreatment on deacetylation
keep constant pH and prevent microbial contamination, and delignication
respectively. The enzyme reaction was conducted in a
shaking incubator at 50
C and 150 rpm for 3 days. The acetyl group was removed very quickly regardless
The concentration of sugars (glucose and xylose) was of temperature and the oxidation condition, whereas lig-
determined by HPLC using Aminex HPX-87P column nin was removed gradually throughout the pretreatment
(BioRad, USA) and RI detector (RefractoMonitor and depended on the pretreatment condition. For exam-
III, Model 1109, LDC/MiltonRoy, USA). ple, deacetylation reached a plateau within 1 week and
there were no signicant dierences between the extremes
(non-oxidative condition at 25
C and oxidative condi-
3. Results and discussion tion at 55
C). However, the delignication trends be-
tween these two conditions were very dierent (Figs. 47).
3.1. Eect of lime consumption on delignication
3.3. Eect of crystallinity
In alkaline pretreatment, much of the lignin in ligno-
cellulosic biomass can be solubilized by hydroxide ion, Corn stover crystallinity increased after lime pretreat-
OH
. As shown in Fig. 3, more lime is needed to get ment. It was related to the solubilization of lignin and

1.0 1.0 1.0

1.0
(g lignin removed/g initial lignin)
(g acetyl removed/g initial acetyl)

(g acetyl removed/g initial acetyl)

(g lignin removed/g initial lignin)

0.8 0.8 0.8 0.8
Delignification
Deacetylation

Deacetylation

Delignification
0.6 0.6 0.6
0.6

0.4 0.4 0.4

0.4

0.2 0.2 0.2 0.2

0.0 0.0 0.0 0.0

0 1 2 3 4 0 1 2 3 4
a
a Pretreatment time (weeks)
Pretreatment time (weeks)

1.0 1.0 1.0

(g lignin removed/g initial lignin)

1.0
(g acetyl removed/g initial acetyl)

(g acetyl removed/g initial acetyl)

(g lignin removed/g initial lignin)

0.8 0.8 0.8

0.8
Delignification
Deacetylation

Delignification
Deacetylation

0.6 0.6
0.6 0.6

0.4 0.4 0.4

0.4

0.2 0.2 0.2

0.2

0.0 0.0 0.0

0.0
0 1 2 3 4 0 1 2 3 4
b Pretreatment time (weeks) b Pretreatment time (weeks)

Fig. 4. The proles of deacetylation (j) and delignication (d) during Fig. 5. The proles of deacetylation (j) and delignication (d) during
the lime pretreatment in non-oxidative (a) and in oxidative (b) the lime pretreatment in non-oxidative (a) and in oxidative (b)
conditions at 25
C. conditions at 35
C.
 S. Kim, M.T. Holtzapple / Bioresource Technology 97 (2006) 583591 587

1.0 1.0 1.0 1.0

(g lignin removed/g initial lignin)

(g acetyl removed/g initial acetyl)

(g lignin removed/g initial lignin)

(g acetyl removed/g initial acetyl)
0.8 0.8 0.8 0.8

Delignification
Deacetylation

Delignification
0.6 0.6

Deacetylation
0.6 0.6

0.4 0.4
0.4 0.4
0.2 0.2
0.2 0.2
0.0 0.0
0 1 2 3 4
0.0 0.0
a Pretreatment time (weeks) a 0 1 2 3 4

1.0 1.0 1.0 1.0

(g lignin removed/g initial lignin)

(g acetyl removed/g initial acetyl)

(g lignin removed/g initial lignin)

(g acetyl removed/g initial acetyl)
0.8 0.8 0.8
Delignification 0.8
Deacetylation

Delignification
Deacetylation
0.6 0.6
0.6 0.6

0.4 0.4
0.4 0.4
0.2 0.2
0.2 0.2
0.0 0.0
0 1 2 3 4
0.0 0.0
b Pretreatment time (weeks) 0 1 2 3 4
b Pretreatment time (weeks)
Fig. 6. The proles of deacetylation (j) and delignication (d) during
the lime pretreatment in non-oxidative (a) and in oxidative (b) Fig. 7. The proles of deacetylation (j) and delignication (d) during
conditions at 45
C. the lime pretreatment in non-oxidative (a) and in oxidative (b)
conditions at 55
C.

hemicelluloseboth are amorphous components.

