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Cellulosecrystallinity–akeypredictorofthe 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 enzymatichydrolysisofcellulose 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]. Thehydrolysisof insoluble,
solid cellulose is a heterogeneous reaction, which does
not match the assumptions of kinetic models based on
Michaelis–Menten kinetics [13,14,20]. After an initial
phase of adsorption of cellulases on cellulose, which is
fast compared to hydrolysis [16,21–26], the enzymes
Keywords
Cel7A; cellulases; cellulose crystallinity;
hydrolysis; Trichoderma reesei
Correspondence
A. Bommarius, School of Chemical and
Biomolecular Engineering, Georgia Institute
of Technology, 311 Ferst Drive, Atlanta,
GA 30332-0100, USA
Fax: +1 404 894 2291
Tel: +1 404 385 1334
E-mail: andreas.bommarius@chbe.gatech.edu
(Received 13 December 2009, revised 16
January 2010, accepted 18 January 2010)
doi:10.1111/j.1742-4658.2010.07585.x
The enzymatichydrolysisofcellulose encounters various limitations that
are both substrate- and enzyme-related. Although thecrystallinityof pure
cellulosic Avicel plays a major role in determining therateofhydrolysis by
cellulases from Trichoderma reesei, we show that it stays constant during
enzymatic conversion. The mode of action of cellulases was investigated by
studying their kinetics on cellulose samples. A convenient method for
reaching intermediate degrees ofcrystallinity with Avicel was therefore
developed and the initial rateofthe cellulase-catalyzed hydrolysisof cellu-
lose was demonstrated to be linearly proportional to thecrystallinity index
of Avicel. Despite correlation with the adsorption capacity of cellulases
onto cellulose, at a given enzyme loading, the initial enzymaticrate contin-
ued to increase with a decreasing crystallinity index, even though the
bound enzyme concentration stayed constant. This finding supports the
determinant role ofcrystallinity 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 ofthe 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 ofthe 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, therateofthe reaction
decreases dramatically, and the final part of cellulose
hydrolysis requires an inordinate fraction ofthe overall
total reaction time [27,28]. Several factors, both
substrate- and enzyme-related, are suggested to be
responsible for this slowdown ofthe reaction rate but,
so far, no mechanistic explanation ofthe slowdown
has been validated. The substrate characteristics
often implied in the slowdown ofthe 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 ofthe slowdown
[14,29,30].
One ofthe most controversial theories concerns the
influence ofcrystallinity and the change ofthe degree
of crystallinity during enzymatic hydrolysis. It is
accepted that the initial degree ofcrystallinityof cellu-
lose plays a major role as arate determinant in the
hydrolysis reaction. A completely amorphous sample is
hydrolyzed much faster than a partially crystalline
cellulose [14,31–33], which has led to the idea that
amorphous domains in a partially crystalline cellulose
sample are hydrolyzed first, leaving crystalline parts to
be hydrolyzed at the end, thus resulting in an increased
crystallinity index (CrI) and explaining the dramatic
drop in rate at higher degrees of conversion [34].
Studies to (dis)prove this phenomenon have differed
in the analytical methods employed (X-ray diffraction
versus solid state
13
C-NMR), the nature ofthe sub-
strate used (complex lignocellulosics versus pure cellu-
lose) and the source ofthe hydrolytic enzymes (mostly
from Trichoderma reesei and other fungal strains) [35].
Several reviews have stated that it is difficult to con-
clude that crystallinity is akey determinant ofthe rate
of enzymatichydrolysis [13,14,29]. Although a correla-
tion between crystallinity and enzymatic hydrolysis
rate has already been demonstrated, controversy
remains [29]. Usually, different types ofcellulose with
different degrees ofcrystallinity are employed in these
studies, such as cotton, cotton linter, Avicel, filter
paper or bacterial cellulose [15,17,36,37]. Their cellu-
lase-catalyzed degradation lead to hydrolysis rates that
were directly related to the CrI ofthecellulose 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. Therateofhydrolysis 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 ofcrystallinityofcellulose 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 thehydrolysisrate in
the case ofcellulose 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
the sample [52] and such treatment was shown to have
no impact on the reducing-end concentration of the
cellulose sample (i.e. its degree of polymerization)
[53,54]. However, the effect of various phosphoric acid
concentrations has only been investigated across a nar-
row range of acid concentrations or mainly at low con-
centrations [46,55–57]. Recently, Zhang et al. [52]
demonstrated that the concentration of phosphoric
acid used to generate swollen cellulose relates to the
rate ofenzymatichydrolysis by controlling the state of
cellulose solubilization. Hydrolysis rates were one
order of magnitude lower for microcrystalline cellulose
compared to amorphous cellulose. This reflects the
composition of highly crystalline and amorphous cellu-
lose at acid concentrations of 0% and above 81%,
respectively. The changes in hydrolysis rate, with varia-
tions in the degree ofcrystallinity as a result of treat-
ment with various phosphoric acid concentrated
solutions, are therefore of significant interest.
