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Industrial Crops and Products 79 (2016) 104109

Contents lists available atScienceDirect

Industrial Crops and Products

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / i n d c r o p

Impact ofdelignification on the morphology and the reactivity ofsteam exploded wheat straw

Mat Huron a, Damien Hudebine a,, Nicolas Lopes Ferreira b, Dominique Lachenal c

a IFP Energies nouvelles, Rond-point de lchangeur de Solaize BP 3, 69360 Solaize, Franceb IFP Energies nouvelles, 1 & 4, avenuede Bois-Prau, 92852 Rueil-MalmaisonCedex, Francec Grenoble INP Pagora, 461 rue de la Papeterie BP65, 38402 Saint MartindHres 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 ofthis article wasto better understand the role oflignin in the recalcitrance oflignocellulosic

biomass during enzymatic hydrolysis. Steam exploded wheat straw was partially delignified with sodium

chlorite to six different grades ofdelignification. Delignification did not have a significant impact on the

enzymatic hydrolysis ofthe studied wheat straw in the experimental conditions tested. Inhibitive impact

oflignin in terms ofnon-productive adsorption was then explored using soda lignin from wheat straw and

kraft lignin from softwood. The addition ofboth 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 ofthe crystalline cellulose Avicel.

2015 Elsevier B.V. All rights reserved.

1. Introduction

Fuels produced from lignocellulosic biomass have a high poten-

tialto partiallyreplacefossil 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 glu-

cose by the combined actions of different specialized enzymes

(endoglucanases, cellobiohydrolases,-glucosidases,etc.).The glu-cose is mainly fermented into ethanol thanks to yeasts in the

same time (SSFSimultaneous Saccharification and Fermentation)

or during a following independent step (SHFSeparate Hydrolysis

and Fermentation). After distillation and rectification, the ethanolis directly incorporatedinto 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 pro-

cess, due to the cost and complexity of the enzymatic cocktail and

to the inherent recalcitrance of the lignocellulosic biomass. This

recalcitrance canbe explained by various characteristics of the sub-

Corresponding author.

E-mail addresses:[email protected], [email protected]

(D. Hudebine).

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

known to hinder the action of enzymes by decreasing accessibil-ity to cellulose. Furthermore, some enzymes can adsorb on the

hydrophobic surface area of lignin and do not participate to the

overall hydrolysis reactionof the cellulose(non-productiveadsorp-

tion). 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 mix-

ture 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 cellu-

lose, but some others did not hinder notably the conversion. For

instance, Nakagame et al. (2010) showed that cellulolytic enzy-

matic lignin from steam exploded corn stover did not affect the

hydrolysis of Avicel, whereas cellulolytic enzymatic lignin fromsteamexploded poplar andfrom organosolv loblollypine decreased

the hydrolysis yieldby respectively 11%and 23%(mixturescontain-

ing20gL1 ofAviceland4gL1 of lignin hydrolyzed at 50 CandpH

4.8. Enzymes loading: 5 FPU and 10CBU per gram of cellulose). The

impactof lignindepends not only on the quantityof lignin, butalso

on its structure and its composition. However, the characteristics of

pure ligninare quite different from those of the original lignin con-

tained 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-

http://dx.doi.org/10.1016/j.indcrop.2015.10.040

0926-6690/ 2015 Elsevier B.V. All rightsreserved.
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M. Huron et al./ Industrial Crops andProducts 79 (2016) 104109 105

not be easily extrapolated to real lignocellulosic substrates. 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., 2010, 2011; Agarwal et al., 2013; Ju et al., 2013).

The delignification method has to be relatively soft in order not to

attack the cellulose, and the chlorite method is often chosen. The

results showed that delignification 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, 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., 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

yeta substratelargely 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

first place, the pretreated wheat straw was delignified by chlorite

method to different grades. The composition and the morphology

of the obtained substrates were characterized in order to evalu-

ate how the delignification impacted them. Then the reactivity ofthe substrates was determined by hydrolyzingthe steam exploded

delignified wheat straws at 48C. The comparison of the results

gave information on the global impact of lignin on the hydrol-

ysis of wheat straw. A second kind of experiments consisted in

studying mixtures of isolated lignin and delignified wheat straw,

in the purpose of focusing on non-productive binding of cellulases

on the lignin surface area. Two different lignins, extracted respec-

tively from wheat straw and softwood, were tested. The results

were compared with mixtures of lignin and Avicel PH101.

