Industrial Crops and Products 79 (2016) 104109

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Industrial Crops and Products

journal homepage: www.elsevier.com/locate/indcrop

Impact of delignication on the morphology and the reactivity of

steam exploded wheat straw
Mat Huron a , Damien Hudebine a, , Nicolas Lopes Ferreira b , Dominique Lachenal c
a
b
c

IFP Energies nouvelles, Rond-point de lchangeur de Solaize BP 3, 69360 Solaize, France

IFP Energies nouvelles, 1 & 4, avenue de Bois-Prau, 92852 Rueil-Malmaison Cedex, France
Grenoble INP Pagora, 461 rue de la Papeterie BP65, 38402 Saint Martin dHres Cedex, France

a r t i c l e

i n f o

Article history:
Received 3 July 2015
Accepted 19 October 2015
Keywords:
Bioethanol
Biofuel
Enzymatic hydrolysis
Lignin
Wheat straw

a b s t r a c t
The purpose of this article was to better understand the role of lignin in the recalcitrance of lignocellulosic
biomass during enzymatic hydrolysis. Steam exploded wheat straw was partially delignied with sodium
chlorite to six different grades of delignication. Delignication did not have a signicant impact on the
enzymatic hydrolysis of the studied wheat straw in the experimental conditions tested. Inhibitive impact
of lignin in terms of non-productive adsorption was then explored using soda lignin from wheat straw and
kraft lignin from softwood. The addition of both lignins had a strong negative inuence on the hydrolysis
of highly crystalline cellulose (Avicel), whereas it impacted only slightly the hydrolysis of delignied
wheat straw. These results are probably linked to the greater accessibility and surface area of steam
exploded wheat straw cellulose, which are much higher than those of the crystalline cellulose Avicel.
2015 Elsevier B.V. All rights reserved.

1. Introduction
Fuels produced from lignocellulosic biomass have a high potential to partially replace fossil fuels, thus contributing to the decrease
of greenhouse gases emissions and to the diversication of energy
sources. During the last decades, many studies have been done in
order to develop cost-effective methods to produce biofuels (Sun
and Cheng, 2002; Zhang and Lynd, 2004; Van Dyk and Pletschke,
2012). In the case of second generation bioethanol, the cellulose
contained in the lignocellulosic substrate is converted into glucose by the combined actions of different specialized enzymes
(endoglucanases, cellobiohydrolases, -glucosidases, etc.). The glucose is mainly fermented into ethanol thanks to yeasts in the
same time (SSFSimultaneous Saccharication and Fermentation)
or during a following independent step (SHFSeparate Hydrolysis
and Fermentation). After distillation and rectication, the ethanol
is directly incorporated into gasoline or used as reactive to produce
some petrochemicals (Ethylene, Ethyl Tert-Butyl Ether, etc.).
The enzymatic hydrolysis is one of the limiting steps of the process, due to the cost and complexity of the enzymatic cocktail and
to the inherent recalcitrance of the lignocellulosic biomass. This
recalcitrance can be explained by various characteristics of the sub-

Corresponding author.
E-mail addresses: damien.hudebine@ifpen.fr, hassanpharmacy@yahoo.com
(D. Hudebine).
http://dx.doi.org/10.1016/j.indcrop.2015.10.040
0926-6690/ 2015 Elsevier B.V. All rights reserved.