The present study aimed to determine the role of
crystallinity and adsorption in the susceptibility of
cellulose to enzymatic degradation. Both
13
C-NMR
solid-state spectroscopy and X-ray crystallography
were applied to investigate thecrystallinityof pure
cellulose (Avicel) at different degrees of conversion by
cellulases from T. reesei, the most commonly studied
cellulase-producing organism. Complementarily, we
generated cellulose (Avicel) with controlled degrees 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 theenzymatichydrolysis rates.
Results
Cellulase hydrolysisrate and cellulose
crystallinity
Various types of (ligno)cellulosic substrate are employed
in current enzymatichydrolysis 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 theenzymatic 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 crystallinityof 60% (measured via solid state
13
C-NMR).
Phosphoric acid pretreatment
First, to validate the efficiency ofthe 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-
cose, and the initial hydrolysis rates were calculated in
terms ofthe production of glucose after a 2 min reac-
tion time. As expected, the more concentrated the phos-
phoric acid solution, the higher the sugar production
(Fig. 1A), so that the pretreatment procedure was con-
sidered to be efficient. Samples treated with pure phos-
phoric acid solution (maximum 85%) resulted in
amorphous cellulose as demonstrated by X-ray diffrac-
tion analysis [58] (Fig. 2). Furthermore, a high amount
of glucose (4.75 gÆL
)1
Æmin
)1
) was produced from the
cellulose sample pretreated with the highest concentra-
tion of acid (85%), and all ofthe Avicel was converted
within 2.5 h compared to the 96 h that was necessary
for untreated Avicel (data not shown).
Phosphoric acid pretreatment has been used to create
cellulose samples of various surface areas and this
parameter was found to be related to theenzymatic rate
[51]. A recent study using phosphoric acid to increase
cellulose accessibility in lignocellulosics suggested the
presence ofa critical point in the phosphoric acid con-
centration below which enzymatichydrolysis was slow,
and above which cellulose was easily dissolved [59]. The
results obtained in the present study (Fig. 1) confirm
that there is a steep change in reactivity (i.e. glucose
A
B
C
Fig. 1. Effect of phosphoric acid concentration on: (A) initial rate of
Avicel enzymatichydrolysis (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.
One ofthe most commonly employed techniques is
X-ray diffraction where the peak height is used to
calculate the CrI [60] (Fig. 2). However, the major
drawback of this analytical method stems from the
formula itself (see Materials and methods) because it
implies that amorphous cellulose gives a main reflec-
tion at 2h =18°, which, upon our analysis, is defi-
nitely not the case for the Avicel used in the present
study (rather, it is shifted to higher angle, $19.5°).
Also, the absolute values thus obtained are extremely
high (> 90% for Avicel), which does not appear to
represent the structure of Avicel well, and deviates
substantially from the NMR analysis (60% for Avicel).
In addition, the literature contains a wide range of
reported values for Avicel using X-ray diffraction, in
the range 62–87.6% using the peak height method
[61–63], and from 39 to 75.3% using various other
methods [61,64,65]. It should be noted, however, that
different drying methods are often being employed,
which also may add to the reported variations in
absolute crystallinity values. Under our conditions, no
satisfactory resolution ofthe C4 carbon signals in
NMR analysis could be obtained below a certain
degree ofcrystallinity and within a reasonable acquisi-
tion time, so that X-ray diffraction was used as an
alternative to map the full crystallinity spectrum.
Given the drawbacks ofthe peak intensity method
[60,66,67], we have developed a new method to obtain
consistent CrI values using multivariate statistical
analysis applied to X-ray diffraction spectra [58].
