Cellulose crystallinity – a key predictor of the enzymatic
hydrolysis rate
Me
´
lanie Hall, Prabuddha Bansal, Jay H. Lee, Matthew J. Realff and Andreas S. Bommarius
School of Chemical and Biomolecular Engineering, Georgia Institute of Technology, Atlanta, GA, USA
Introduction
The enzymatic hydrolysis of cellulose to glucose has
received increased interest over the last 10 years,
and growing demand for economically sustainable
biofuels indicates an urgent need for reducing the
costs associated with their production. Cellulose,
a polysaccharide made by most plants, is one of the
most abundant organic compounds on Earth and
represents a major potential feedstock for the biofuels
industry. However, the current enzymatic degradation
of cellulose faces major issues that prevent its wide
utilization in the production of economically competi-
tive biofuels [1–4].
Cellulose is hydrolyzed to glucose via the synergistic
action of several enzymes. Endoglucanases (EC 3.2.1.4)
break down cellulose chains at random positions
within the chains, whereas exoglucanases (i.e. cello-
biohydrolases, EC 3.2.1.91) cleave off cellobiose speci-
fically from the chain ends in a processive manner
[5–10]. Cellobiose is subsequently converted into glucose
by b-glucosidase (EC 3.2.1.21) [7,11–14]. The exo-endo
synergism is easily expained by the fact that endo-
glucanases provide more chain ends for cellobiohydro-
lases to act upon [15–19]. The hydrolysis of insoluble,
solid cellulose is a heterogeneous reaction, which does

bound enzyme concentration stayed constant. This finding supports the
determinant role of crystallinity rather than adsorption on the enzymatic
rate. Thus, the cellulase activity and initial rate data obtained from various
samples may provide valuable information about the details of the mecha-
nistic action of cellulase and the hydrolysable ⁄ reactive fractions of cellulose
chains. X-ray diffraction provides insight into the mode of action of Cel7A
from T. reesei. In the conversion of cellulose, the (021) face of the cellulose
crystal was shown to be preferentially attacked by Cel7A from T. reesei.
Abbreviations
CP ⁄ MAS, cross polarization ⁄ magic angle spinning; CrI, crystallinity index; DNS, dinitrosalicylic acid; PASC, phosphoric acid swollen cellulose.
FEBS Journal 277 (2010) 1571–1582 ª 2010 The Authors Journal compilation ª 2010 FEBS 1571
cleave off cellobiose and move along the same chain,
hydrolyzing glycosidic bonds until an event occurs that
terminates cleavage. As the reaction proceeds to inter-
mediate degrees of conversion, the rate of the reaction
decreases dramatically, and the final part of cellulose
hydrolysis requires an inordinate fraction of the overall
total reaction time [27,28]. Several factors, both
substrate- and enzyme-related, are suggested to be
responsible for this slowdown of the reaction rate but,
so far, no mechanistic explanation of the slowdown
has been validated. The substrate characteristics
often implied in the slowdown of the reaction rate
include surface area, porosity, the degree of poly-
merization, crystallinity, and the overall composition
(complex substrates such as lignocellulosics versus
pure cellulose). For enzyme-related features, deactiva-
tion, inhibition, jamming, clogging and imperfect
processivity are often cited as causes of the slowdown
[14,29,30].

lase-catalyzed degradation lead to hydrolysis rates that
were directly related to the CrI of the cellulose sample
[17,31,37–39]. To correctly relate the CrI with hydro-
lysis rate, it is of prime importance to study samples
that have the same basic composition and provenance.
For this reason, pure cellulose may be preferable to
complex substrates because the presence of lignin or
hemicellulose may interfere with the action of cellulase
and reduce accessibility, and therefore the hydrolysis
rates [29,40,41].
Another important criterion related to hydrolysis
rate involves the adsorption capacity of cellulases onto
cellulose. The rate of hydrolysis was shown to be pro-
portional to the amount of adsorbed enzymes
[22,25,42–44]. Additionally, the difference in reactivity
between a crystalline and an amorphous cellulose was
found to be related to the adsorption capacity of endo-
glucanases on both types of substrate [45]. Further-
more, the degree of crystallinity of cellulose influences
adsorption at a given protein loading and the maxi-
mum adsorption constant was shown to be greatly
enhanced at low crystallinity indices [46]. The same
study concluded that the effective binding was the lim-
iting parameter with respect to the hydrolysis rate in
the case of cellulose with low degrees of crystallinity,
despite a high adsorption constant.
Amorphous cellulose has been widely used to inves-
tigate cellulase activity [35,47–51]. Treatment with
85% phosphoric acid to produce phosphoric acid swol-
len cellulose (PASC) results in complete dissolution of

