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Industrial Crops and Products 79 (2016) 104–109

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Impact of delignification on the morphology and the reactivity of
steam exploded wheat straw
Maïté Huron a , Damien Hudebine a,∗ , Nicolas Lopes Ferreira b , Dominique Lachenal c

IFP Energies nouvelles, Rond-point de l’échangeur de Solaize – BP 3, 69360 Solaize, France
IFP Energies nouvelles, 1 & 4, avenue de Bois-Préau, 92852 Rueil-Malmaison Cedex, France
Grenoble INP Pagora, 461 rue de la Papeterie BP65, 38402 Saint Martin d’Hères Cedex, France

a r t i c l e

i n f o

Article history:
Received 3 July 2015
Accepted 19 October 2015
Enzymatic hydrolysis
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 delignified with sodium
chlorite to six different grades of delignification. Delignification did not have a significant 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 influence on the hydrolysis
of highly crystalline cellulose (Avicel), whereas it impacted only slightly the hydrolysis of delignified
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 diversification 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 (SSF—Simultaneous Saccharification and Fermentation)
or during a following independent step (SHF—Separate Hydrolysis
and Fermentation). After distillation and rectification, 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:,
(D. Hudebine).
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 fibers 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 L−1 of Avicel and 4 g L−1 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 modified 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-