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 thecellulose 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 ofcellulose 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
sample with 85% moisture content can theoretically
accommodate 49 water molecules per glucose unit,
whereas, at a 60% moisture content, this ratio is
reduced to 13 (based on the observation that 1 g of
Avicel yields 1.15 g of glucose at 100% conversion).
Cellulose enzymatic hydrolysis
There have been numerous, and sometimes controver-
sial, studies on the change ofcellulose crystallinity
I
am
20 30 40
I
002
Fig. 2. X-ray diffraction pattern of microcrystalline cellulose Avicel
(multiple peaks) and amorphous Avicel (single smooth peak) gener-
ated with 85% phosphoric acid (reflection around 20° is attributed
to amorphous parts and gives a CrI of 0% based on peak intensity
method) [60]. x-axis: Bragg angle (2h). I
002
represents the maximum
intensity at 2h = 22.5°, I
am
shows the minimum intensity at
2h =18° used to calculate crystallinity in the peak height method,
and the straight line represents the background (see Materials and
methods).
Cellulose crystallinity M. Hall et al.
1574 FEBS Journal 277 (2010) 1571–1582 ª 2010 The Authors Journal compilation ª 2010 FEBS
upon enzymatic hydrolysis. Both trends (i.e. increased
degree ofcrystallinity over conversion and no change
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 ofa clear
understanding ofthe mechanistic action of cellulase on
partially crystalline cellulose. Furthermore, in situ
measurements ofcellulose structure under reacting
conditions (i.e. in aqueous buffers) are difficult to
perform because all current methods require the prior
isolation ofcellulose 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 ofa mixture of cellulases, no change
in crystallinity was observed; however, variations in
relative peak intensity in X-ray diffraction patterns
showed that Cel7A attacked preferentially the (021)
plane ofthe crystal because the peak corresponding to
this face (centered around 21°) disappeared after 20%
conversion (Fig. 4). Overall, peak intensity ratios for
the other peaks were conserved [planes (101), (10
1),
(002) and (040) at 15, 16, 22.5 and 35°, respectively].
The same trend was observed with the commercial
cellulase mixture, implying no competition for this
plane from the other enzymes (endoglucanases, Cel6A
and b-glucosidase) or any dominant behavior from
Cel7A. The implications of this preferential attack
need to be investigated further because this may
provide options for engineering Cel7A and thus enable
overall faster hydrolysis.
Adsorption
Adsorption studies were conducted using cellulose
samples generated with various amounts of phosphoric
acid and thus displaying intermediate degrees of crys-
tallinity (Fig. 1B). Adsorption experiments were car-
ried out at 4 °C to prevent thehydrolysisof cellulose
and the resulting loss of adsorbent material that would
ultimately bias the results. Furthermore, the adsorp-
tion profile at 4 °C was found to be similar to that at
50 °C after 30 min [46]. The adsorption step has been
shown to be rapid, with half ofthe maximally
adsorbed enzyme being bound with 1–2 min and the
adsorption equilibrium being reached after 30 min [22].
Adsorption experiments were first performed using
the same degree of loading as employed during a com-
mon enzymatichydrolysis run (175 lgÆmg
)1
cellulose;
Figs 1–3). Surprisingly, a maximum value of adsorbed
enzyme concentration (150 lgÆmg
)1
cellulose) was
reached for thecellulose 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) ofthe crystal (centered around
21°) is visible only for untreated Avicel.
M. Hall et al. Cellulose crystallinity
FEBS Journal 277 (2010) 1571–1582 ª 2010 The Authors Journal compilation ª 2010 FEBS 1575
appeared to increase inversely and linearly with the
CrI at higher crystallinity values (i.e. > 45%). A con-
stant amount of adsorbed enzymes ($ 150 lgÆmg
)1
cellulose) led to faster hydrolysis at lower degrees of
crystallinity (i.e. < 45% CrI; Fig. 6B), whereas, at
crystallinity indices above 45%, the adsorption capac-
ity increased and was linearly proportional to the
initial rate.
