Effect of the Pre-Treatment Severity on the Antioxidant Properties of Ethanol Organosolv Miscanthus x giganteus Lignin

doi:10.4236/nr.2012.32005

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Natural Resources, 2012, 3, 29-34

http://dx.doi.org/10.4236/nr.2012.32005 Published Online June 2012 (http://www.SciRP.org/journal/nr)

Effect of the Pre-Treatment Severity on the Antioxidant

Properties of Ethanol Organosolv Miscanthus x giganteus

Lignin

Roland El Hage1,2, Dominique Perrin3, Nicolas Brosse3*

1Centre des Matériaux de Grande Diffusion (CMGD), Ecole des Mines d’Alès, France; 2INNOBAT, Cap Alpha, Clapiers, France;

3Laboratoire d’Etude et de Recherche sur le MAteriau Bois, Faculté des Sciences et Techniques, Nancy-Université, Vandœuvre-

lès-Nancy, France.

Email: *[email protected]

Received November 7th, 2011; revised December 29th, 2011; accepted January 8th, 2012

ABSTRACT

The effect of the severity of an organosolv treatment of Miscanthus x giganteus on antioxidant capacity of the obtained

lignin was studied. Four organosolv lignins extracted with different severity conditions were chosen and tested. Results

obtained using the methyl linoleate method have shown a correlation between oxygen uptake index and the combined

severity. It was found that lignin extracted at higher severity pre-treatment and with a higher phenolic hydroxyl content,

lower aliphatic hydroxyl content, molecular weight and polydispersity has the highest antioxidant capacity.

Keywords: Miscanthus x giganteus; Organosolv Lignin; Antioxidant; Combined Severity; Molecular Weight;

Phenolic and Aliphatic Hydroxyl

1. Introduction

The organosolv pulping has been the subject of con-

siderable research activity and has generated increasing

interest as the pulp and paper industry is moving toward

minimization of environmental impact [1]. In the 1990’s,

this technology was developed at the industrial scale in

Canada (Alcell® Process). One of the advantages of the

organosolv process is the fractionation of the lignocel-

lulosic materials into three major components: cellulosic

fibers, hemicelluloses and lignin. As a result, with the

increasing attention devoted to the biorefinery concept, a

renewed interest in the organosolv treatment is currently

observed and this technology seems to be promising for

the production of ethanol and high value chemicals and

materials from lignin, hemicelluloses and extractives [2].

Indeed, production of high-quality lignin is one of the

unique advantages of the organosolv treatment over alter-

native processes which generally produce a degraded

lignin employed in low added value applications and

energy production. Oganosolv lignins are high-purity,

low molecular weight and sulfur-free products. Moreover,

they are soluble in many organic solvents, possess low

glass transition temperatures, and are easier to thermally

process than kraft lignins. Thus, availability of such high-

quality lignin in large quantities should stimulate deve-

lopment in new lignin applications in the fields of fibers,

biodegradable polymers, adhesives... Lignins as well as

other polyphenols are potent free radical scavengers [3]

and organosolv lignins are considered to be a valuable

source of antioxidant phenolic compounds, which could

be recovered as functional food or feed ingredients. A

large number of biochemical or chemical methods are

used to measure antioxidant capacity. One class of those

methods is based upon inhibition of oxidation of organic

substrates: styrene [4], linoleic acid, methyl or ethyl

linoleate [5], canola oil [6]. The oxygen uptake measure-

ment is the most direct method used to measure the

reaction extent. Moreover, the induced oxidation of methyl

linoleate and its radical long chain reaction is well

documented and well known to be inhibited by various

antioxidants [7,8].

Pan et al. [9] have examined the radical scavenging

effect of organosolv lignins from hybrid poplar and the

relationship between the antioxidant capacity, lignin struc-

ture and extraction conditions. The applicability of lignins

from different sources as antioxidants has been also suc-

cessfully tested [10]. Recently Garcia et al. [11] have

studied the effect of pre-treatment processes on the antio-

xidant capacity of miscanthus sinensis lignins.

Miscanthus x giganteus (MxG) is one of the biomass

resources which has attracted considerable attention as a

possible dedicated energy crop [12,13]. Indeed, MxG

*Corresponding author.

Effect of the Pre-Treatment Severity on the Antioxidant Properties of Ethanol Organosolv Miscanthus x giganteus Lignin

presents some valuable advantages: it is a perennial grass

which requires little nitrogen fertilizer or herbicide, it can

grow to over 3 m tall per year to produce from 20 to 25

tons of dry matter per hectare, and it is non invasive.

