Optimization of a formic/acetic acid delignification treatment on beech wood and its influence on the structural characteristics of the extracted lignins

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Research ArticleReceived: 7 January 2013 Revised: 3 May 2013 Accepted article published: 17 May 2013 Published online in Wiley Online Library: 20 June 2013

(wileyonlinelibrary.com) DOI 10.1002/jctb.4123

Optimization of a formic/acetic aciddelignification treatment on beech woodand its influence on the structuralcharacteristics of the extracted ligninsMathilde Simon,a Yves Brostaux,b Caroline Vanderghem,a Benoit Jourez,c

Michel Paquota and Aurore Richela∗

Abstract

BACKGROUND: In order to replace petrochemicals by bio-based lignin products in high value-added applications, a formic/aceticacid treatment was adapted to beech wood (Fagus sylvatica L.) for lignin extraction.

RESULTS: Beech wood particles were delignified at atmospheric pressure by a formic acid/acetic acid/water mixture. Cookingtime and temperature were optimized for delignification, pulp yield and 2-furfural concentration. Response surface designanalysis revealed that delignification yield increased with cooking time and temperature.

CONCLUSION: The multi-criteria optimization of delignification was used to find the ideal cooking conditions (5 h 07 min,104.2 ◦C) to maximize delignification (70.5%) and pulp yield (58.7%) and, to a lesser extent, minimize 2-furfural production.Treatment conditions were found to influence the chemical structure of extracted lignins. Cooking time and temperatureinversely influenced lignin molecular weights.c© 2013 Society of Chemical Industry

Keywords: chemical structure; Fagus sylvatica L; formic/acetic acid treatment; lignin; response surface methodology

INTRODUCTIONThe sustainable production of energy, fuels and chemicalsfrom renewable raw materials is a challenge. Lignocellulosicsubstrates, including hardwood, softwood and grasses, are anabundant renewable resource mainly composed of cellulose,hemicelluloses and lignin. They constitute a promising alternativeto petrochemical fossil resources for the production of biofuelsand bio-based products.

The first steps of biorefinery processes are centered on theextraction and recycling of cellulose (into fermentable glucose).These processes used in the production of bioenergy leavedegraded hemicelluloses and lignins recovered as side-productswith no possibilities of high value-added applications. Within thecontext of an integrated biorefinery, and for economic reasons,the recovery and the non-energetic transformation of lignins haveopened up new opportunities.

Lignin is an aromatic cross-linked heteropolymer composedof three phenylpropane units (p-hydroxyphenyl, guaiacyl andsyringyl units) linked together via radical coupling reactions by spe-cific ether or carbon–carbon bonds.1,2 With its phenylpropanoidstructure and its abundance of lignocellulosic substrates, lignincould replace some petrochemicals in industrial applicationssuch as polymer reinforcement, functional additives in plastics,pesticides and phenol-formaldehyde resins production.3,4 Ligninswith high phenolic contents have antioxidant properties of

interest in the food and non-food sectors.5 When the total amountof free aliphatic alcohols is also high, lignin derivatives havethe potential for lignin–polyolefins composites production, asdispersants or compatibilizers.4,6

As the final application of lignin is dependent on both itschemical structure and extraction process from lignocellulosicsources, the development of fast and efficient physicochemicalcharacterization methods is a prerequisite to optimize extractionprocessing conditions. The formic acid/acetic acid/water processat atmospheric pressure was found effective for the delignificationof lignocellulosic raw materials, and especially for herbaceous

biomass.7–10 This acid process offered good separation of major

∗ Correspondence to: Aurore Richel, Department of Industrial BiologicalChemistry, Gembloux Agro-Bio Tech, University of Liege, Passage des Deportes2, B-5030 Gembloux, Belgium. Email:[email protected]

a Department of Industrial Biological Chemistry, Gembloux Agro-Bio Tech,University of Liege, Passage des Deportes 2, B-5030 Gembloux, Belgium

b Department of Applied Statistics, Computer Science and Mathematics,Gembloux Agro-Bio Tech, University of Liege, Passage des Deportes 2, B-5030Gembloux, Belgium

c Laboratory of Wood Technology, Direction of Forest Environment, Departmentof Natural and Agricultural Environment Studies, Public Service of Wallonia, Av.Marechal Juin 23, B-5030, Gembloux, Belgium

J Chem Technol Biotechnol 2014; 89: 128–136 www.soci.org c© 2013 Society of Chemical Industry