2. Materials andmethods

2.1. Materials

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 character-

ized byJoffres et al. (2014).

Softwood kraft lignin (SO Lignin) was provided by Mead-

Westvaco (MWV, Richmond, United States), and a complete

characterization can be found in the work ofDuval et al. (2013).

The study was performed on steam exploded Expert wheat straw

whichwas grown on chalky soil in Lavannes, France and harvested

in July 2011. It was supplied by Procthol 2G (France) and washedand neutralized with KOH until pH 5.

The enzymatic cocktail called K619 was obtained from a crude

enzyme preparation usingthe hyper cellulolyticmutant 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 con-

taining 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 final

cocktail was estimated by the Lowry method to be 53g L1 (filter

paper activity: 360 FPU per gram of proteins). This cocktail was

supplemented with a commercial -glucosidase cocktail (SP188

(64gL1)) secreted byAspergillusnigerandprovidedby Novozymes

(Novo Nodisk A/S, Bagsvaerd, Denmark).

2.2. Chlorite delignification

The delignification of pretreated wheat straw was done follow-

ing the chlorite protocol firstly described by Wise et al. (1946) and

Timell (1961), andadapted by Ahlgren and Goring (1971). The sub-

strate was first washed andneutralized at pH 5. Thereactivecharge

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 5L of pure water

and recovered by vacuum filtration through glass crucibles (poros-

ity 1). Several grades of delignified 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 (delignified 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 12andgrinded(

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106 M. Huron et al./ Industrial Crops andProducts 79 (2016) 104109

Table 1

Chemical composition of partially delignified steam exploded wheat straws.

Pretreated wheat straw 0 h 1 h 1 h30 2 h30 4 h 7 h

Cellulose mass fraction (%) 57.2 62.8 70.8 72.0 84.3 84.1 85.3

Standard deviation 0.6 0.6 0.4 0.9 1.1 0.2 0.4

Xylan mass fraction (%) 4.0 3.6 3.8 4.1 4.3 4.2 5.3


Dry residue mass fraction (%) 36.2 33.5 23.1 22.2 10.9 9.6 7.5

Lignin 34.3 32.3 Nda 21.2 8.6 6.5 4.4Ashes 1.9 1.2 Nda 1.0 2.3 3.1 3.1


Total (%) 97.3 99.8 97.7 98.2 99.4 97.8 98.1


a Nd: not determined.

Assayswere performedin duplicatefor a total periodof 72h and

samples of 1 mLwere taken periodically for analysis. The samples

were heated at 90C for 10min to stop the reaction, before being

centrifuged at 3600gduring 20min. The supernatant was recov-

ered to determine the concentration of glucose and calculate the

hydrolysis conversion of the cellulose.

Glucose was measured using YSI Model 2700 SELECT. Mea-

surements were done in duplicate. When the concentration ofglucose was too high (>4.5gL1), 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 defined as the glucose

amount in the liquid phaseproductdividedby the cellulose content

(as glucose equivalent) in the substrate.

3. Results and discussion

3.1. Delignification of steam explodedwheat straw

In order to study the global impact of lignin on the enzymatic

hydrolysis, steam exploded wheat straw was delignified followingthe chlorite protocol. Six different reaction times, ranging from 0

to 7 h, were used to obtained different delignification grades. The

composition of eachsubstrate (WS 0h toWS 7 h) was measured by

acid hydrolysis. The results were compared with those of original

steam exploded wheat straw (WS PTT). Glucose and xylose con-

tent 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 anddry 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 pretreatmentstep. The composition of WS PTT and WS 0h was the same, which

showed that the composition of the substrate was not modified

by the chlorite method when the reaction step was skipped. Then,

dueto thedelignification,the delignifiedsubstratescontained more

cellulose and xylan. Finally, three main delignification grades were

obtained corresponding to approximately the following mass frac-

tions:

- 33% lignin initially (Ref. WS PTT)

- 22% lignin at1 h and 1h30 (Ref. WS 1h30)

- 6% lignin after 2 h30 (Ref. WS 4 h)

Even after 7h of delignification, the substrate still contained

some traces of lignin, which may be non-extractable because

0

0.2

0.4

0.6

0.8

1

0 25 50 75 100 125

dV/dD(L.g-1)

Mean Diameter (nm)

Fig. 1. Pore size distribution in the delignified wheat straws from BET measure-

ments: WS PTT (), WS 1 h 30 (),WS 4 h ( ).

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

become altered by longdelignification times(Ishizawa et al., 2009),

and since no compositionaldifferences were observed between the

three last substrates, WS 4h was considered in this article as the

most delignified one. It was further used as a representation ofwheat straw pure cellulose.

The substrate reactivity is known to be strongly dependent on

its morphological characteristics such as crystallinity, polymeriza-

tion degree andsurface area (Zhang and Lynd, 2004). Consequently

the evolution of these properties during the delignification step is

an important parameter to follow. Measures of crystallinity, sur-

face area, density and SEM photos were performed for the three

main delignification grades i.e., WS PTT, WS 1h30and WS 4h. The

analysis results are shown in Table 2.

Crystallinity increased slightly during chlorite delignification.

However, XRD method is strongly influenced by the biomass com-

position. It measures indeed the relative amount of crystalline

cellulose in the total solid, including lignin. As a consequence, this

resultis probably primarilydue to elimination of amorphous mate-rials, and it is uncertain whether the cellulose structure has really

been altered by delignification (Kim et al., 2003; Yu et al., 2011).

The surface area of steam exploded wheat straw dropped at

the beginning of the delignification. It was almost divided by two

during the first 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 fibers are known to swell in water,

the measured values were only qualitative and the available sur-

face area during the hydrolysis was probably bigger. Nevertheless,

it gave some insights on the evolution of the surface area during

the delignification. Fig. 1 shows the pore size distribution of the

three substrates. WS PTT presented a mesoporous surface similar

to the results ofPiccolo et al. (2010), probably due to the steam

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Table 2

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

Avicel WS PTT WS 1 h30 WS 4 h

Crystallinity (XRD) % 83 4 61 4 67 4 69 4

BET surface area m2 g1 0.8 6.1 3.9 3.0

Density g mL 1 1.55 0.02 1.48 0.02 1.48 0.02 1.51 0.02

Fig. 2. SEM photosof thedelignified wheat straws (WS PTT, WS 1h30,WS 4 h) at three magnifications (500, 10,000, 30,000).

explosion pretreatment. The pores seemed to disappear duringdelignification, 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 ofAgarwal et al.

(2013) who supposed that delignification 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 con-

firm their assumption. Furthermore, 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.

The SEM photos are presented in Fig. 2. The morphology of the

delignified substrates was very similar to the morphology of steam

exploded wheat straw before delignification. Some cellulosic fiberswere embedded into an amorphous matrix composed of disorga-

nized cellulosic microfibrils. At high magnification, the mesopores

were visible on WS PTT but not on the other substrates, which was

in accordance with the BET results.

The reactivity of delignified steam exploded wheat straws

(Fig. 3) was compared in order to study the impact of delignifica-

tion on the enzymatic hydrolysis rate. Enzymatic hydrolysis were

carried on in the following conditions:

Enzyme loading (K619): 40mg L1

Solid content:10g L1

Thereactivityof WS PTTand WS 0 h was thesame, which shows

that chlorite method did not affect the reactivity of the substrates

0

20

40

60

80

100

0 12 24 36 48 60 72

Celluloseconversion(%)

Time (h)

Fig.3.Hydrolysis of delignifiedwheat straws: WS 0 h ( ), WS 1 h ( ), WS 2h30

( ),WS 4 h ( ), WS 7 h ().pH 4.7,48 C, cellulosemassconcentration: 10g L1,

K619 mass concentration: 40mg L1.