At higher enzyme loading (seven-fold greater than
the original loading; i.e. 1230 lgÆmg
)1
cellulose), the
initial rates were found to be generally higher
(Fig. 6C, filled circles), confirming the findings
obtained in previous studies [22,25,42–44], although
this trend was especially true at lower degrees of crys-
tallinity. By contrast, untreated Avicel (CrI = 60%)
displayed similar rates at both enzyme concentrations,
and little difference in rate for the two enzyme con-
centrations was observed up to a CrI of 50%. Also
at high enzyme loading, the profile of adsorbed
enzyme versus the degree ofcrystallinity ⁄ initial rate
was similar to that at low enzyme loading, except
that constant adsorption was observed only for CrI
in the range 0–35%.
Discussion
Cellulase hydrolysisrate 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 ofcrystallinity (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 ofthe reaction with
cellulases). The results shown are the average of quadruplicates.
Cellulose crystallinity M. Hall et al.
1576 FEBS Journal 277 (2010) 1571–1582 ª 2010 The Authors Journal compilation ª 2010 FEBS
The latter is supported by the data obtained from
moisture content measurement (Fig. 1C). Most aque-
ous reagents can only penetrate the amorphous parts
of cellulose; therefore, these domains are also termed
the accessible regions of cellulose, and crystallinity and
accessibility are closely related [68]. It is likely that
crystallinity and accessibility are related; however,
moisture content (i.e. the capacity to retain water) by
itself is not directly related to enzyme accessibility
because water molecules are three orders of magnitude
smaller than cellulases [72]. A highly crystalline cellu-
lose sample has a tight structure with cellulose chains
closely bound to each other, leaving too little space for
enzymes to initiate thehydrolysis process anywhere
within thecellulose crystal.
Overall, thehydrolysisrate versus the phosphoric
acid concentration profile resembles a very steep and
sharp sigmoid curve (Fig. 1A), which led to an evalua-
tion ofthe concentration range corresponding to the
sigmoid region. In their review, Zhang et al. [35]
stressed that the CrI ofcellulose was not strongly asso-
ciated with hydrolysis rates. By contrast, the results
obtained in the present study show a very close and lin-
ear relationship between the CrI and initial hydrolysis
rate for samples of same origin obtained after pretreat-
ment with phosphoric acid (R
2
= 0.96; Fig. 5), demon-
strating that crystallinity is a good predictorof the
hydrolysis rate. More precisely, in a phosphoric acid
concentration range of 75–80%, thehydrolysis rate,
crystallinity and phosphoric acid concentration are
mutually dependent parameters resulting from the
structural changes that take place upon acid pretreat-
ment ofcellulose and are also linearly related. The
degree of phosphoric acid addition enables the tight
control ofthe overall structure ofcellulose in the Avicel
sample. This convenient method for reaching intermedi-
ate degrees ofcrystallinity 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 therateof 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 ofcrystallinity data is not trivial, looking at
initial hydrolysis rates may be an elegant alternative to
estimating the degree ofcrystallinityof pure cellulose.
No significant change was observed in the degree of
crystallinity during theenzymatichydrolysisof 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-
tal are disrupted, therefore leaving an overall
unchanged crystallinity but a structure that is reduced
in size.
Thus, the belief that mixed cellulose samples have
their amorphous components hydrolyzed first is not
consistent with the results obtained in the present
study. The change in crystallinity cannot account for
the sharp decrease in reaction rate observed, and thus
another explanation is required for the slowdown.
A number of studies reporting an increasing crystal-
linity along enzymatichydrolysis have attributed the
slowdown in therate to this crystallinity change
[25,33,34,39,71]. However, the changes reported were
often modest. Figure 3 shows that a 10% increase in
CrI at high CrI values leads to a 40% decrease in ini-
tial rate; therefore, it does not appear physically possi-
ble that a change in CrI by some percentage points
results in such dramatic drops in the rate. Constant
crystallinity and decreased rates indicate surface
changes on cellulose that start rapidly after the begin-
ning of hydrolysis. Factors other than crystallinity
impeding enzymatic action (both enzyme- and sub-
strate-related) require closer attention.