The comparison of the results gave information on the global impact of lignin on the hydrolysis of wheat straw.. (2010) observed that removal of lignin by chlorite method doubled the hydrolysis degree of the steam exploded spruce. In the first place. Morphological analysis Microcrystalline cellulose (Avicel PH101) was purchased from Sigma–Aldrich (Lyon. 2. A second kind of experiments consisted in studying mixtures of isolated lignin and delignified wheat straw. Enzymatic hydrolysis Reactivity tests were performed in 100 mL Schott Duran laboratory glass bottles at pH 4.05◦ and a count rate per step of 50 s.M. acid hydrolysis were performed following the procedure developed by NREL (Sluiter et al. All buffer components and salts used were reagent grade and purchased from Sigma–Aldrich and GE Healthcare (Saclay. Then the reactivity of the substrates was determined by hydrolyzing the steam exploded delignified wheat straws at 48 ◦ C. delignification affected only slightly the hydrolysis of already very reactive substrates such as steam-exploded Douglas fir wood chips or green liquor pretreated hardwood (Esteghlalian et al. the pretreated wheat straw was delignified by chlorite method to different grades. which is yet a substrate largely studied for the production of 2nd generation bioethanol in Europe. Then. / Industrial Crops and Products 79 (2016) 104–109 not be easily extrapolated to real lignocellulosic substrates. However.. It was supplied by Procéthol 2G (France) and washed and neutralized with KOH until pH 5. The samples were laid between two Kapton films. in the purpose of focusing on non-productive binding of cellulases on the lignin surface area. which corresponded to the summation of insoluble lignin and ashes.3 g of sodium chlorite and 0. The study was performed on steam exploded Expert wheat straw which was grown on chalky soil in Lavannes. 2008).. The crystallinity index (CrI) was calculated according to the method of Segal et al. the sugars produced were analyzed using a High Performance Liquid Chromatography (ICS3000-Dionex). The composition and the morphology of the obtained substrates were characterized in order to evaluate how the delignification impacted them. France). The delignification method has to be relatively soft in order not to attack the cellulose. and the chlorite method is often chosen. Richmond.. The amount of other sugars was negligible. this kind of research has never been carried on wheat straw. Several authors thus compared the reactivity of delignified substrates in order to see if the elimination of lignin enhances the hydrolysis rate (Varnai et al. The surface area of the substrates was determined with the Brunauer–Emmett–Teller (BET) method which is based on nitrogen adsorption at different pressures. (2014). Several grades of delignified substrates were produced by increasing the reaction time from 0 to 7 h. 2002.1.3. Compositional analysis In order to measure the quantity of cellulose. Zeiss. and the xylan content from the quantity of xylose. On the contrary. 2. Morphology of the substrates was observed with a scanning electron microscope (Supra 40. Chloramphenicol was added (50 mg L−1 ) to prevent any bacterial contamination. The various lignocellulosic substrates used in this study were previously lyophilized with a freeze dryer Alpha 1–2 and grinded (<1 mm). The total mass of the solution was 70 g. Bagsvaerd. extracted respectively from wheat straw and softwood.5. Two different lignins. (1946) and Timell (1961). Yu et al. Softwood kraft lignin (SO Lignin) was provided by MeadWestvaco (MWV. (ii) fed-batch with a mixture containing several inducers including lactose as carbon source. After the acid hydrolysis. (2008) was performed in fermentors using two steps: (i) growth on lactose. All samples were sputter-coated with 5 nm platinum–palladium before analysis. Agarwal et al. 2013). 2013. United States).4. (1959). These studies allow to better understand the role of lignin in the recalcitrance of lignocellulose. Crystalline structure of the substrates was analyzed by a X’Pert Pro PANalytical X-ray diffractometer with a copper anode.2. The enzymatic cocktail called K619 was obtained from a crude enzyme preparation using the hyper cellulolytic mutant strain from Trichoderma reesei CL847. The reaction’s temperature was set to 70 ◦ C. were tested. The angular range varied between 5 and 70◦ 2 with an analysis step of 0. The cellulose content was calculated from the quantity of glucose released. The substrate content was 10 g L−1 and the K619 enzyme concentration was 40 mg L−1 for wheat straw (delignified or not) and 100 mg L−1 for Avicel. Prior to this specific analysis. to our knowledge. extracted by alkaline treatment.1 mL of acetic acid per gram of dry biomass (wheat straw). and adapted by Ahlgren and Goring (1971). France).8 in a heating body (48 ◦ C) using a magnetic agitation. The substrate was first washed and neutralized at pH 5. and a complete characterization can be found in the work of Duval et al. 2. The results showed that delignification had a great positive impact on the reactivity of most of the woody substrates. Chlorite delignification The delignification of pretreated wheat straw was done following the chlorite protocol firstly described by Wise et al. The reactive charge was composed of 0. Percentages of cellulose.. Materials and methods 105 2. This lignin was deeply characterized by Joffres et al. Huron et al. Ashes content was determined by calcination of the substrate in a Thermo Scientific muffle following the NREL procedure (Sluiter et al. the secreted enzymes were recovered after separation from the mycelia by centrifugation. was purchased from Green Value SA (Protobind 1000). and the initial Liquid to Biomass mass ratio was 15. Denmark). This article focuses on the impact of lignin on enzymatic hydrolysis of steam exploded wheat straw. At the end of the reaction. 2. acceleration voltage of 1 kV). Materials 2. The protein concentration of the final cocktail was estimated by the Lowry method to be 53 g L−1 (filter paper activity: 360 FPU per gram of proteins). France and harvested in July 2011. (2013). This analysis was carried out with a Micromeritics ASAP 2420 system. the solid was washed in 5 L of pure water and recovered by vacuum filtration through glass crucibles (porosity 1). A fresh charge of reactants (sodium chlorite and acetic acid) was added at hourly intervals without withdrawal of any liquor. 2011). For instance. Varnai et al. The production previously described by Herpoel-Gimbert et al. 2011. 2011). 2010. The added volume of N188 was half of the volume of K619. lignin and ashes were calculated on a dry weight basis. This cocktail was supplemented with a commercial ␤-glucosidase cocktail (SP188 (64 g L−1 )) secreted by Aspergillus niger and provided by Novozymes (Novo Nodisk A/S. Commercial wheat straw lignin (WS Lignin). the lyophilized samples were dried again at 40 ◦ C during 6 h. The remaining solid after acid hydrolysis was dried in an oven at 45 ◦ C until it reached a constant weight... xylan. The buffer used was citrate buffer at 50 mmol L−1 . . Ju et al. hemicellulose and lignin in the wheat straw (delignified or not). The results were compared with mixtures of lignin and Avicel PH101.