unless delignification occurred. The hydrolysis rate of the different

delignified wheat straws was not significantly different, reaching

7080% of conversion after 48h. This result shows that delignifica-

tionof steamexploded wheat strawdid notenhancethe conversion

of the cellulosic fraction, on the contrary of several studies carried

onwoodysubstrates(Varnai et al.,2010; Agarwalet al.,2013). Inthe

present case, delignification 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 influence on

hydrolysis by decreasing the amount of non-productively adsorbed

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108 M. Huron et al./ Industrial Crops andProducts 79 (2016) 104109

0

20

40

60

80

100

0

12

24

36

48

60

72


Time (h)

Fig. 4. Hydrolysis of steam exploded wheat straw and Avicel: WS PTT (), Avicel

(); pH4.7,48 C, cellulosemassconcentration: 10g L1, K619 massconcentration:

100mgL1.

enzymes and increasing the amount of accessible cellulose. On the

opposite, reduction of specific 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 cellu-

lose. It is likely that lignin does not hinder much the hydrolysisof 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 Dou-

glas fir, both substrates being more reactive than Avicel. Likewise,

Yu et al. (2011) observed that lignin did not hinder the conver-

sion of easily hydrolysable pretreated wood. The impact of lignin

on theenzymatic hydrolysis seems thus stronglydependent on the

properties and reactivity of the substrate before delignification.

3.2. Influence of isolated lignin on cellulose hydrolysis

The experiments on steam exploded and delignified wheat

straw showed that delignification does not impact the enzymatic

hydrolysis of this substrate. This point was further studied by

hydrolyzing mixtures of delignified wheat straw (WS 4 h) and

wheat straw lignin (WS Lignin). Cellulose content was fixed to

10g L1, and enzyme concentration was 40mg 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 delignification considerably enhances the hydrolysis of woody

substrate, it wasinteresting to compare theresults 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 strawcellulose wasonly slightlydecreasedby theadditionof

WS Lignin and the maximal conversion was not affected (Fig. 5A).

SO Ligninhad noeffect on hydrolysis of WS 4heither(Fig.5B). This

supports the fact that the relative absence of delignification effect

was notinfluenced by the compositionaldifferences between straw

lignin and wood lignin.

In order to makesure thatnon-productiveadsorption may occur

on the studied lignins, similar tests were carried on using Avi-cel PH101 as cellulosic substrate. As pretreated wheat straw is

much more reactive than Avicel, a greater enzyme concentration

(100mg L1) was used in the tests. The results are shown in Fig. 6A

and B. The hydrolysis of Avicel was strongly inhibited by the addi-

tion 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

0

20

40

60

80

100

0 24 48 72 96 120 144


Time (h)

A0

20

40

60

80

100

0 24 48 72 96 120 144


Time (h)

B

Fig. 5. Hydrolysis of delignified wheat straw with lignin:WS 4h and W S Lignin(A), WS 4h and SO Lignin(B);mass ratio Lignin/Substrate= 0% (), 25% () , 50% (). pH4.7,

48 C, cellulosemass concentration: 10g L1, K619 mass concentration: 40mg L1.

0

20

40

60

80

100

0 24 48 72 96 120 144


Time (h)

A0

20

40

60

80

100

0 24 48 72 96 120 144


Time (h)

B

Fig. 6. Hydrolysis of Avicelwith lignin:Avicel andWS Lignin(A),Aviceland SO Lignin(B);mass ratio Lignin/Substrate= 0% (),25% (),50% ().pH 4.7,48 C, cellulose mass

concentration: 10 g L1

, K619 mass concentration: 100mg L1

.

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Table 3

Surface area repartition in the mixtures cellulose-WS Lignin.

Avicel+ WS Lignin WS 4 h + W S Lignin

Avicel WS Lignin WS 4 h WS Lignin

Specific surface area m2 g1 1 13 3 13

Fraction of total area (25% lignin) % 18.0 82.0 39.7 60.3

Fraction of total area (50% lignin) % 7.1 92.9 18.8 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 Avi-

cel. 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 affinity of enzymes to cellulose. On the contrary of

WS Lignin, the specific surface area of SO Lignin was too small to

be measured by BET adsorption (

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