crystallinity using phosphoric acid solutions of precisely
Cellulose crystallinity M. Hall et al.
1572 FEBS Journal 277 (2010) 1571–1582 ª 2010 The Authors Journal compilation ª 2010 FEBS
calibrated concentration. These pretreated cellulose
samples were employed to investigate and elucidate the
relationship between the degree of crystallinity, adsorp-
tion and the enzymatic hydrolysis rates.
Results
Cellulase hydrolysis rate and cellulose
crystallinity
Various types of (ligno)cellulosic substrate are employed
in current enzymatic hydrolysis studies and thus are
a source of discrepancies in the results obtained and
the potential confusion regarding the challenging
problem of understanding the mode of action of cellu-
lase [35]. The presence of hemicellulose, and especially
lignin, a strong adsorbent on cellulase, in lignocellulo-
sics, interferes with the enzymatic activity of cellulases
on cellulose [14,29,41]. To avoid such interference, we
used Avicel, a commonly used, commercially and
reproducibly obtainable pure cellulose substrate with a
well-characterized structure and an average degree
of crystallinity of 60% (measured via solid state
13
C-NMR).
Phosphoric acid pretreatment
First, to validate the efficiency of the phosphoric acid
pretreatment, acid-pretreated samples were hydrolyzed
with cellulases and an excess of b-glucosidase to remove
product inhibition and fully convert cellobiose to glu-

C
Fig. 1. Effect of phosphoric acid concentration on: (A) initial rate of
Avicel enzymatic hydrolysis (glucose produced in the first 2 min of
the reaction with cellulases); (B) CrI obtained from X-ray diffraction
data and multivariate statistical analysis; (C) moisture content of
cellulose samples after treatment with phosphoric acid (measure-
ment performed after tightly controlled filtration and subsequent
drying at 60 °C). The results shown are the average of at least
triplicates (duplicates for crystallinity).
M. Hall et al. Cellulose crystallinity
FEBS Journal 277 (2010) 1571–1582 ª 2010 The Authors Journal compilation ª 2010 FEBS 1573
production) from 1 to 4.75 gÆL
)1
Æmin
)1
(Fig. 1A) over a
narrow range of phosphoric acid content (75–80%),
and not as a step change but as a steep continuum. No
further increase was observed in the range 80–85%,
which is the maximum possible phosphoric acid con-
centration, close to the 81% obtained by Moxley et al.
[59] for maximum glucan digestibility. Below 75%, the
glucose production rate tends to level off, with a mini-
mum being obtained with untreated Avicel (0.6 gÆL
)1
Æ
min
)1
glucose at 0% phosphoric acid).
There are several ways to measure cellulose CrI.

Figure 1B shows that the CrI closely tracks the
breakthrough behavior of reactivity (Fig. 1A) when
employing the same amount of phosphoric acid that
was used to pretreat the cellulose sample: the degree of
crystallinity remains fairly unchanged at approximately
55–60% over a wide range of phosphoric acid concen-
trations but decreases linearly to almost 0% in a con-
centration range of 75–80% phosphoric acid. Thus,
the phosphoric acid effect is clearly evident: not only is
it related to dissolution capacity [59], but also it dis-
rupts the crystalline structure of cellulose and can turn
partially crystalline cellulose amorphous. Avicel, a mi-
crocrystalline type of cellulose, has a mixed composi-
tion (amorphous and crystalline) and the results
obtained in the present study suggest that the more
concentrated the acid solution, the more crystalline
regions are turned amorphous. The capacity of cellu-
lose samples to retain water relative to the proportion
of amorphous parts has been postulated [68,69], and
was verified with the acid-treated samples. Figure 1C
shows the tight relationship between moisture content
and acid concentration, supporting the conclusion with
respect to structural changes derived from crystallinity
measurement occurring in the 75–80% acid concentra-
tion range. Upon treatment at higher acid concentra-
tions, cellulose samples have a higher capacity to
retain water, owing to the higher number of hydroxyl
groups that are available to bind to (and adsorb) water
molecules because these hydroxyl groups are no longer
hydrogen bonded to other glucose moieties. A cellulose