steam exploded wheat straw was delignified following the chlorite protocol. Yu et al.5 9. a complementary measure of glucose. Then. The composition of each substrate (WS 0 h to WS 7 h) was measured by acid hydrolysis.9 84. WS PTT) .5 3.5 4. 2004). The cellulose mass fraction of steam exploded wheat straw was 57%.1 0. That indicated that the measures of glucose.7 0.2 85.6 0. When the concentration of glucose was too high (>4. Fig. WS PTT presented a mesoporous surface similar to the results of Piccolo et al. 3.22% lignin at 1 h and 1 h30 (Ref. xylose and dry residue were representative of the overall composition of the substrate. WS 1 h30 and WS 4 h. The samples were heated at 90 ◦ C for 10 min to stop the reaction.5 32. due to the delignification. were used to obtained different delignification grades.4 3.9 0. 2009).1 84.0 0.4 72. and since no compositional differences were observed between the three last substrates. the total mass balance reached almost 100%.3 1. ranging from 0 to 7 h.1 0.8 1. The analysis results are shown in Table 2.e. WS 4 h ( ). which may be non-extractable because embedded in the cellulose matrix.2 21.2 0.0 0. The supernatant was recovered to determine the concentration of glucose and calculate the hydrolysis conversion of the cellulose. Pretreated wheat straw 0h 1h 1 h30 2 h30 4h 7h 57.2 34.8 0.2 4.8 0.. WS 1 h30) .3 1.6 6.2 5. XRD method is strongly influenced by the biomass composition. Delignification of steam exploded wheat straw In order to study the global impact of lignin on the enzymatic hydrolysis.1 0. before being centrifuged at 3600×g during 20 min. The substrate reactivity is known to be strongly dependent on its morphological characteristics such as crystallinity. the delignified substrates contained more cellulose and xylan. and it is uncertain whether the cellulose structure has really been altered by delignification (Kim et al. It was almost divided by two during the first hour and a half and decreased at a much slower rate during the following hours.0 3.8 0.0 Dry residue mass fraction (%) Lignin Ashes Standard deviation 36. However. The xylan content was low.9 8. 2003. density and SEM photos were performed for the three main delignification grades i.3 0.2 0 0 25 50 75 100 Mean Diameter (nm) 125 Fig.5 33. The surface area of steam exploded wheat straw dropped at the beginning of the delignification. As the cellulosic fibers are known to swell in water. Glucose was measured using YSI Model 2700 SELECT.1.3 7. Results and discussion 3.2 22. The results were compared with those of original steam exploded wheat straw (WS PTT). It was further used as a representation of wheat straw pure cellulose.0 97. the measured values were only qualitative and the available surface area during the hydrolysis was probably bigger.3 98.33% lignin initially (Ref. which showed that the composition of the substrate was not modified by the chlorite method when the reaction step was skipped.4 0.6 98.7 Cellulose mass fraction (%) Standard deviation Xylan mass fraction (%) Standard deviation Nd: not determined.3 0.6 62.3 1. Crystallinity increased slightly during chlorite delignification. Measurements were done in duplicate. At the end of the enzymatic reaction. it gave some insights on the evolution of the surface area during the delignification. as hemicelluloses were mainly eliminated during the pretreatment step.8 0.2 0.0 0.3 0.6% lignin after 2 h30 (Ref. is the remaining solid at the end of the acid hydrolysis. Glucose and xylose content was measured by HPLC. Pore size distribution in the delignified wheat straws from BET measurements: WS PTT (). The cellulose-to-glucose conversion yield is defined as the glucose amount in the liquid phase product divided by the cellulose content (as glucose equivalent) in the substrate.2 1. For each substrate.4 4. polymerization degree and surface area (Zhang and Lynd. The composition of WS PTT and WS 0 h was the same..6 70. these results have to be used with caution since the analyses were performed on dry substrates. Results are shown in Table 1. probably due to the steam . 2011). However.. / Industrial Crops and Products 79 (2016) 104–109 Table 1 Chemical composition of partially delignified steam exploded wheat straws.1 Nda Nda 0. (2010).6 0.8 0.g-1) a 3. cellobiose and xylose was done using a High Performance Liquid Chromatography (ICS3000-Dionex).1 99. WS PTT.0 4. composed of lignin and ashes.2 1. including lignin.6 97.1 0.0 0. As a consequence. the substrate still contained some traces of lignin.4 0. WS 4 h) Even after 7 h of delignification.3 0.3 0.0 4.3 Total (%) Standard deviation 97.1 0. this result is probably primarily due to elimination of amorphous materials.1 23. WS 1 h30 (䊏).5 g L−1 ). three main delignification grades were obtained corresponding to approximately the following mass fractions: . Huron et al. Assays were performed in duplicate for a total period of 72 h and samples of 1 mL were taken periodically for analysis. Six different reaction times. Finally.2 10. surface area. and the lignin mass fraction 34%.6 2.2 0. Nevertheless. 1 shows the pore size distribution of the three substrates. Measures of crystallinity. Consequently the evolution of these properties during the delignification step is an important parameter to follow. 1. WS 4 h was considered in this article as the most delignified one.106 M.2 99. It measures indeed the relative amount of crystalline cellulose in the total solid.. The dry residue. As the cellulose is likely to become altered by long delignification times (Ishizawa et al. 1 dV/dD (µL. samples were diluted in pure water.