Moreover, it is a rhizomatous C4 grass species, which

has a high carbon dioxide fixation rate. These properties

make miscanthus an interesting raw material for industrial

bioconversion processes.

In the present work ethanol organosolv lignins were

extracted from Miscanthus x giganteus over a chosen

range of severity. The influence of the treatment severity

on the antioxidant capacity was studied using the oxygen

uptake index method in presence of methyl linoleate.

2. Experimental

2.1. Materials

The raw Miscanthus x giganteus (MxG) was harvested in

spring 2008 in Trier (Germany). The air-dried MxG was

milled to a particle size of 1 - 3 mm using a Wiley mill

and stored at room temperature during the course of this

study. The untreated feedstock contained 25% Klason

lignin, 37% cellulose and 36% hemicelluloses [12]. All

chemical reagents used in this study were purchased from

Sigma Aldrich and VWR (France) and used as received.

2.2. Ethanol Organosolv Lignin (EOL)

Extraction

25 g (dry weight, dry matter content about 90%) of

Miscanthus was treated with aqueous ethanol (EtOH/ H2O

= 0.5 - 0.65) in presence of sulfuric acid as a catalyst. The

solid to liquid ratio used was 1:8. The pre-treatments were

carried out in a 1.0 L glasslined pressure Parr reactor

equipped with a 4842 temperature controller (Parr

Instrument Company, Moline, IL). The pre-treated Mis-

canthus was washed with warm (60˚C) ethanol/water (8:1,

3 × 50.00 mL). The washes were combined and 3

volumes of water were added to precipitate the Ethanol

Organosolv Lignin, which was collected by centrifuga-

tion and air dried. EOL characterizations were previously

described [14,15].

2.3. Evaluation of Lignin Antioxidant Properties

Antioxidant properties of MxG organosolv lignins were

investigated by evaluating oxygen uptake inhibition dur-

ing oxidation of methyl linoleate. The induced oxida-

tion by molecular oxygen was performed in a gas-tight

borosilicate glass apparatus [5,16]. Butan-1-ol was used

as solvent for lignin dissolution. Temperature was set to

60˚C, initial conditions inside the vessel were as follows;

methyl linoleate (Fluka, 99%) concentration: 0.32 mol/L;

2,2’-azobisisobutyronitrile(AIBN) (Fluka, 98%) concen-

tration: 7.2 × 10–3 mol·L–1; lignin concentration: 0.2

g·L–1; oxygen pressure: 145 Torr. Oxygen uptake was

monitored continuously by a pressure transducer (Viatron

model 104). Without any additive, oxygen uptake is

roughly linear and constitutes the control. In the presence

of an antioxidant, oxygen consumption is slower, and we

estimated the antioxidative capacity of extract by com-

paring oxygen uptake at a chosen time (4 h), in the pres-

ence of this compound (pressure variation ΔPsample)

and in the absence of the compound (ΔPcontrol) accord-

ing to:

OUI = (ΔPcontrol – ΔPsample)/ΔPcontrol

This ratio defines antioxidative capacity as an oxygen

uptake inhibition index (OUI); it should spread from 0 to

100%, for poor and strong antioxidants, respectively, and

may be negative for proxidants. Do not add any kind of

pagination anywhere in the paper.

2.4. Error Analysis

For all EOL samples, oxygen update index values were

calculated from 2 independent experiments performed

under the same conditions. Error values (standard devia-

tion) were also estimated following the method described

by Mounanga et al. 2008 [16] and are about ~5% for all

the essays.

3. Results and Discussion

In recent work we have successfully developed an etha-

nol organosolv process for the pretreatment of MxG [12]

and we have confirmed that this process leads us to pro-

duce little degraded and relatively pure ethanol or-

ganosolv lignin (EOL) from miscanthus [14]. The isolation

of EOL fractions from MxG is illustrated in Figure 1.

Lignin was extracted using various experimental condi-

tions (Table 1), a temperature range between 170˚C and

190˚C, sulfuric acid (SA) concentration between 0.5%

Miscanthus x Giganteus

Organosolv treatment

t = 170˚C, 180˚C,190˚C

SA = 0.5%, 1.0%, 1.2%,1.6%

EtOH/H

O = 0.5, 0.65

t = 60 min

Black liquor

Filtration

Liquid

Phase

MxG

lignin

Figure 1. Schematic of the or ganosolv process.

Effect of the Pre-Treatment Severity on the Antioxidant Properties of Ethanol Organosolv Miscanthus x giganteus Lignin

Table 1. Lignin oxygen uptake index, phenolic content, molecular weight and polydispersity values.