Adsorption
There are multiple substrate-related factors that can
influence the reaction rate in theenzymatic hydrolysis
of cellulose (see Introduction). From the results
obtained in the present study with respect to determin-
ing the role ofcrystallinity in enzymatic activity, it is
logical to ask whether crystallinity might not be mask-
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 ofcrystallinity (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 ofa 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 enzymatichydrolysis rates [72]
and its increase at lower degrees ofcrystallinity was
proposed as a reason for enhanced digestibility [59]. It
has also been suggested that rendering the substrate
more amorphous increases access to the reducing ends
of cellulose, thus enhancing reaction rates [53]. These
data support these hypotheses only partially and,
importantly, demonstrate that the effect of improved
access on thehydrolysisrate is limited to higher
degrees of crystallinity, whereas, at low degrees of
crystallinity, rate enhancement is strictly the result of a
dynamic cause that is independent ofthe adsorption
phase (favored enzymatic motion as result ofthe larger
free space available at lower degrees of crystallinity),
and is also directly related to the enzyme concentra-
tion. This can be related to recent work demonstrating
that the overcrowding of enzymes on the cellulose
surface lowers their activity [74]. Surface area may also
play a role in the various rate profiles observed. Some
studies have focused on the relationships between
surface area and crystallinity [75]; overall, a reduction
in crystallinity would relate to a higher surface area.
In the present study, this would easily explain the
higher adsorption capacity observed at lower degrees
of crystallinity but not why the adsorption reaches a
plateau (in an undersaturated regime) below a certain
CrI and the rates keep increasing. Also, the internal
surface of highly crystalline cellulose is poorly acces-
sible to enzymes, leading to such low adsorption, pos-
sibly in contrast to more amorphous samples. An
accessible surface area has been the subject of numer-
ous studies [40] but, in view ofthe results obtained in
the present study, this does not appear to be the only
critical parameter with respect to controlling hydro-
lysis rates.
Avicel hydrolysis rates were not significantly chan-
ged upon the addition ofa 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 ofcellulose 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 ofthe 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 ofthe hydrolysable fractions are covered. The
role of adsorption for a given cellulose sample appears
to be more important to theenzymaticrate at lower
degrees ofcrystallinity (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
reached ($ 600 lgÆmg
)1
cellulose), whereas the initial
rates were still increasing (Fig. 6). The breakpoint below
which crystallinity is the only determining factor for the
reaction rate is expected to decrease as enzyme loading
increases because it becomes comparatively harder to
attain the maximum adsorption capacity (saturation) at
low degrees ofcrystallinity (open cellulose structure) as
well as the maximum coverage of hydrolysable fractions
(investigations underway). Examining various enzyme
concentrations and hydrolysisrate ⁄ adsorption profiles
on substrates with different degrees ofcrystallinity may
thus provide an effective way of quantifying cellulose
hydrolyzability.
Finally, future trends for the application of cellu-
lases in biofuel technology should focus on efficient
ways of disrupting cellulosecrystallinity and thus
render the overall process economically more viable by
reducing the time required to reach full conversion.
Materials and methods
Materials
All chemicals and reagents were purchased from Sigma
(St Louis, MO, USA) unless otherwise stated. Avicel PH-101,
cellulases from T. reesei (159 FPUÆmL
)1
) and b-glucosidase
(from almonds, 5.2 UÆmg
)1
) were obtained from Sigma and
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 theenzymatic 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 hydrolysisof 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
)1
, 3.4 gÆL
)1
total protein) were added and the
mixture was stirred at 50 °C. At the desired time points,
samples were centrifuged, and glucose content in the super-
natant was measured via the dinitrosalicylic acid (DNS)
assay. For crystallinity measurements at various conversion
levels using CP ⁄ MAS
13
C- NMR [and the corresponding
Eqn (2)], reactions were run on a 15 mL scale (one reaction
tube per time point, ranging from 10 min to 92 h) and,
after centrifugation and washing with buffer and water,
recovered cellulose was either freeze-dried, oven-dried
(60 °C) or air-dried. When Cel7A was used as single cellu-
lase component, 92 lg of purified enzyme per mg of Avicel
were added to the reaction mixture.
Determination of glucose content
Glucose released from cellulose was measured using the
DNS assay, as described previously [28]. The calibration
curve was generated with pure glucose standards. DNS
assay was compared with HPLC analysis and found to
yield identical conversion results.
Determination ofthe degree ofcrystallinity of
cellulose
X-ray diffraction
X-ray diffraction patterns ofcellulose samples obtained
after freeze-drying were recorded with an X’Pert PRO
X-ray diffractometer (PANanalytical BV, Almelo, the
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 ofthe 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 thecrystallinity of
untreated Avicel.