K619 mass concentration: 40 mg L−1 . Agarwal et al.55 ± 0. Some cellulosic fibers were embedded into an amorphous matrix composed of disorganized cellulosic microfibrils.000). reaching 70–80% of conversion after 48 h.M. The hydrolysis rate of the different delignified wheat straws was not significantly different. the elimination of lignin has usually a positive influence on hydrolysis by decreasing the amount of non-productively adsorbed ... However. which was in accordance with the BET results. At high magnification. steam exploded wheat straw and loblolly pine are very different substrates and it is likely that delignification does not affect their structure in the same way. which shows that chlorite method did not affect the reactivity of the substrates 100 Cellulose conversion (%) explosion pretreatment. WS 1 h30. Crystallinity (XRD) BET surface area Density % m2 g−1 g mL−1 Avicel WS PTT WS 1 h30 WS 4 h 83 ± 4 0.48 ± 0. 3. The SEM photos are presented in Fig. WS 4 h ( ). (2013) who supposed that delignification of loblolly pine led to the creation of new pores and enlargement of the present pores.02 Fig.7. 2. WS 1 h30. unless delignification occurred.0 1. In the present case. It is probable that the elimination of lignin allows the cellulose to swell or recombine (hydrogen bonds). cellulose mass concentration: 10 g L−1 . This result shows that delignification of steam exploded wheat straw did not enhance the conversion of the cellulosic fraction.8 1. WS 7 h (). This result is in contradiction with those of Agarwal et al. 3) was compared in order to study the impact of delignification on the enzymatic hydrolysis rate. WS 4 h) at three magnifications (×500. Huron et al. WS 4 h). which could explain the surface area drop. – Enzyme loading (K619): 40 mg L−1 – Solid content:10 g L−1 The reactivity of WS PTT and WS 0 h was the same. The reactivity of delignified steam exploded wheat straws (Fig. thus increasing the internal surface area of the substrate. Hydrolysis of delignified wheat straws: WS 0 h ( 60 72 ).51 ± 0.02 61 ± 4 6.000.48 ± 0. 2013).02 69 ± 4 3. The morphology of the delignified substrates was very similar to the morphology of steam exploded wheat straw before delignification. ×30. 2010. 48 ◦ C. reduction of the total surface area on the other hand. leading to the obstruction of the pores. The pores seemed to “disappear” during delignification. delignification seemed to have two opposite effects which may counterbalance each other: elimination of lignin on the one hand. the mesopores were visible on WS PTT but not on the other substrates.1 1. ×10. Indeed. Enzymatic hydrolysis were carried on in the following conditions: 80 60 40 20 0 0 12 24 36 48 Time (h) Fig. Furthermore. on the contrary of several studies carried on woody substrates (Varnai et al. 2. WS 2 h30 ( ). pH 4.02 67 ± 4 3. SEM photos of the delignified wheat straws (WS PTT. surface area measurement of their substrates are required to confirm their assumption. WS 1 h ( ).9 1. / Industrial Crops and Products 79 (2016) 104–109 107 Table 2 Structural properties of Avicel and delignified wheat straws (WS PTT.