Experiment T (˚C) t (min) EtOH/ H2O (v/v) SA1 (%) CS2OUI3 (%)Phenolic OH

(mmol·g–1)

Aliphatic OH

(mmol·g–1) Mw (×103)Ip

EOL1 170 60 0.65 0.5 1.7558 2.34 3.11 6.5 2.6

EOL2 170 60 0.65 1.0 2.0862 2.72 1.78 6 1.6

EOL3 190 60 0.65 1.2 2.8686 4.04 1.07 3.6 1.4

EOL4 190 60 0.50 1.6 2.9391 3.93 1.26 3.2 1.4

1SA = Sulfuric Acid; 2CS = Combined Severity; 3OUI = Oxygen Uptake Index.

0 1 2 3 4 5 6

Time

[

]

Control

Oxygen uptake (hpa)

EOL1

EOL2

EOL3

EOL4

and 1.6%, and ethanol concentration of 50% or 65%, and

a reaction time of one hour. The investigation covered a

range of combined severity [(CS) = Log((t exp(T - Tref))-

pH] of 1.75 - 2.93; CS describes the severity of the pre-

treatment as a function of treatment time (t = min), tem-

perature (T˚C) and the pH of the medium[17]. The effect

of severity conditions on EOL chemical structure was

investigated and published recently [15]. The effect of

the treatment severity on miscanthus lignin structure has

been also studied and it was demonstrated that the in-

creasing in severity of the organosolv treatment was ac-

companied by (1) a decrease in the aliphatic OH groups,

(2) an increase of phenolic OH groups, (3) a decrease of

the weight average (Mw) molecular weight and polydis-

persity and (4) a strong increase in the degree of condensa-

tion and a cleavage of α or β aryl ether bonds [15].

The study of the antioxidant capacity of EOLs was re-

alized using the induced oxidation method of methyl

linoleate. Figure 2 shows the autoxidation of methyl

linoleate induced by AIBN-Azobisisobutyronitrile in ab-

sence (control) and in presence of lignin (EOL1, EOL2,

EOL3, EOL4) extracted at different pre-treatment sever-

ities. Antioxidant essays were realized under the same

conditions in presence of same lignin concentration (0.2

g·L–1). It appears that the autoxidation of methyl li-

noleate alone is almost linear [Figure 2 (control)]. By

comparing the obtained values in methyl linoleate oxi-

dation, it appears that lignin samples exhibited antioxi-

dant activity by slowing down oxidation of linoleate.

This behavior indicates that organosolv lignin acts as a

potential anti-oxidant that inhibits the oxidation of

methyl linoleate.

Figure 2. Oxygen uptake during the autoxidation of methyl

linoleate induced by AIBN in the presence of MxG organo-

solv lignins.

86% and EOL4, OUI = 91%) seem to be more efficient

than catechin (OUI = 76%). As catechin is a very effi-

cient antioxidant in this system [18], more efficient than

most of wood extracts, EOLs are very efficient antioxi-

dants.

Several comparisons were performed in order to under-

stand differences of lignin antioxidative capacities and to

examine links with lignin structures and experimental con-

dition of processes. Data of combined severity (CS), total

phenolic hydroxyl content, total aliphatic hydroxyl con-

tent determined by 31P NMR, average molecular weight

(Mw) and polydispersity (Ip) determined by GPC [15]

are also compiled in Table 1.

Antioxidative capacities [OUI (%)] defined as the ratio

of oxygen uptakes at 4 h in presence of lignin are also

reported in Table 1. As can be seen in this table, results

of OUI are not the same for each lignin. These values

lead us to classify the antioxidant properties of EOLs

from the lowest (OUI = 58%) to the highest (OUI = 91%)

as follow EOL1 < EOL2 < EOL3 < EOL4. Furthermore

the oxidation of methyl linoleate in presence of a flavon-

oid polyphenolic antioxidant (catechin) was performed in

the same conditions. As a result, miscanthus organosolv

lignins recovered at high severity level (EOL3, OUI =

Figure 3 shows the Oxygen uptake index (%) as a

function of the combined severity. It can be observed that

OUI is positively correlated with the combined severity;

the correlation coefficient (R2) is about 0.98. The anti-

oxidant activity increases with the combined severity of

the organosolv pretreatment process. Thus the effect of

lignin extraction process on its antioxidant properties is

confirmed [9-19]. Lignins extracted at elevated tempera-

ture, longer reaction time, increased catalyst amount, and

dilute ethanol showed higher antioxidant activity.