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Formic/acetic acid delignification of beech wood www.soci.org

lignocellulosic components (cellulose, hemicelluloses and lignin)by the hydrolysis and the subsequent dissolution of lignin andhemicellulose fragments in the ‘black liquor’.10 In this process,acetic acid acted as the dissolution solvent for hemicelluloses andlignin, while formic acid behaved as the catalyst for the cleavageof lignin–carbohydrate complexes.7 The possible recycling oforganic acids at the end of the process gave the opportunity of aneco-friendly strategy.9

In this study, formic acid/acetic acid/water treatment atatmospheric pressure was adapted to beech wood particles (Fagussylvatica L.). The effect of ‘cooking time and temperature’ on bothdelignification and chemical structure of lignins obtained afterprecipitation from the black liquor was evaluated. A two-factorcentral composite design and response surface methodology wereused for the optimization of treatment parameters. A relationbetween treatment parameters and physicochemical features ofextracted lignins was proposed.

MATERIAL AND METHODSRaw materialThe woody material used in this experiment was collected on 31January 2011. Three young beech trees (Fagus sylvatica L.) wereharvested in an oak stand with natural regeneration of beech andmaple. The stand was located in the extreme south of Belgium in aregion of the Gaume (49 ◦ 40.403’ N; 5 ◦ 19.372’ E) at an altitude of345 m. The relief is a plateau inclined slightly south. The maritimetemperate climate is characterized by a mean temperature of 16◦C in July and 0 ◦C in January. The substrate is clay with a loamtexture and moderate drainage.

The three trees had a DBH over bark of 14.3 cm at 1.3 mabove ground level. Two logs 1.3 m long were collected fromthe bottom of each tree and debarked manually. They were thencrushed to white chips of dimensions about 2.3 × 1.5 × 0.3cm. These were oven dried at 50 ◦C for 24 h to reduce theirmoisture content to less than 18% before being conditionedin a climatic chamber (22 ◦C and 65% RH) until constant massto stabilize at a final moisture content of 12 ± 2%. Chips wereground to 4 mm diameter particles (Tecator Cyclotech 1093) beforechemical extractions.

Compositional analysisRaw material (Fagus sylvatica L.) was analyzed for total solids,ashes, proteins, extractives, glucuronic acids, acetyl and methylgroups, monosaccharides and lignin content. Total solids andashes were determined gravimetrically.11,12 A Kjeldahl procedurewith a conversion factor of 6.25 was used for the determination ofprotein content.13 Water and ethanol extractives were assessed ina soxhlet apparatus by two successive extractions with water andethanol.14 Glucuronic acids were assayed according to the pro-cedure K-URONIC 03/11 developed by Megazyme after hydrolysis(6 h, 100 ◦C) of beech particles with sulfuric acid (2 mol L-1). Thedegree of acetylation and methylation was evaluated by highperformance liquid chromatography (HPLC) after saponificationof beech wood particles according to a published procedure.15

Monosaccharides composition was found by gas chromatographyafter hydrolysis and derivatization (reduction and acetylation) ofmonosaccharides in alditol acetates.16 Total lignin content talliedwith the sum of Klason lignin and acid soluble lignin. Klason ligninor acid insoluble lignin was determined gravimetrically whileacid soluble lignin was estimated spectrophotometrically.17 Allanalyses were performed at least in triplicate.

Formic/acetic acid treatmentA formic/acetic acid treatment of Fagus sylvatica L. was carried outaccording to the procedure presented by Vanderghem et al.10 Theexperimental domain of the two independent variables (time andtemperature) was chosen on the basis of preliminary experimentsand extended from 87 to 107 ◦C for cooking temperature andfrom 1 h 30 min to 4 h 30 min (abbreviated to 1h30–4h30from here on) for cooking time. Prior to the acid treatment atselected temperature and reaction time, beech wood particleswere soaked at 50 ◦C for 30 min in a formic acid/acetic acid/watermixture with a volume ratio 30/50/20. The liquid/dry mattermass ratio was 25/1. At the end of treatments, pulps andblack liquors were separated by filtration. Pulps were washedthree times with the formic/acetic acid mixture and then withdistilled water. Lignins were precipitated from black liquors byadding nine volumes of distilled water. Precipitates were washedby acidified distilled water (pH 2). Recovered pulps and ligninswere freeze-dried.

Characterization of pulps and black liquorsThe characterization of pulps and black liquors after treatmentswas done on the basis of total solids, total lignin content andmonosaccharides composition. The total solid residue was drieduntil constant weight prior to gravimetrical determination of pulpyield. The degradation product 2-furfural (2-F) was quantified inthe black liquors by modified methods.18,19 Typically, this protocolconsisted of the direct injection (20 µL) of black liquors in aHPLC system after filtration by a 0.45 µm filter. All analyses wereperformed at least in duplicate.