Solid state
13
C-NMR
The solid-state CP ⁄ MAS
13
C-NMR experiments were per-
formed on a Bruker Avance ⁄ DSX-400 spectrometer
(Bruker Instuments, Inc., Bellerica, MA, USA) operating at
frequencies of 100.55 MHz for
13
C. All the experiments
were carried out at ambient temperature using a Bruker
4-mm MAS probe. The samples ($35% moisture content)
were packed in 4 mm zirconium dioxide rotors and spun at
10 kHz. Acquisition was carried out with a CP pulse
sequence using a 5 ls pulse and a 2.0 ms contact pulse over
4 h. CrI was calculated according to standard methods [28]:
CrI ¼ A
86À92 p:p:m:
=ðA
79À86 p:p:m:
þ A
86À92 p:p:m:
ÞÂ100 ð2Þ
where A
86–92 p.p.m.
and A
79–86 p.p.m.
are the areas ofthe crys-
talline and amorphous C4 carbon signal of cellulose,
respectively.
Oven-drying (60 °C) showed no impact on the crystallin-
ity of untreated Avicel.
Multivariate statistical analysis of X-ray data
The CrI ofcellulose 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 ofthe 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.
^
f
j
,the least square estimate of f
j
, was used to estimate the
crystallinity by multiplying the contribution of Avicel
ð1 À
^
f
j
Þ by its crystallinity (calculated by CP ⁄ MAS
13
C-
NMR as 60%):
CrI
j
¼ð1 À f
j
ÞÂCrI
c
ð4Þ
where CrI
j
is thecrystallinity (in percentage) ofthe j
th
sample of Avicel AND CrI
c
is thecrystallinityof Avicel
(calculated by CP ⁄ MAS
13
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
.H
2
O 1.5 mgÆL
)1
, FeSO
4
.7H
2
O
5mgÆL
)1
, COCl
2
2mgÆL
)1
, ZnSO
4
1.5 mgÆL
)1
) supple-
mented with glucose (2%). After 3 days at 28 °C and
150 r.p.m., the fungus was grown on lactose (2%) in mini-
M. Hall et al. Cellulose crystallinity
FEBS Journal 277 (2010) 1571–1582 ª 2010 The Authors Journal compilation ª 2010 FEBS 1579
mal medium for up to 12 days at 28 °C and 150 r.p.m.
After filtration over glass-microfiber filter (1.6 lmGF⁄ A;
Whatman, Maidstone, UK), the filtrate was diafiltered by
repeated concentration and dilution with sodium acetate
buffer (50 mm, pH 5.5) using a polyethersulfone membrane
(molecular weight cut-off of 10 kDa). The concentrate was
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 thecrystallinity measure-
ments.
References
1 Himmel ME, Ding SY, Johnson DK, Adney WS,
Nimlos MR, Brady JW & Foust TD (2007) Biomass
recalcitrance: engineering plants and enzymes for
biofuels production. Science 315, 804–807.
2 Himmel ME, Ruth MF & Wyman CE (1999) Cellulase
for commodity products from cellulosic biomass. Curr
Opin Biotechnol 10, 358–364.
3 Wyman CE (2007) What is (and is not) vital to advanc-
ing cellulosic ethanol. Trends Biotechnol 25, 153–157.
4 Lynd LR, Laser MS, Brandsby D, Dale BE, Davison
B, Hamilton R, Himmel M, Keller M, McMillan JD,
Sheehan J et al. (2008) How biotech can transform
biofuels. Nat Biotechnol 26, 169–172.
5 Divne C, Sta
˚
hlberg J, Reinikainen T, Ruohonen L,
Pettersson G, Knowles JKC, Teeri TT & Jones TA
(1994) The 3-dimensional crystal-structure of the
catalytic core of cellobiohydrolase-i from Trichoderma
reesei. Science 265, 524–528.
6 Divne C, Sta
˚
hlberg J, Teeri TT & Jones TA (1998)
High-resolution crystal structures reveal how a cellulose
chain is bound in the 50 angstrom long tunnel of cello-
biohydrolase I from Trichoderma reesei. J Mol Biol 275,
309–325.
7 Teeri TT (1997) Crystalline cellulose degradation: new
insight into the function of cellobiohydrolases. Trends
in Biotechnology 15, 160–167.