Regardless of the oxidative treatment, as delignication to more than 0.40 g holocellulose hydrolyzed/g holo-
proceeded due to lime pretreatment, the xylan (hemicel- cellulose in treated biomass. Later in the treatment,
lulose) contents slightly decreased causing the glucan to complete deacetylation was achieved, and the 3-d enzy-
xylan ratio (G/X) to increase (Fig. 8). This means that matic digestibility increased linearly with delignication.
lignin and hemicellulose are selectively removed (or sol- Linear relationships between delignication and 3-d
ubilized), but cellulose is not signicantly aected by enzymatic digestibility at 15 FPU/g cellulose were better
lime pretreatment at mild temperatures (2555
C), even for glucan (R2 = 0.7551) than for xylan (R2 = 0.4321), as
though corn stover was contacted with alkali for a long shown in Fig. 10. The low value of linear regression
time, 16 weeks. coecient for xylan could be due to insucient xylanase
The degree of crystallinity slightly increased with de- activity in the cellulase enzyme preparation, as discussed
lignication due to the increase of glucan content in the in Kim and Holtzapple (2005).
pretreated solid fraction of corn stover (Fig. 9). These linear relationships were successfully identied
between 3-d enzymatic digestibilities of glucan and/or
3.4. Eect of deacetylation and delignication xylan and delignication.
on 3-d sugar yield of enzymatic hydrolysis
3.5. Proposed model for corn stover
Pretreated corn stover showed high levels of deacety-
lation and delignication, which increased its enzymatic Chang and Holtzapple (2000) reported that lignin
digestibility. and acetyl groups in hemicellulose are signicant barri-
In the early stages of lime pretreatment, when corn ers for cellulase enzymes to access the lignocellulosic
stover was highly deacetylated but with little delignica- ber matrix. Also, they reported that crystallinity aects
tion, its 3-d hydrolysis yield of holocellulose at 15 FPU/ the eciency of enzyme contacted with cellulose and
g cellulose of enzyme loading increased from 0.25 g hemicellulose. Lime pretreatment signicantly removes
holocellulose hydrolyzed/g holocellulose in raw biomass the acetyl and lignin barriers allowing enzyme to access
588 S. Kim, M.T. Holtzapple / Bioresource Technology 97 (2006) 583591

60 sigmoidal as a function of lignin content remaining in

lime-treated woody biomass. By their empirical model,
50 3-d hydrolysis yields increase sigmoidally rather than
linearly at the low range of lignin residuals. In our case,
Glucan and xylan contents
(g/100 g treated biomass)

40 Glucan lower lignin residuals (higher delignication) were only

obtained from the oxidative lime-pretreatment.
30 In this study, with lime-treated corn stover, the crys-
tallinity and acetyl contents did not vary. The acetyl
20 group content was almost 0% and CrI did not change
Xylan
signicantly; therefore, it is expected that the 3-d hydro-
10 lysis proles would be sigmoidal with residual lignin (L).
The following empirical equations generate the requisite
0 sigmoidal relationships:
0 20 40 60 80 100
Delignification a0
Yg   4
g lignin removed a1
L
1 exp
a 100 g lignin in raw biomass a2
4 and
b0
Yg   5
b1
L
3 1 exp
in lime-pretreated biomass

b2
Ratio of glucan/xylan

2
70

1 60
Crystallinity (%)

0 50
0 20 40 60 80 100
Delignification
Treated
g lignin removed 40

b 100 g lignin in raw biomass Untreated

Fig. 8. Correlation of delignication with holocellulose (glucan and 30
xylan) content (a) and with the ratio of glucan to xylan (b) of lime- 0 20 40 60 80 100
Delignification
pretreated solid in non-oxidative (m) and oxidative (d) conditions.
g lignin removed
a 100 g lignin in raw biomass
the substrates, cellulose and hemicellulose. Even though
the crystallinity is high, the amount of adsorbed enzyme 70
is sucient to achieve high digestibility in a 3-d enzy-
matic hydrolysis.
60
Oxidative lime pretreatment lowers the acetyl and lig-
Crystallinity (%)