over conversion) were observed at different levels of
intensity [14,31,33,70,71]. As mentioned above, the dif-
ferent types of substrate as well as the analytical meth-
ods employed contributed to the absence of a clear
understanding of the mechanistic action of cellulase on
partially crystalline cellulose. Furthermore, in situ
measurements of cellulose structure under reacting
conditions (i.e. in aqueous buffers) are difficult to
perform because all current methods require the prior
isolation of cellulose and drying [29].
The CrI of Avicel was monitored via X-ray diffrac-
tion during its hydrolysis by a commercial mixture of
cellulases from T. reesei and an excess of b-glucosidase
to prevent cellobiose inhibition. The X-ray diffraction
data obtained gave an artificially high degree of crys-
tallinity for untreated Avicel (92%) using the method
of Segal et al. [60]. Small variations at such high values
are challenging to monitor; therefore, cross polariza-
tion ⁄ magic angle spinning (CP ⁄ MAS)
13
C-NMR spec-
troscopy was employed as an alternate method. The
CrI of untreated Avicel (calculated as described previ-
ously) [28] averaged 61% and was found to be
constant over the course of hydrolysis, until
approximately 90% conversion (Fig. 3). Similarly,
using purified Cel7A from T. reesei (see Materials and
methods) instead of a mixture of cellulases, no change
in crystallinity was observed; however, variations in
relative peak intensity in X-ray diffraction patterns

the same degree of loading as employed during a com-
mon enzymatic hydrolysis run (175 lgÆmg
)1
cellulose;
Figs 1–3). Surprisingly, a maximum value of adsorbed
enzyme concentration (150 lgÆmg
)1
cellulose) was
reached for the cellulose samples with a CrI below a
threshold value of approximately 45% (Fig. 6A, open
triangles), whereas the amount of adsorbed enzyme
Fig. 3. CrI of Avicel monitored during hydrolysis with cellulases via
CP ⁄ MAS
13
C-NMR [reactions were run at 50 °C in sodium acetate
buffer (50 m
M,pH5)at20gÆL
)1
Avicel with the addition of b-gluco-
sidase (15 kUÆL
)1
) and cellulases (24 mLÆL
)1
, 3.4 gÆL
)1
total
protein)]. The results shown are the average of duplicates.
Fig. 4. X-ray diffraction patterns of untreated Avicel and partially
converted cellulose in the range 10–40° (2h). x-axis: Bragg angle
(2h). The reflection of face (021) of the crystal (centered around

Cellulase hydrolysis rate and cellulose crystallinity
The correlation between the CrI and the initial hydro-
lysis rate (Fig. 5) shows a continuous decrease in
rate as crystallinity increases. At higher degrees of
crystallinity, cellulose samples are less amenable to
enzymatic hydrolysis, less reactive and less accessible.
A
B
C
Fig. 6. Adsorption, CrI and initial rates at two cellulases loadings:
D, 175 lgÆmg
)1
cellulose; •, 1230 lgÆmg
)1
cellulose. Initial rates
correspond to the amount of glucose produced over a 2 min reac-
tion (20 mgÆmL
)1
cellulose, cellulases at 175 resp. 1230 lgÆmg
)1
cellulose and an excess of b-glucosidase, 50 °C). Adsorption stud-
ies were conducted at 4 °C over 30 min. (A) Adsorption versus CrI;
(B) initial rate versus adsorption; (C) initial rate versus CrI, where
the grey shaded area represents the importance and role of adsorp-
tion on enzymatic rate. Dotted lines are added for clarity to help
identify trends. The results shown are the average of quadrupli-
cates.
Fig. 5. Effect of crystallinity (obtained from X-ray diffraction data and
multivariate statistical analysis) on the initial rate in Avicel enzymatic
hydrolysis (glucose produced in the first 2 min of the reaction with