8 Kipper K, Va
¨
ljama
¨
e P & Johansson G (2005) Processive
action of cellobiohydrolase Cel7A from Trichoderma
reesei is revealed as ‘burst’ kinetics on fluorescent poly-
meric model substrates. Biochem J 385, 527–535.
9 Li YC, Irwin DC & Wilson DB (2007) Processivity,
substrate binding, and mechanism ofcellulose hydroly-
sis by Thermobifida fusca Ce19A. Appl Environ
Microbiol 73, 3165–3172.
10 Davies G & Henrissat B (1995) Structures and mecha-
nisms of glycosyl hydrolases. Structure 3 , 853–859.
11 Henrissat B (1994) Cellulases and their interaction with
cellulose. Cellulose 1, 169–196.
12 Knowles J, Lehtovaara P & Teeri T (1987) Cellulase
families and their genes. Trends in Biotechnology 5,
255–261.
13 Lynd LR, Weimer PJ, van Zyl WH & Pretorius IS
(2002) Microbial cellulose utilization: fundamentals and
biotechnology. Microbiol Mol Biol Rev 66 , 506–577.
14 Zhang YHP & Lynd LR (2004) Toward an aggregated
understanding ofenzymatichydrolysisof cellulose:
noncomplexed cellulase systems. Biotechnol Bioeng 88,
797–824.
15 Va
¨
ljama
¨
e P, Sild V, Nutt A, Pettersson G & Johansson
G (1999) Acid hydrolosis of bacterial cellulose reveals
different modes of synergistic action between cellobio-
hydrolase I and endoglucanase I. Eur J Biochem 266,
327–334.
16 Medve J, Karlsson J, Lee D & Tjerneld F (1998)
Hydrolysis of microcrystalline cellulose by cellobiohy-
drolase I and endoglucanase II from Trichoderma reesei:
adsorption, sugar production pattern, and synergism of
the enzymes. Biotechnol Bioeng 59, 621–634.
17 Hoshino E, Shiroishi M, Amano Y, Nomura M &
Kanda T (1997) Synergistic actions of exo-type cellulas-
es in thehydrolysisofcellulose with different crystallini-
ties. J Ferment Bioeng 84, 300–306.
18 Nidetzky B, Hayn M, Macarron R & Steiner W (1993)
Synergism of Trichoderma reesei cellulases while degrad-
ing different celluloses. Biotechnol Lett 15, 71–76.
19 Henrissat B, Driguez H, Viet C & Schulein M (1985)
Synergism of cellulases from Trichoderma reesei in the
degradation of cellulose. Biotechnology 3, 722–726.
20 Bansal P, Hall M, Realff MJ, Lee JH & Bommarius AS
(2009) Modeling cellulase kinetics on lignocellulosic
substrates. Biotechnol Adv 27, 833–848.
21 Sta
˚
hlberg J, Johansson G & Pettersson G (1991) A new
model for enzymatic-hydrolysis ofcellulose based on
the 2-domain structure of cellobiohydrolase-I. Biotech-
nology 9, 286–290.
Cellulose crystallinity M. Hall et al.