Effect of the Pre-Treatment Severity on the Antioxidant Properties of Ethanol Organosolv Miscanthus x giganteus Lignin

1.5 2 2.5 3

Combined severity

100

y = 31x

= 0.9759

Oxygen uptake index (%)

Figure 3. Oxygen uptake index as a function of organosolv

pre-treatment combined severity.

The antioxidant efficiency of isolated lignin fractions

is described to be related to their structure, purity and

polydispersity [3,9,20]. Figures 4(a) and (b) show the

oxygen uptake index as a function of phenolic hydroxyl

and aliphatic hydroxyl content respectively. As we pre-

viously demonstrated, aliphatic hydroxyl moieties in

MxG organosolv lignin decreased with the severity of the

treatment while phenolic hydroxyl moieties increased

[15]. In our case it can be observed in Figures 4(a) and

(b) that OUI is correlated to phenolic hydroxyl group

(ArOH) and to aliphatic hydroxyl group (AlkOH) re-

spectively. OUI increases with the lignin phenolic OH

content and with the reduction of aliphatic OH. It is clear

from Figures 4(c) and (d) that lignin with high molecu-

lar weight and polydispersity had low antioxidant prop-

erties. Low molecular weights result from cleavage of

some inter-unit bonds in lignin and this degradation is

accompanied by an increasing of OH aliphatic content

and decreasing in OH phenolic group.

The radical scavenging activity of lignin phenolic com-

pounds depends not only on the hydrogen atom withdraw-

ing but also on stability of the radical formed [9]. Figure 5

shows the mechanism generally invoked for induced

oxidation of polyunsaturated acids [7-21]. It was also

reported that methoxyl groups ameliorate the antioxidant

activity [3-9]. In our recent published work results [15],

we have shown using 13C NMR that EOLs contain

methoxyl group (about 1.4 MeO per aromatic unit) and

that no demethoxylation was observed during the or-

ganosolv treatment, even at high severity. So the in-

creasing in the antioxidant activity observed in this study

could be rationalized by a simultaneous effect: 1) in-

creasing donating hydrogen atom and phenoxyl radical

formation due to higher phenolic group contents and 2)

stability of the radicals formed in presence of methoxy

group in ortho positions (Figure 5).

1.5 2 2.5 3 3.5 4 4.5

Phenolic OH (mmol·g

–1

)

100

y = 1.9741x2 + 6.3787x+31.44

R2 = 0.9635

OUI (%)

(a)

0.5 1 1.5 2 2.5 3 3.5

Aliphatic OH (mmol·g

–1

)

100

y = 15.096x2 – 76.826x+150.92

= 0.97

OUI (%)

(b)

3 3.5 4 4.5 5 5.5 6 6.5 7

Average molecular weight (Mw × 103)

100

y = –9.994x + 122.47

= 0.9988

OUI (%)

(c)

1 1.5 2 2.5 3

Polydispersity (Ip)

100

OUI (%)

(d)

Figure 4. Oxygen uptake index as a function of EOL pheno-

lic OH and aliphatic OH content, average molecular we ight

and polydispersity.

Effect of the Pre-Treatment Severity on the Antioxidant Properties of Ethanol Organosolv Miscanthus x giganteus Lignin 33

Products

Coupling reactions

MeO

H+

OMe

MeO OMe

Products

LOOH + LO

AIBN + LH + O

+ LH + O

Figure 5. Mechanism for induced oxidation of polyunsa-

turated aci ds.

4. Conclusion

In this work it has been shown that Miscanthus x giganteus

organosolv lignin have a potential application as antioxi-

dant. Lignin extraction process parameters are important

factors that can influence the antioxidant properties. In

this way, the obtained results of oxygen updated index

have confirmed that lignin antioxidant activity increased

positively with the severity treatment. Lignin fractions

extracted at elevated temperature and high catalyst con-

tents have revealed a better antioxidant behavior than

catechin a well known antioxidant. The present study has

also confirmed the close correlation between lignin che-

mical structure and its antioxidant activities; lignin frac-

tions with higher phenolic hydroxyl groups content, lower

molecular weight, polydispersity and aliphatic hydroxyl

groups content gave higher values of antioxidant activity.

5. Acknowledgements

The authors gratefully acknowledge the financial support

of the CPER 2007-2013 “Structuration du Pôle de Compé-

titivité Fibres Grand’Est” (Competitiveness Fibre Cluster),

through regional (Région Lorraine), national (DRRT and

FNADT) and European (FEDER) funds.

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