strate such as its lignin content. The presence of lignin is indeed

known to hinder the action of enzymes by decreasing accessibility to cellulose. Furthermore, some enzymes can adsorb on the
hydrophobic surface area of lignin and do not participate to the
overall hydrolysis reaction of the cellulose (non-productive adsorption). Lignin is also suspected of decreasing the thermal stability
of proteins and preventing bers swelling (Mooney et al., 1998;
Borjesson et al., 2007). In order to shed light on the inhibitive role
of lignin on enzymatic hydrolysis, many teams studied some mixture of cellulose (mainly Avicel) and isolated wood lignins (Berlin
et al., 2006; Nakagame et al., 2010; Rahikainen et al., 2011; Kim,
2012). Some lignins had a strong impact on the hydrolysis of cellulose, but some others did not hinder notably the conversion. For
instance, Nakagame et al. (2010) showed that cellulolytic enzymatic lignin from steam exploded corn stover did not affect the
hydrolysis of Avicel, whereas cellulolytic enzymatic lignin from
steam exploded poplar and from organosolv loblolly pine decreased
the hydrolysis yield by respectively 11% and 23% (mixtures containing 20 g L1 of Avicel and 4 g L1 of lignin hydrolyzed at 50 C and pH
4.8. Enzymes loading: 5 FPU and 10CBU per gram of cellulose). The
impact of lignin depends not only on the quantity of lignin, but also
on its structure and its composition. However, the characteristics of
pure lignin are quite different from those of the original lignin contained in the substrate, as they are modied during the extraction
step. Although this kind of study is very useful to study the impact
of different lignins in terms of non-productive adsorption, it can-

M. Huron et al. / Industrial Crops and Products 79 (2016) 104109

not be easily extrapolated to real lignocellulosic substrates. Several

authors thus compared the reactivity of delignied substrates in
order to see if the elimination of lignin enhances the hydrolysis
rate (Varnai et al., 2010, 2011; Agarwal et al., 2013; Ju et al., 2013).
The delignication method has to be relatively soft in order not to
attack the cellulose, and the chlorite method is often chosen. The
results showed that delignication had a great positive impact on
the reactivity of most of the woody substrates. For instance, Varnai
et al. (2010) observed that removal of lignin by chlorite method
doubled the hydrolysis degree of the steam exploded spruce. On
the contrary, delignication affected only slightly the hydrolysis of
already very reactive substrates such as steam-exploded Douglas
r wood chips or green liquor pretreated hardwood (Esteghlalian
et al., 2002; Yu et al., 2011).
These studies allow to better understand the role of lignin in
the recalcitrance of lignocellulose. However, to our knowledge, this
kind of research has never been carried on wheat straw, which is
yet a substrate largely studied for the production of 2nd generation
bioethanol in Europe. This article focuses on the impact of lignin
on enzymatic hydrolysis of steam exploded wheat straw. In the
rst place, the pretreated wheat straw was delignied by chlorite
method to different grades. The composition and the morphology
of the obtained substrates were characterized in order to evaluate how the delignication impacted them. Then the reactivity of
the substrates was determined by hydrolyzing the steam exploded
delignied wheat straws at 48 C. The comparison of the results
gave information on the global impact of lignin on the hydrolysis of wheat straw. A second kind of experiments consisted in
studying mixtures of isolated lignin and delignied wheat straw,
in the purpose of focusing on non-productive binding of cellulases
on the lignin surface area. Two different lignins, extracted respectively from wheat straw and softwood, were tested. The results
were compared with mixtures of lignin and Avicel PH101.