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. 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. This pretreatment is known to decrease the negative impact of lignin by increasing accessibility to cellulose.7. 48 ◦ C. a greater enzyme concentration (100 mg L−1 ) was used in the tests. The same result was obtained by Esteghlalian et al. Hydrolysis of steam exploded wheat straw and Avicel: WS PTT (). pH 4. 5B). Fig. cellulose mass concentration: 10 g L−1 . / Industrial Crops and Products 79 (2016) 104–109 3. 25% (䊏). 50% (). Avicel (䊏). K619 mass concentration: 100 mg L−1 . cellulose mass concentration: 10 g L−1 . As pretreated wheat straw is much more reactive than Avicel. 50% (). The impact of lignin on the enzymatic hydrolysis seems thus strongly dependent on the properties and reactivity of the substrate before delignification. 25% (䊏). Avicel and SO Lignin (B). which decreased the maximal hydrolysis yield by as far as 25% at 144 h. As a consequence. pH 4.7. Likewise.108 M. This point was further studied by hydrolyzing mixtures of delignified wheat straw (WS 4 h) and wheat straw lignin (WS Lignin). Yu et al. WS 4 h and SO Lignin (B). The hydrolysis of Avicel was strongly inhibited by the addition of both lignins. 6. Cellulose content was fixed to 10 g L−1 . 25 and 50%. similar tests were carried on using Avicel PH101 as cellulosic substrate. . SO Lignin had no effect on hydrolysis of WS 4 h either (Fig. The cellulose/lignin mass ratio of the 25% mixture is similar to the cellulose/lignin mass ratio determined for the substrate WS PTT. 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. which results in a drop of the enzymatic cocktail activity. and that impacts the interaction with enzymes. On the opposite. mass ratio Lignin/Substrate = 0% (). who did not notice any difference in the reactivity of bleached and non-bleached steam exploded Douglas fir. Hydrolysis of delignified wheat straw with lignin: WS 4 h and WS Lignin (A). 6A and B. As delignification considerably enhances the hydrolysis of woody substrate. The results are shown in Fig. 5A). Huron et al. cellulose mass concentration: 10 g L−1 . K619 mass concentration: 100 mg L−1 . 48 ◦ C. It validates the hypothesis that enzymes can be adsorbed on the lignin in a non-productive way. 4. Hydrolysis of Avicel with lignin: Avicel and WS Lignin (A). Influence of isolated lignin on cellulose hydrolysis 80 60 40 20 0 0 12 24 36 48 Time (h) 60 72 Fig. It is likely that lignin does not hinder much the hydrolysis of WS PTT. K619 mass concentration: 40 mg L−1 . elimination of lignin does not enhance the cellulose conversion. which is a very reactive substrate compared to Avicel PH101 (cf. 5.7. 4). enzymes and increasing the amount of accessible cellulose. 48 ◦ C. This supports the fact that the relative absence of delignification effect was not influenced 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. (2002). pH 4. The steam explosion step is probably also partly responsible for the present result. reduction of specific surface area decreases the amount of productively adsorbed enzymes. As expected. (2011) observed that lignin did not hinder the conversion of easily hydrolysable pretreated wood. mass ratio Lignin/Substrate = 0% (). These results show that the impact of lignin addition depends on the properties of the cellulose. and enzyme concentration was 40 mg L−1 . 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. both substrates being more reactive than Avicel. 100 100 Cellulose conversion (%) The experiments on steam exploded and delignified wheat straw showed that delignification does not impact the enzymatic hydrolysis of this substrate.2. Three different lignin/substrate mass ratios (dry basis) were used: 0.

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