nin contents to obtain high digestibility, regardless of

crystallinity. This result agrees with Chang and Holtzap- 50
ples (2000) observations of lime pretreatment on poplar Treated
wood.
Using 147 data sets of pretreated poplar wood, 40
Chang and Holtzapple (2000) suggested an empirical Untreated
formula for hydrolysis yields (Yg,Yx, and Ygx) for glu-
30
cose, xylose, and total sugar (glucose + xylose) that is 30 40 50 60
a function of the contents of lignin (L), acetyl (A), Glucan content
glucan (G), xylan (X), and crystallinity (CrI). b (g glucan/100 g treated biomass)
Their data are summarized in Fig. 11, which shows, Fig. 9. Correlation of crystallinity with delignication (a) and glucan
for xed values of acetyl content and crystallinity, the content (b) of lime-pretreated corn stover in non-oxidative (h) and
proles of 3-d hydrolysis yield from holocellulose are oxidative (j) conditions.
 S. Kim, M.T. Holtzapple / Bioresource Technology 97 (2006) 583591 589

100 100

Ygx, 3-d hydrolysis yield of holocellulose

3-d hydrolysis yield of glucan, Yg 100

100 g glucan in treated biomass

g holocellulose hydrolyzed
80 80

100 g treated biomass

g glucan hydrolyzed

60 60

R2 = 0.7551
40 40
Acetyl (%)

20 20
0
3
0
0 0 5 10 15 20 25 30
0 20 40 60 80 100 L, Lignin content
Delignification g lignin remaining
g lignin removed 100 g treated biomass

a 100 g lignin in raw biomass

Fig. 11. 3-d hydrolysis yield of holocellulose as a function of lignin
content in lime-treated woody biomass with air (Chang and Holtzap-
ple, 2000).
100
3-d hydrolysis yield of xylan, Yx100

Table 1
100 g xylan in treated biomass

80 Parameters of correlations for 3-d hydrolysis yields of glucan (Yg, g

glucan hydrolyzed/100 g glucan in treated biomass) and xylan (Yx, g
g gxylan hydrolyzed

xylan hydrolyzed/100 g xylan in treated biomass)

60
Parameters Yg (Eq. (4)) Yx (Eq. (5))
R2 = 0.4321 a0 or b0 120 90
40 a1 or b1 13.3 16.3
a2 or b2
5.7
5.3

20
lysis yield of total sugar (g holocellulose hydrolyzed/
0 100 g treated biomass); a0a2 and b0b2 are constants.
0 20 40 60 80 100 The constants (ai and bi, i = 0, 1, and 2) of the simpli-
Delignification
ed models listed in Table 1 were empirically determined
g lignin removed from the oxidative lime-pretreatment data by using non-
b 100 g lignin in raw biomass linear regression for parameter estimation by minimiz-
ing the root mean square errors in Excel. The plots of
Fig. 10. Eect of delignication on the hydrolysis yields of glucan (a)
and xylan (b) in 3-d enzymatic digestibility at 15 FPU/g cellulose.
Eqs. (4)(6) are shown as solid lines in Fig. 12. Thus,
for oxidative lime-pretreatment of corn stover, the
hydrolysis yields of glucan (Yg), xylan (Yx), and holocel-
lulose (Ygx) were well described by the simplied non-
and linear models with the single parameter (L).
Y g  G=0.90 Y x  X =0.88
Y gx
G=0.90 X =0.88
4. Conclusion
Yg Yx
6
0.90X 0.88G
1 1 Lime is a very eective chemical for deacetylation and
0.88G 0.90X delignication of corn stover.
where G is the glucan content in lime-treated corn stover Acetyl groups were removed within 1 week regard-
(g glucan/100 g treated biomass); X is the xylan content less of temperature and the oxidation condition for lime
in lime-treated corn stover (g xylan/100 g treated bio- pretreatment, whereas lignin was removed gradually
mass); L is the lignin content in lime-treated corn stover through the whole period of pretreatment and depended
(g lignin/100 g treated biomass); Yg is the 3-d hydrolysis on the pretreatment conditions.
yield of glucan (g glucan hydrolyzed/100 g treated bio- The hydrolysis yield of glucan and xylan to glucose
mass); Yx is the 3-d hydrolysis yield of xylan (g xylan and xylose was aected by deacetylation and linearly de-
hydrolyzed/100 g treated biomass); Ygx is the 3-d hydro- pended on delignication.
590 S. Kim, M.T. Holtzapple / Bioresource Technology 97 (2006) 583591