2. Materials and methods

105

2.2. Chlorite delignication

The delignication of pretreated wheat straw was done following the chlorite protocol rstly described by Wise et al. (1946) and
Timell (1961), and adapted by Ahlgren and Goring (1971). The substrate was rst washed and neutralized at pH 5. The reactive charge
was composed of 0.3 g of sodium chlorite and 0.1 mL of acetic acid
per gram of dry biomass (wheat straw), and the initial Liquid to
Biomass mass ratio was 15. The reactions temperature was set to
70 C. A fresh charge of reactants (sodium chlorite and acetic acid)
was added at hourly intervals without withdrawal of any liquor. At
the end of the reaction, the solid was washed in 5 L of pure water
and recovered by vacuum ltration through glass crucibles (porosity 1). Several grades of delignied substrates were produced by
increasing the reaction time from 0 to 7 h.
2.3. Compositional analysis
In order to measure the quantity of cellulose, hemicellulose and
lignin in the wheat straw (delignied or not), acid hydrolysis were
performed following the procedure developed by NREL (Sluiter
et al., 2011). The various lignocellulosic substrates used in this
study were previously lyophilized with a freeze dryer Alpha 12
and grinded (<1 mm). After the acid hydrolysis, the sugars produced
were analyzed using a High Performance Liquid Chromatography
(ICS3000-Dionex). The cellulose content was calculated from the
quantity of glucose released, and the xylan content from the quantity of xylose. The amount of other sugars was negligible. The
remaining solid after acid hydrolysis was dried in an oven at 45 C
until it reached a constant weight, which corresponded to the summation of insoluble lignin and ashes. Ashes content was determined
by calcination of the substrate in a Thermo Scientic mufe following the NREL procedure (Sluiter et al., 2008). Percentages of
cellulose, xylan, lignin and ashes were calculated on a dry weight
basis.

2.1. Materials

2.4. Morphological analysis

Microcrystalline cellulose (Avicel PH101) was purchased from

SigmaAldrich (Lyon, France). All buffer components and salts
used were reagent grade and purchased from SigmaAldrich and
GE Healthcare (Saclay, France). Commercial wheat straw lignin
(WS Lignin), extracted by alkaline treatment, was purchased from
Green Value SA (Protobind 1000). This lignin was deeply characterized by Joffres et al. (2014).
Softwood kraft lignin (SO Lignin) was provided by MeadWestvaco (MWV, Richmond, United States), and a complete
characterization can be found in the work of Duval et al. (2013).
The study was performed on steam exploded Expert wheat straw
which was grown on chalky soil in Lavannes, France and harvested
in July 2011. It was supplied by Procthol 2G (France) and washed
and neutralized with KOH until pH 5.
The enzymatic cocktail called K619 was obtained from a crude
enzyme preparation using the hyper cellulolytic mutant strain from
Trichoderma reesei CL847. The production previously described by
Herpoel-Gimbert et al. (2008) was performed in fermentors using
two steps: (i) growth on lactose, (ii) fed-batch with a mixture containing several inducers including lactose as carbon source. Then,
the secreted enzymes were recovered after separation from the
mycelia by centrifugation. The protein concentration of the nal
cocktail was estimated by the Lowry method to be 53 g L1 (lter
paper activity: 360 FPU per gram of proteins). This cocktail was
supplemented with a commercial -glucosidase cocktail (SP188
(64 g L1 )) secreted by Aspergillus niger and provided by Novozymes
(Novo Nodisk A/S, Bagsvaerd, Denmark).

Morphology of the substrates was observed with a scanning

electron microscope (Supra 40, Zeiss, acceleration voltage of 1 kV).
All samples were sputter-coated with 5 nm platinumpalladium
before analysis.
Crystalline structure of the substrates was analyzed by a XPert
Pro PANalytical X-ray diffractometer with a copper anode. The samples were laid between two Kapton lms. The angular range varied
between 5 and 70 2 with an analysis step of 0.05 and a count
rate per step of 50 s. The crystallinity index (CrI) was calculated
according to the method of Segal et al. (1959).
The surface area of the substrates was determined with the
BrunauerEmmettTeller (BET) method which is based on nitrogen
adsorption at different pressures. This analysis was carried out with
a Micromeritics ASAP 2420 system. Prior to this specic analysis,
the lyophilized samples were dried again at 40 C during 6 h.
2.5. Enzymatic hydrolysis
Reactivity tests were performed in 100 mL Schott Duran laboratory glass bottles at pH 4.8 in a heating body (48 C) using
a magnetic agitation. The total mass of the solution was 70 g.
The buffer used was citrate buffer at 50 mmol L1 . The substrate
content was 10 g L1 and the K619 enzyme concentration was
40 mg L1 for wheat straw (delignied or not) and 100 mg L1 for
Avicel. The added volume of N188 was half of the volume of K619.
Chloramphenicol was added (50 mg L1 ) to prevent any bacterial
contamination.