= 0.96; Fig. 5), demon-
strating that crystallinity is a good predictor of the
hydrolysis rate. More precisely, in a phosphoric acid
concentration range of 75–80%, the hydrolysis rate,
crystallinity and phosphoric acid concentration are
mutually dependent parameters resulting from the
structural changes that take place upon acid pretreat-
ment of cellulose and are also linearly related. The
degree of phosphoric acid addition enables the tight
control of the overall structure of cellulose in the Avicel
sample. This convenient method for reaching intermedi-
ate degrees of crystallinity allows the exclusion of addi-
tional parameters that might influence the enzymatic
action on cellulose, such as the type and source of cellu-
lose or mixed components, and yields an explicit proof
of the tight relationship between initial cellulose crystal-
linity and the rate of degradation by cellulases from
T. reesei. The use of this method could support kinetics
studies where the estimation of intrinsic parameters for
cellulose is needed. Furthermore, because the interpre-
tation of crystallinity data is not trivial, looking at
initial hydrolysis rates may be an elegant alternative to
estimating the degree of crystallinity of pure cellulose.
No significant change was observed in the degree of
crystallinity during the enzymatic hydrolysis of Avicel
up to 90% conversion (Fig. 3). Despite their ability to
distinguish different degrees of crystallinity, cellulases
are not efficient at reducing ⁄ disrupting overall cellulose
crystallinity, most likely because cellulose chains are
hydrolyzed as soon as their interactions with the crys-

ing another phenomenon, specifically adsorption.
A constant adsorption profile at different enzyme
concentrations was found to relate to increasing hydro-
lysis rates at decreasing degrees of crystallinity (Fig. 6)
and supports our previous conclusion. This is in
contrast to studies stating that increased hydrolysis
rates were likely the result of an increasing adsorptive
capacity rather than substrate reactivity [14]. The
observed phenomenon is most likely the result of a
difference in the amount of productively bound enzyme
and the percentage of surface coverage. Indeed, at low
degrees of crystallinity, adsorbed enzymes are more
active at the same overall concentration (i.e. initial rates
are higher; Fig. 6C), most likely because of a more
open cellulose structure that prevents enzyme molecules
residing on neighboring chains from hindering one
another [73]. At a very low CrI and constant adsorbed
enzyme concentration, the percentage of surface cover-
age is smaller because the surface area is larger at
M. Hall et al. Cellulose crystallinity
FEBS Journal 277 (2010) 1571–1582 ª 2010 The Authors Journal compilation ª 2010 FEBS 1577
lower crystallinity indices [14]. Exoglucanases may also
locate a chain end faster on an open structure and thus
be able to start hydrolysis immediately after binding
(initial rates were determined after only a 2 min reac-
tion time). Accessibility was suggested to be an impor-
tant factor that affects enzymatic hydrolysis rates [72]
and its increase at lower degrees of crystallinity was
proposed as a reason for enhanced digestibility [59]. It
has also been suggested that rendering the substrate

lysis rates.
Avicel hydrolysis rates were not significantly chan-
ged upon the addition of a much higher enzyme con-
centration for samples displaying a degree of
crystallinity in the range 60–50% (Fig. 6C), demon-
strating that all hydrolysable fractions of cellulose were
already covered by enzymes at lower loading, despite
an increase in the amount of adsorbed cellulase at
higher loading. High enzyme loading (1230 lgÆmg
)1
cellulose) resulted in saturation of the Avicel surface,
whereas low enzyme loading (175 lgÆmg
)1
cellulose)
led to less than full but more than half-saturation
(adsorption isotherms not shown). In other words,
a higher cellulose surface coverage (in an undersaturated
regime) does not necessarily lead to higher rates
because it might simply result in unproductive binding
once all of the hydrolysable fractions are covered. The
role of adsorption for a given cellulose sample appears
to be more important to the enzymatic rate at lower
degrees of crystallinity (Fig. 6C).
At higher enzyme loading, crystallinity appears to
play a minor role (Fig. 6A). At degrees of crystallinity
in the range 60–35%, the amount of adsorbed enzyme
increases linearly, whereas adsorption is constant below
a breakpoint that can be estimated at approximately
35% CrI (compared to 45% at lower enzyme loading).
Below 35% CrI, a maximum of absorbed cellulases was