120 100

100 g glucan in treated biomass

100 g xylan in treated biomass

100 80
g glucan hydrolyzed

g xylan hydrolyzed
80
60
Yg

60

Yx
40
40

20
20

0 0
0 5 10 15 20 25 0 5 10 15 20 25
L L
g lignin remaining g lignin remaining
100 g treated biomass 100 g treated biomass
a b

120
100 g holocellulose in treated biomass

100
g holocellulose hydrolyzed

80

60
Ygx

40

20

0
0 5 10 15 20 25
L
g lignin remaining

c 100 g treated biomass

Fig. 12. Correlations between the weight fraction of the residual lignin (WL) and 3-d hydrolysis yields: (a) Yg; (b) Yx; (c) Ygx, for corn stover
pretreated with lime and air. The enzyme loading rate is 15 FPU/g cellulose. The solid lines show plots of non-linear regressions using Eqs. (4)(6),
respectively.

The degree of crystallinity increased with delignica- References

tion due to the increase of glucan content in the pre-
treated solid fraction of corn stover. Chang, V.S., Holtzapple, M.T., 2000. Fundamental factors aecting
Oxidative lime pretreatment lowers the acetyl and biomass enzymatic reactivity. Applied Biochemistry and Biotech-
nology 8486, 537.
lignin contents to obtain high digestibility, regardless Garrote, G., Domnguez, H., Parajo, J.C., 2002. Autohydrolysis
of crystallinity. of corncob: study of non-isothermal operation for xylooligosac-
Non-linear models for 3-d hydrolysis yields of glucan charide production. Journal of Food Engineering 52, 211
(Yg), xylan (Yx), and holocellulose (Ygx) were established 218.
Kaar, W.E., Holtzapple, M.T., 2000. Using lime pretreatment to
as a function of the residual lignin content (L) for the
facilitate the enzyme hydrolysis of corn stover. Biomass and
corn stover pretreated with lime and air. Bioenergy 18 (3), 189199.
Kim, S., Holtzapple, M.T., 2005. Lime pretreatment and enzymatic
hydrolysis of corn stover. Bioresource Technology, in press,
Acknowledgement doi:10.1016/j.biortech.2005.01.014.
Kong, F., Engler, C.R., Soltes, E.J., 1992. Eects of cell-wall acetate,
This work was supported by the US Department of xylan backbone, and lignin on enzymatic hydrolysis of aspen wood.
Agriculture, contract number 00-52104-9663. Applied Biochemistry and Biotechnology 34/35, 2335.
 S. Kim, M.T. Holtzapple / Bioresource Technology 97 (2006) 583591
Lee, Y.H., Fan, L.T., 1982. Kinetic studies of enzymatic hydrolysis of
insoluble cellulose: analysis of the initial rates. Biotechnology and
Bioengineering 24, 23832406.
Mitchell, D.J., Grohmann, K., Himmel, M.E., 1990. Eect of the
degree of acetylation of the enzymatic digestion of acetylated
xylans. Journal of Wood Chemistry and Technology 10 (1), 111
121.
NREL standard procedure no. 03. Determination of acid-insoluble
lignin in biomass.
NREL standard procedure no. 04. Determination of acid-soluble
lignin in biomass.
NREL standard procedure no. 06. Measurements of cellulase
activities.
591
Puri, V.P., 1984. Eect of crystallinity and degree of polymerization of
cellulose on enzymatic saccharication. Biotechnology and Bio-
engineering 26, 12191222.
Rivers, D.B., Emert, G.H., 1989. Factors aecting the enzymatic
hydrolysis of municipal-solid-waste components. Biotechnology
and Bioengineering 31, 278281.
Sinitsyn, A.P., Gusakov, A.V., Vlasenko, E.Y., 1991. Eect of
structural and physico-chemical features of cellulosic substrates
on the eciency of enzymatic hydrolysis. Applied Biochemistry
and Biotechnology 30, 4359.
Whistler, R.L., Jeans, A., 1943. Quantitative estimation of acetyl in
carbohydrate acetates. Industrial and Engineering Chemistry 15
(5), 317318.