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M. Huron et al. / Industrial Crops and Products 79 (2016) 104109

Table 1
Chemical composition of partially delignied steam exploded wheat straws.
Pretreated wheat straw

0h

1h

1 h30

2 h30

4h

7h

57.2
0.6

62.8
0.6

70.8
0.4

72.0
0.9

84.3
1.1

84.1
0.2

85.3
0.4

4.0
0.0

3.6
0.2

4.1
0.0

4.3
0.0

4.2
0.2

5.3
0.0

Dry residue mass fraction (%)

Lignin
Ashes
Standard deviation

36.2
34.3
1.9
0.5

33.5
32.3
1.2
0.1

23.1
Nda
Nda
0.2

22.2
21.2
1.0
0.2

10.9
8.6
2.3
0.5

9.6
6.5
3.1
0.3

7.5
4.4
3.1
0.3

Total (%)
Standard deviation

97.3
0.2

99.8
1.0

97.7
0.6

98.2
1.1

99.4
0.6

97.8
0.3

98.1
0.7

Cellulose mass fraction (%)

Standard deviation
Xylan mass fraction (%)
Standard deviation

Nd: not determined.

Assays were performed in duplicate for a total period of 72 h and

samples of 1 mL were taken periodically for analysis. The samples
were heated at 90 C for 10 min to stop the reaction, before being
centrifuged at 3600g during 20 min. The supernatant was recovered to determine the concentration of glucose and calculate the
hydrolysis conversion of the cellulose.
Glucose was measured using YSI Model 2700 SELECT. Measurements were done in duplicate. When the concentration of
glucose was too high (>4.5 g L1 ), samples were diluted in pure
water. At the end of the enzymatic reaction, a complementary
measure of glucose, cellobiose and xylose was done using a
High Performance Liquid Chromatography (ICS3000-Dionex). The
cellulose-to-glucose conversion yield is dened as the glucose
amount in the liquid phase product divided by the cellulose content
(as glucose equivalent) in the substrate.

1
dV/dD (L.g-1)

3.8
0.0

0.8

0.6
0.4
0.2
0
0

25
50
75
100
Mean Diameter (nm)

125

Fig. 1. Pore size distribution in the delignied wheat straws from BET measurements: WS PTT (), WS 1 h30 (), WS 4 h (

).

3. Results and discussion

3.1. Delignication of steam exploded wheat straw
In order to study the global impact of lignin on the enzymatic
hydrolysis, steam exploded wheat straw was delignied following
the chlorite protocol. Six different reaction times, ranging from 0
to 7 h, were used to obtained different delignication grades. The
composition of each substrate (WS 0 h to WS 7 h) was measured by
acid hydrolysis. The results were compared with those of original
steam exploded wheat straw (WS PTT). Glucose and xylose content was measured by HPLC. The dry residue, composed of lignin
and ashes, is the remaining solid at the end of the acid hydrolysis.
Results are shown in Table 1.
For each substrate, the total mass balance reached almost 100%.
That indicated that the measures of glucose, xylose and dry residue
were representative of the overall composition of the substrate.
The cellulose mass fraction of steam exploded wheat straw was
57%, and the lignin mass fraction 34%. The xylan content was low,
as hemicelluloses were mainly eliminated during the pretreatment
step. The composition of WS PTT and WS 0 h was the same, which
showed that the composition of the substrate was not modied
by the chlorite method when the reaction step was skipped. Then,
due to the delignication, the delignied substrates contained more
cellulose and xylan. Finally, three main delignication grades were
obtained corresponding to approximately the following mass fractions:
- 33% lignin initially (Ref. WS PTT)
- 22% lignin at 1 h and 1 h30 (Ref. WS 1 h30)
- 6% lignin after 2 h30 (Ref. WS 4 h)
Even after 7 h of delignication, the substrate still contained
some traces of lignin, which may be non-extractable because