phosphoric acid (85%) was obtained from EMD (Gibbs-
town, NJ, USA). Trichoderma reesei QM9414 strain was
obtained from ATCC (#26921; American Type Culture
Collection, Manassas, VA, USA). The BCA protein assay
kit was obtained from Thermo Fischer Scientific (Rockford,
IL, USA).
Phosphoric acid pretreatment
One gram of slightly moistened Avicel was added to 30 mL of
an ice-cold aqueous phosphoric acid solution (concentration
range 42–85% weight) and allowed to react over 40 min with
occasional stirring. After the addition of 20 mL of ice-cold
Cellulose crystallinity M. Hall et al.
1578 FEBS Journal 277 (2010) 1571–1582 ª 2010 The Authors Journal compilation ª 2010 FEBS
acetone and subsequent stirring, the resulting slurry was
filtered over a fritted filtered-funnel and washed three times
with 20 mL of ice-cold acetone, and four times with 100 mL
of water. The resulting cellulose obtained after the last
filtration was used as such in the enzymatic hydrolysis
experiments, and the moisture content was estimated upon
oven-drying at 60 °C overnight. Samples were freeze-dried
prior to X-ray diffraction measurement.
Enzymatic hydrolysis of cellulose
A suspension of Avicel (20 gÆL
)1
) in sodium acetate buffer
(1 mL, 50 mm, pH 5) was hydrated for 1 h with stirring
at 50 °C. b-Glucosidase (15 kUÆL
)1
) and cellulases
(24 mLÆL

Netherlands) at room temperature from 10 to 60 °C, using
Cu ⁄ Ka
1
irradiation (1.54 A
˚
) at 45 kV and 40 mA. The scan
speed was 0.021425°Æs
)1
with a step size of 0.0167°. CrI was
calculated using the peak intensity method [60]:
CrI ¼ðI
002
À I
am
Þ=I
002
 100 ð1Þ
where I
002
is the intensity of the peak at 2h = 22.5° and
I
am
is the minimum intensity corresponding to the amor-
phous content at 2h =18°.
Freeze-drying showed no impact on the crystallinity of
untreated Avicel.
Solid state
13
C-NMR
The solid-state CP ⁄ MAS

The CrI of cellulose samples was also calculated by quanti-
fying the contribution of amorphous cellulose (PASC) and
Avicel to its (normalized) X-ray diffraction spectra [58]:
I
j
ð2hÞ¼f
j
I
p
ð2hÞþð1 À f
j
ÞI
c
ð2hÞþe ð3Þ
where I
j
(2h) is the intensity of the j
th
sample at diffraction
angle 2h, I
p
(2h) is the intensity of PASC at diffraction
angle 2h, I
C
(2h) is the intensity of untreated Avicel at dif-
fraction angle 2h, f
j
is the contribution of PASC to the
spectrum and e is the random error.
^

C-NMR as 60%).
Cel7A purification
Trichoderma reesei QM9414 was grown on potato dextrose
agar plate under light illumination. Spores were harvested
and used to inoculate the liquid medium (minimal medium:
(NH
4
)
2
SO
4
5gÆL
)1
, CaCl
2
0.6 gÆL
)1
, MgSO
4
0.6 gÆL
)1
,
KH
2
PO
4
15 gÆL
)1
, MnSO
4

purified by means of anion-exchange chromatography using
a Q-Sepharose Fast Flow with a 10–500 mm sodium acetate
gradient (pH 5.5). Cel7A was eluted in the last peak, and
purity was confirmed by SDS-PAGE, where only one single
protein band was observable ($ 67 kDa). Enzyme concen-
trations were estimated by the Bradford assay, using BSA
as standard.
Adsorption study
Cellulose samples (20 mgÆmL
)1
) in NaOAc buffer (50 mm,
pH 5) were incubated at 50 °C for 1 h at 900 r.p.m., and
then were cooled down to 4 °C. Cellulases were added in
various amounts and the mixture was further agitated for
30 min. After centrifugation, the supernatant was collected
and protein content analysis was performed using the BCA
protein assay kit (Thermo Fischer Scientific).
Acknowledgements
Chevron Corporation is acknowledged for their fund-
ing. Dr J. Leisen and Dr J. I. Hong are thanked for
their technical assistance with the crystallinity measure-
ments.
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