cellulose crystallinity – a key predictor of the enzymatic hydrolysis rate

Cellulose crystallinity – a key predictor of the enzymatichydrolysis rateMelanie 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

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: [email protected]

(Received 13 December 2009, revised 16

January 2010, accepted 18 January 2010)

doi:10.1111/j.1742-4658.2010.07585.x

The enzymatic hydrolysis of cellulose encounters various limitations that

are both substrate- and enzyme-related. Although the crystallinity of pure

cellulosic Avicel plays a major role in determining the rate of hydrolysis 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 of crystallinity with Avicel was therefore

developed and the initial rate of the cellulase-catalyzed hydrolysis of cellu-

lose was demonstrated to be linearly proportional to the crystallinity index

of Avicel. Despite correlation with the adsorption capacity of cellulases

onto cellulose, at a given enzyme loading, the initial enzymatic rate contin-

ued to increase with a decreasing crystallinity index, even though the

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].

One of the most controversial theories concerns the

influence of crystallinity and the change of the degree

of crystallinity during enzymatic hydrolysis. It is

accepted that the initial degree of crystallinity of cellu-

lose plays a major role as a rate 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 13C-NMR), the nature of the sub-

strate used (complex lignocellulosics versus pure cellu-

lose) and the source of the 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 a key determinant of the rate

of enzymatic hydrolysis [13,14,29]. Although a correla-

tion between crystallinity and enzymatic hydrolysis

rate has already been demonstrated, controversy

remains [29]. Usually, different types of cellulose with

different degrees of crystallinity 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 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

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 of enzymatic hydrolysis 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 of crystallinity 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 13C-NMR

solid-state spectroscopy and X-ray crystallography

were applied to investigate the crystallinity of 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 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 state13C-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-

cose, and the initial hydrolysis rates were calculated in

terms of the 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)1Æmin)1) was produced from the

cellulose sample pretreated with the highest concentra-

tion of acid (85%), and all of the 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 the enzymatic rate

[51]. A recent study using phosphoric acid to increase

cellulose accessibility in lignocellulosics suggested the

presence of a critical point in the phosphoric acid con-

centration below which enzymatic hydrolysis 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 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


production) from 1 to 4.75 gÆL)1Æmin)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)1Æmin)1 glucose at 0% phosphoric acid).

There are several ways to measure cellulose CrI.

One of the 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 of the C4 carbon signals in

NMR analysis could be obtained below a certain

degree of crystallinity 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 of the 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 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

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 of cellulose crystallinity

Iam

20 30 40

I002

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). I002 represents the maximum

intensity at 2h = 22.5�, Iam 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).



upon enzymatic hydrolysis. Both trends (i.e. increased

degree of crystallinity 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 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-glucosidaseto 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) 13C-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

showed that Cel7A attacked preferentially the (021)

plane of the 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 the hydrolysis of 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 of the 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 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 13C-NMR [reactions were run at 50 �C in sodium acetate

buffer (50 mM, pH 5) at 20 gÆL)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

21�) is visible only for untreated Avicel.



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 of crystallinity ⁄ 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 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

cellulases). The results shown are the average of quadruplicates.



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 the hydrolysis process anywhere

within the cellulose crystal.

Overall, the hydrolysis rate versus the phosphoric

acid concentration profile resembles a very steep and

sharp sigmoid curve (Fig. 1A), which led to an evalua-

tion of the concentration range corresponding to the

sigmoid region. In their review, Zhang et al. [35]

stressed that the CrI of cellulose 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 (R2 = 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-

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 enzymatic hydrolysis have attributed the

slowdown in the rate 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 the enzymatic hydrolysis

of cellulose (see Introduction). From the results

obtained in the present study with respect to determin-

ing the role of crystallinity 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 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



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

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 the hydrolysis rate 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 of the adsorption

phase (favored enzymatic motion as result of the 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 of the 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 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

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 of crystallinity (open cellulose structure) as

well as the maximum coverage of hydrolysable fractions

(investigations underway). Examining various enzyme

concentrations and hydrolysis rate ⁄ adsorption profiles

on substrates with different degrees of crystallinity 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 cellulose crystallinity 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



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)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 13C- 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 of the degree of crystallinity of

cellulose

X-ray diffraction

X-ray diffraction patterns of cellulose 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, usingCu ⁄Ka1 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 wascalculated using the peak intensity method [60]:

CrI ¼ ðI002 � IamÞ=I002 � 100 ð1Þ

where I002 is the intensity of the peak at 2h = 22.5� and

Iam 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 13C-NMR

The solid-state CP ⁄MAS 13C-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 13C. 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 A86�92 p:p:m:=ðA79�86 p:p:m: þ A86�92 p:p:m:Þ � 100 ð2Þ

where A86–92 p.p.m. and A79–86 p.p.m. are the areas of the 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 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]:

Ijð2hÞ ¼ fjIpð2hÞ þ ð1� fjÞIcð2hÞ þ e ð3Þ

where Ij (2h) is the intensity of the j th sample at diffraction

angle 2h, Ip (2h) is the intensity of PASC at diffraction

angle 2h, IC (2h) is the intensity of untreated Avicel at dif-

fraction angle 2h, fj is the contribution of PASC to the

spectrum and e is the random error.

f j,the least square estimate of fj, was used to estimate the

crystallinity by multiplying the contribution of Avicel

ð1� fjÞ by its crystallinity (calculated by CP ⁄MAS 13C-

NMR as 60%):

CrIj ¼ ð1� fjÞ � CrIc ð4Þ

where CrIj is the crystallinity (in percentage) of the j th

sample of Avicel AND CrIc is the crystallinity of Avicel

(calculated by CP ⁄MAS 13C-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:

(NH4)2SO4 5 gÆL)1, CaCl2 0.6 gÆL)1, MgSO4 0.6 gÆL)1,

KH2PO4 15 gÆL)1, MnSO4.H2O 1.5 mgÆL)1, FeSO4.7H2O

5 mgÆL)1, COCl2 2 mgÆL)1, ZnSO4 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-



mal medium for up to 12 days at 28 �C and 150 r.p.m.

After filtration over glass-microfiber filter (1.6 lm GF ⁄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 the crystallinity measure-

ments.

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cellulose crystallinity – a key predictor of the enzymatic hydrolysis rate

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