Experimental design and analysisOptimum values of the two independent variables – cookingtime and temperature – were identified by response surfacemethodology. The two factors were optimized for three responses:delignification, pulp yield and 2-F concentration in the black liquor.The model coefficients were estimated by carrying out a two-factorcentral composite design. This consisted of 13 randomized runsincluding five repetitions of the center point (0,0), four cube pointsat (1,1), (–1,1), (1,–1) and (−1,–1) and four axial points (α,0), (0,α),(−α,0) and (0,–α). The value of α was set to

√2, such that all

peripheral experimental points were equidistant from the centerpoint (0,0). Each coded level (−1,0,1) of the independent variablescorresponded with an experimental design level equivalent i.e.87, 97 and 107◦C for cooking time and to 1h30, 3h and 4h30 fortreatment temperature.

Multiple regressions through the least-square method wereperformed for data analysis. A second-order polynomial equationwas adjusted to the responses expressed as a function of theindependent variables:

Y = β0 +∑k

i=1βixi +

∑k

i=1βiix

2i +

∑k

i=1

∑k

j=i+1βijxixj (1)

where Y is the predicted response, k the number of independentvariables, xi and xj the coded independent variables, β0 theconstant coefficient, β i , β ii and β ij the coefficients of the linear,quadratic and interaction term.

Minitab 15 statistical software was used as statistical support forthe generation of the two-factor central composite design and thefitting of response surface models.

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www.soci.org M Simon et al.

Characterization of formic/acetic acid ligninsNuclear magnetic resonance (NMR) analyses were performedusing a Varian Unity 600 MHz spectrometer at 25 ◦C. Ligninsamples (75 mg) were dissolved in deuterated dimethyl sulfoxideprior to analyses. Adiabatic broadband {1H-13C} bidimensionalheteronuclear single quantum coherence (g-HSQC AD) experi-ments were conducted using a well-established protocol.19 Gelpermeation chromatography (GPC) was applied on acetylatedlignins using a HPLC system equipped with three Styragel columnsHR1, HR2 and HR3 and a differential refractometer connected inseries. Acetylation of lignins, essential to allow the dissolutionof samples in tetrahydrofuran (THF), was achieved according toa published protocol.20 The concentration of acetylated ligninsamples in THF amounted to 5 mg mL-1. The elution solvent wasTHF at a flow rate of 1 mL min-1. Polystyrene standards were usedfor the calibration curve construction.

RESULTS AND DISCUSSIONChemical composition of raw materialThe chemical composition of raw Fagus sylvatica L. is presented inTable 1. The amount of Klason lignin and acid soluble lignin was,respectively, 19.6% and 2.0%. These results were in agreementwith the conclusions of Dapia et al.21 (Klason lignin 23.7% andASL 2.7%) and with the lignin content reported by Kosikova et al.(22.3%),22 Demirbas (21.9%),23 and Popescu et al. (18–22%).24

Total monosaccharides represented 64.6% of total dry matterwith glucose (46.1%) and xylose (15.5%) as major constituents.The other residual monosaccharides were, in order of abundance,mannose (1.8%), galactose (0.8%) and arabinose (0.3%). Accordingto the amount of xylose, glucuronic acids (2.3%), acetyl groups(4.2%) and methyl groups (0.1%), beech wood hemicelluloseswere O-acetyl-4-O-methylglucuronoxylans. The backbone of thisxylan consisted of β-1,4-D-xylopyranosyl residues which weresubstituted by 4-O-methylglucuronic acid attached to the C2

position by α-1,2 linkages and by O-acetyl groups attachedto the C2 or C3 positions.25 The small amounts of mannoseconfirmed the presence of glucomannans, another hemicellulosefound in lower quantities than glucuronoxylans. Glucomannanswere composed of a linear backbone of β-D-mannopyranosyl andβ-D-glucopyranosyl residues linked by β-1,4 glycosidic bonds.26

Extractives corresponding mainly to tannins, resins and fatty acidsrepresented 4.3 and 0.9% of total dry matter, respectively, forwater and ethanol extractives. Proteins (0.6%) and ashes (0.4%)completed the beech wood composition.

Response surface design and modelsThe 13 runs of the experimental design and the results obtainedfor each response studied, namely delignification, pulp yield and2-F concentration, are presented in Table 2. Treatment 12 wasnot performed because the temperature of the cooking mixturecould not exceed 107 ◦C. Indeed, at atmospheric pressure, 107 ◦Ccorresponded to the boiling point of the mixture.