1580 FEBS Journal 277 (2010) 1571–1582 ª 2010 The Authors Journal compilation ª 2010 FEBS
[...]... confined) enzymatic catalysis: contributions from the fractal and jamming (overcrowding) effects Appl Catal A- Gen 317, 7 0–8 1 31 Fan LT, Lee YH & Beardmore DH (1980) Mechanism ofthe enzymatic- hydrolysisofcellulose– effects of major structural features ofcellulose on enzymatichydrolysis Biotechnol Bioeng 22, 17 7–1 99 32 Fan LT, Lee YH & Beardmore DR (1981) The influence of major structural features of cellulose. .. concentration on therateofthehydrolysisofcellulose Biotechnol Bioeng 33, 122 1–1 234 43 Lee YH & Fan LT (1982) Kinetic-studies of enzymatichydrolysis of insoluble cellulose– analysis ofthe initial rates Biotechnol Bioeng 24, 238 3–2 406 44 Ryu DDY & Lee SB (1986) Enzymatic- hydrolysisofcellulose– determination of kinetic-parameters Chem Eng Commun 45, 11 9–1 34 45 Klyosov AA, Mitkevich OV & Sinitsyn AP... kinetics ofhydrolysis by cellobiohydrolases I and II is consistent with acellulose surfaceerosion model Eur J Biochem 253, 46 9–4 75 Igarashi K, Wada M, Hori R & Samejima M (2006) Surface density of cellobiohydrolase on crystalline celluloses –a critical parameter to evaluate enzymatic kinetics at a solid-liquid interface FEBS Journal 273, 286 9–2 878 Walker LP & Wilson DB (1991) Enzymatic- hydrolysisof cellulose. .. Szijarto N, Siika-aho M, Tenkanen M, Alapuranen M, Vehmaanpera J, Reczeya K & Viikari L (2008) Hydrolysisof amorphous and crystalline cellulose by heterologously produced cellulases of Melanocarpus albomyces J Biotechnol 136, 14 0–1 47 51 Stone JE, Scallan AM, Donefer E & Ahlgren E (1969) Digestibility as a simple function ofa molecule of similar size to a cellulase enzyme Adv Chem Ser 95, 21 9–2 41... lignocellulose fractionation technology and enzymaticcellulosehydrolysis J Agric Food Chem 56, 788 5–7 890 Segal L, Creely JJ, Martin AE & Conrad CM (1959) An empirical method for estimating the degree ofcrystallinityof native cellulose using the x-ray diffractometer Text Res J 29, 78 6–7 94 Thygesen A, Oddershede J, Lilholt H, Thomsen AB & Stahl K (2005) On the determination ofcrystallinity and cellulose. .. (1986) Role ofthe activity and adsorption of cellulases in the efficiency ofthe enzymatic- hydrolysisof amorphous and crystalline cellulose Biochemistry 25, 54 0–5 42 46 Lee SB, Shin HS, Ryu DDY & Mandels M (1982) Adsorption of cellulase on cellulose– effect of physicochemical properties ofcellulose on adsorption and rateofhydrolysis Biotechnol Bioeng 24, 213 7–2 153 47 Schulein M (1997) Enzymatic properties... Vertova A (1999) Microcrys- 1582 65 66 67 68 69 70 71 72 73 74 75 talline cellulose powders: structure, surface features and water sorption capability Cellulose 6, 5 7–6 9 Soltys J, Lisowski Z & Knapczyk J (1984) X-Ray diffraction study ofthecrystallinity index and the structure ofthe microcrystalline cellulose Acta Pharm Technologica 30, 17 4–1 80 Park S, Johnson DK, Ishizawa CI, Parilla PA & Davis... on crystallinity and average chain length for bacterial and microcrystalline celluloses Cellulose 14, 28 3– 293 Hong J, Ye XH & Zhang YHP (2007) Quantitative determination ofcellulose accessibility to cellulase based on adsorption ofa nonhydrolytic fusion protein containing CBM and GFP with its applications Langmuir 23, 1253 5–1 2540 Valjamae P, Sild V, Pettersson G & Johansson G ¨ ¨ (1998) The initial... screening and selection strategies Biotechnol Adv 24, 45 2–4 81 36 McLean BW, Boraston AB, Brouwer D, Sanaie N, Fyfe CA, Warren RAJ, Kilburn DG & Haynes CA (2002) Carbohydrate-binding modules recognize fine substructures ofcellulose J Biol Chem 277, 5024 5–5 0254 Cellulosecrystallinity 37 Puri VP (1984) Effect ofcrystallinity and degree of polymerization ofcellulose on enzymatic saccharification Biotechnol... Hatakeya T & Nakano J (1974) Dsc study on recrystallization of amorphous cellulose with water J Appl Polym Sci 18, 306 9–3 076 Ramos LP, Zandona A, Deschamps FC & Saddler JN (1999) The effect of Trichoderma cellulases on the fine structure ofa bleached softwood kraft pulp Enzyme Microb Technol 24, 37 1–3 80 Chen Y, Stipanovic AJ, Winter WT, Wilson DB & Kim YJ (2007) Effect of digestion by pure cellulases . Cellulose crystallinity – a key predictor of the enzymatic
hydrolysis rate
Me
´
lanie Hall, Prabuddha Bansal, Jay H. Lee, Matthew J. Realff and Andreas. Avicel.
Multivariate statistical analysis of X-ray data
The CrI of cellulose samples was also calculated by quanti-
fying the contribution of amorphous cellulose (PASC)