embedded in the cellulose matrix. As the cellulose is likely to

become altered by long delignication times (Ishizawa et al., 2009),
and since no compositional differences were observed between the
three last substrates, WS 4 h was considered in this article as the
most delignied one. It was further used as a representation of
wheat straw pure cellulose.
The substrate reactivity is known to be strongly dependent on
its morphological characteristics such as crystallinity, polymerization degree and surface area (Zhang and Lynd, 2004). Consequently
the evolution of these properties during the delignication step is
an important parameter to follow. Measures of crystallinity, surface area, density and SEM photos were performed for the three
main delignication grades i.e., WS PTT, WS 1 h30 and WS 4 h. The
analysis results are shown in Table 2.
Crystallinity increased slightly during chlorite delignication.
However, XRD method is strongly inuenced by the biomass composition. It measures indeed the relative amount of crystalline
cellulose in the total solid, including lignin. As a consequence, this
result is probably primarily due to elimination of amorphous materials, and it is uncertain whether the cellulose structure has really
been altered by delignication (Kim et al., 2003; Yu et al., 2011).
The surface area of steam exploded wheat straw dropped at
the beginning of the delignication. It was almost divided by two
during the rst hour and a half and decreased at a much slower
rate during the following hours. However, these results have to
be used with caution since the analyses were performed on dry
substrates. As the cellulosic bers are known to swell in water,
the measured values were only qualitative and the available surface area during the hydrolysis was probably bigger. Nevertheless,
it gave some insights on the evolution of the surface area during
the delignication. Fig. 1 shows the pore size distribution of the
three substrates. WS PTT presented a mesoporous surface similar
to the results of Piccolo et al. (2010), probably due to the steam

M. Huron et al. / Industrial Crops and Products 79 (2016) 104109

107

Table 2
Structural properties of Avicel and delignied wheat straws (WS PTT, WS 1 h30, WS 4 h).

Crystallinity (XRD)
BET surface area
Density

%
m2 g1
g mL1

Avicel

WS PTT

WS 1 h30

WS 4 h

83 4
0.8
1.55 0.02

61 4
6.1
1.48 0.02

67 4
3.9
1.48 0.02

69 4
3.0
1.51 0.02

Fig. 2. SEM photos of the delignied wheat straws (WS PTT, WS 1 h30, WS 4 h) at three magnications (500, 10,000, 30,000).

Enzyme loading (K619): 40 mg L1

Solid content:10 g L1
The reactivity of WS PTT and WS 0 h was the same, which shows
that chlorite method did not affect the reactivity of the substrates

100
Cellulose conversion (%)

explosion pretreatment. The pores seemed to disappear during

delignication, which could explain the surface area drop. It is
probable that the elimination of lignin allows the cellulose to swell
or recombine (hydrogen bonds), leading to the obstruction of the
pores. This result is in contradiction with those of Agarwal et al.
(2013) who supposed that delignication of loblolly pine led to
the creation of new pores and enlargement of the present pores,
thus increasing the internal surface area of the substrate. However,
surface area measurement of their substrates are required to conrm their assumption. Furthermore, steam exploded wheat straw
and loblolly pine are very different substrates and it is likely that
delignication does not affect their structure in the same way.
The SEM photos are presented in Fig. 2. The morphology of the
delignied substrates was very similar to the morphology of steam
exploded wheat straw before delignication. Some cellulosic bers
were embedded into an amorphous matrix composed of disorganized cellulosic microbrils. At high magnication, the mesopores
were visible on WS PTT but not on the other substrates, which was
in accordance with the BET results.
The reactivity of delignied steam exploded wheat straws
(Fig. 3) was compared in order to study the impact of delignication on the enzymatic hydrolysis rate. Enzymatic hydrolysis were
carried on in the following conditions:

80
60
40
20
0
0

12

24
36
48
Time (h)

Fig. 3. Hydrolysis of delignied wheat straws: WS 0 h (

60

72

), WS 1 h (

), WS 2 h30

( ), WS 4 h (
), WS 7 h (). pH 4.7, 48 C, cellulose mass concentration: 10 g L1 ,
K619 mass concentration: 40 mg L1 .

unless delignication occurred. The hydrolysis rate of the different

delignied wheat straws was not signicantly different, reaching
7080% of conversion after 48 h. This result shows that delignication of steam exploded wheat straw did not enhance the conversion
of the cellulosic fraction, on the contrary of several studies carried
on woody substrates (Varnai et al., 2010; Agarwal et al., 2013). In the
present case, delignication seemed to have two opposite effects
which may counterbalance each other: elimination of lignin on the
one hand, reduction of the total surface area on the other hand.
Indeed, the elimination of lignin has usually a positive inuence on
hydrolysis by decreasing the amount of non-productively adsorbed

108

M. Huron et al. / Industrial Crops and Products 79 (2016) 104109

3.2. Inuence of isolated lignin on cellulose hydrolysis

80
60
40

20
0
0

12

24

36
48
Time (h)

60

72

Fig. 4. Hydrolysis of steam exploded wheat straw and Avicel: WS PTT (), Avicel
(); pH 4.7, 48 C, cellulose mass concentration: 10 g L1 , K619 mass concentration:
100 mg L1 .

enzymes and increasing the amount of accessible cellulose. On the

opposite, reduction of specic surface area decreases the amount
of productively adsorbed enzymes.
The steam explosion step is probably also partly responsible
for the present result. This pretreatment is known to decrease
the negative impact of lignin by increasing accessibility to cellulose. It is likely that lignin does not hinder much the hydrolysis
of WS PTT, which is a very reactive substrate compared to Avicel
PH101 (cf. Fig. 4). As a consequence, elimination of lignin does not
enhance the cellulose conversion. The same result was obtained
by Esteghlalian et al. (2002), who did not notice any difference in
the reactivity of bleached and non-bleached steam exploded Douglas r, both substrates being more reactive than Avicel. Likewise,
Yu et al. (2011) observed that lignin did not hinder the conversion of easily hydrolysable pretreated wood. The impact of lignin
on the enzymatic hydrolysis seems thus strongly dependent on the
properties and reactivity of the substrate before delignication.

100

100

Cellulose conversion (%)

The experiments on steam exploded and delignied wheat

straw showed that delignication does not impact the enzymatic
hydrolysis of this substrate. This point was further studied by
hydrolyzing mixtures of delignied wheat straw (WS 4 h) and
wheat straw lignin (WS Lignin). Cellulose content was xed to
10 g L1 , and enzyme concentration was 40 mg L1 . Three different
lignin/substrate mass ratios (dry basis) were used: 0, 25 and 50%.
The cellulose/lignin mass ratio of the 25% mixture is similar to the
cellulose/lignin mass ratio determined for the substrate WS PTT.
As delignication considerably enhances the hydrolysis of woody
substrate, it was interesting to compare the results with a softwood
lignin (SO Lignin). The composition of woody lignin is indeed very
different from wheat straw lignin, and that impacts the interaction
with enzymes. As expected, the hydrolysis rate of steam exploded
wheat straw cellulose was only slightly decreased by the addition of
WS Lignin and the maximal conversion was not affected (Fig. 5A).
SO Lignin had no effect on hydrolysis of WS 4 h either (Fig. 5B). This
supports the fact that the relative absence of delignication effect
was not inuenced by the compositional differences between straw
lignin and wood lignin.
In order to make sure that non-productive adsorption may occur
on the studied lignins, similar tests were carried on using Avicel PH101 as cellulosic substrate. As pretreated wheat straw is
much more reactive than Avicel, a greater enzyme concentration
(100 mg L1 ) was used in the tests. The results are shown in Fig. 6A
and B. The hydrolysis of Avicel was strongly inhibited by the addition of both lignins, which decreased the maximal hydrolysis yield
by as far as 25% at 144 h. It validates the hypothesis that enzymes
can be adsorbed on the lignin in a non-productive way, which
results in a drop of the enzymatic cocktail activity.
These results show that the impact of lignin addition depends
on the properties of the cellulose. Non-productive adsorption of

80
60
40
20

A
0
0

24

48

72
96
Time (h)

120

Cellulose conversion (%)

Cellulose conversion (%)

100

80
60
40
20

B
0
0

144

24

48

72
96
Time (h)

120

144

Fig. 5. Hydrolysis of delignied wheat straw with lignin: WS 4 h and WS Lignin (A), WS 4 h and SO Lignin (B); mass ratio Lignin/Substrate = 0% (), 25% (), 50% (). pH 4.7,
48 C, cellulose mass concentration: 10 g L1 , K619 mass concentration: 40 mg L1 .

Cellulose conversion (%)

80
60
40
20

A
0
0

24

48

72
96
Time (h)

120

144

Cellulose conversion (%)

100

100

80
60
40
20

B
0
0

24

48

72
96
Time (h)

120

144

Fig. 6. Hydrolysis of Avicel with lignin: Avicel and WS Lignin (A), Avicel and SO Lignin (B); mass ratio Lignin/Substrate = 0% (), 25% (), 50% (). pH 4.7, 48 C, cellulose mass
concentration: 10 g L1 , K619 mass concentration: 100 mg L1 .

M. Huron et al. / Industrial Crops and Products 79 (2016) 104109

109

Table 3
Surface area repartition in the mixtures cellulose-WS Lignin.
Avicel + WS Lignin

Specic surface area

Fraction of total area (25% lignin)
Fraction of total area (50% lignin)

m2 g1
%
%

WS 4 h + WS Lignin

Avicel

WS Lignin

WS 4 h

WS Lignin

1
18.0
7.1

13
82.0
92.9

3
39.7
18.8

13
60.3
81.3

enzymes on lignin seems to be negligible in the case of WS 4 h

but not in the case of Avicel. This is probably due to the greater
accessibility of steam exploded straw cellulose compared to Avicel. The surface area ratios for mixture of cellulose and WS Lignin
are calculated in Table 3. The low surface area of Avicel limited
the proportion of potentially actively adsorbed enzymes compared
to WS 4 h. The high crystallinity of Avicel may also contribute to
decrease the afnity of enzymes to cellulose. On the contrary of
WS Lignin, the specic surface area of SO Lignin was too small to
be measured by BET adsorption (<0.5 m2 g1 ). However, softwood
lignins are known to have high afnity with cellulases (Mooney
et al., 1998; Yu et al., 2012). As a consequence, non-productive
adsorption may become signicant even if low surface area is developed by lignin.
The results of this study prove that the effect of isolated lignins
depends strongly from the properties of the cellulose. The quantity of non-productively adsorbed enzymes depends not only on
the afnity they have for both lignin and cellulose, but also on
the specic surface area ratio between lignin and cellulose. The
determination of this surface area in wet conditions has yet to be
improved in order to know the exact values of accessible surface
for enzymes.
4. Conclusions
Delignication of steam exploded wheat straw did not impact
its hydrolysis rate. This could be explained by the high accessibility of cellulose in this substrate, due to steam explosion. Addition
of lignin did not affect the hydrolysis of wheat straw cellulose
either but impacted strongly the hydrolysis of Avicel, which conrms that lignin is not an obstacle for the enzyme conversion of
the previous substrate. These results complete other works carried on pretreated wood and highlight the necessity to characterize
properly the physico-chemical properties of pretreated substrates
in order to improve their enzymatic hydrolysis.
Acknowledgment
This study was part of Projet Futurol, a project supported by BPI
France.
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