For each dependent variable, regression coefficients of thesecond-order polynomial equation were calculated on the basis ofexperimental data (Table 3). Coded variables were used during theanalysis of response surface design. In order to simplify the second-order polynomial equations, statistically non-significant modelterms at α = 0.05 – with a P-value higher than 0.05 – were removedfrom the model and a new adjustment of the parameters wasperformed. A Student t test and P-value were used for estimation

Table 1. Chemical composition of raw Fagus sylvatica L

Components Dry matter, %1

Lignin 21.6 ± 0.3

Klason lignin 19.6 ± 0.3

Acid soluble lignin 2.0 ± 0.0

Monosaccharides 64.6 ± 1.1

Glucose 46.1 ± 0.6

Xylose 15.5 ± 0.2

Mannose 1.8 ± 0.2

Galactose 0.8 ± 0.1

Arabinose 0.3 ± 0.0

Glucuronic acid 2.3 ± 0.2

Acetyl groups 4.2 ± 0.1

Methyl groups 0.1 ± 0.0

Extractives 5.1 ± 0.1

Water extractives 4.3 ± 0.1

Ethanol extractives 0.9 ± 0.0

Protein 0.6 ± 0.0

Ash 0.4 ± 0.0

a Each analysis was performed in triplicate.

Table 2. Experimental design and observed responses for the fourdependent variables

Independent variables Dependent variables

Run

Time

(h)

Temperature

( ◦C)

Delignification

(%)

Pulp yield

(%)

2-F

(ppm)

1 5h07 97 44.8 67.5 0.2

2 3h 83 12.3 87.3 0.2

3 3h 97 27.5 76.6 0.7

4 1h30 87 7.0 88.8 0.1

5 0h53 97 14.8 86.6 0.2

6 1h30 107 41.5 68.0 2.0

7 4h30 87 16.0 84.2 0.1

8 3h 97 30.0 79.7 0.3

9 3h 97 33.9 74.9 0.2

10 3h 97 23.1 80.0 0.3

11 3h 97 34.2 74.1 1.8

12 3h 111 * * *

13 4h30 107 75.2 55.3 20.0

of the significance (Table 3) of each regression coefficient. Themost significant regression term presents a high value of tand low value of P. Table 3 also shows for each second-orderpolynomial equation the P-value of lack-of-fit. A P-value for lack-of-fit higher than 0.05 indicates that the model adequately fitsthe data. Coefficients of multiple determination (R2) of 97.40,94.51 and 87.04%, respectively, for delignification, pulp yield and2-F concentration show that the second-order polynomial modelequations fit correctly with the experimental data.

Delignification was expressed as the ratio (%) between theamount of lignin removed from the pulp during the acidictreatment and the amount of lignin in the initial material. Briefly,delignification corresponded to lignin removal and rangedfrom 7.0 to 75.2% (Table 2). It was strongly affected by thetemperature. At 83–87 ◦C, delignification only reached values

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Table 3. Regression coefficients of the fitted second-order polynomials

Delignification Pulp yield 2-F production

Term

Regression

coefficient t P

Regression

coefficient t p

Regression

coefficient t P

Constant β0 29.47 21.49 0.000 77.12 77.74 0.000 0.67 0.72 0.496

Linear

time β1 10.63 8.06 0.000 −5.54 −5.81 0.000 2.25 2.49 0.042

temperature β2 22.71 13.52 0.000 −12.24 −10.08 0.000 5.82 5.07 0.001

Quadratic

temperature * temperature β22 6.49 3.58 0.009 −3.29 −2.51 0.037 4.38 3.53 0.010

Interaction

time * temperature β12 6.18 3.31 0.013 4.48 3.51 0.010

Lack-of-fit 0.916 0.507 0.002

between 7 and 16%. At a moderate temperature of 97 ◦C thedelignification was increased to between 15% and 34%. Furtherincrease in temperature led to significant increase in percentagedelignification, reaching values of 75% at 107 ◦C after 4h30.Equation (2) describes delignification as a function of cookingtime and temperature expressed in coded values:

Y1 = 29.47 + 10.63x1 + 22.71x2 + 6.49x22 + 6.18x1x2

(R2 = 97.40%, R2

adj = 95.92%)

(2)

where Y1 corresponds to delignification, x1 to the cooking timeand x2 to the cooking temperature.

Delignification was affected by both cooking time andtemperature. Increase of cooking time and temperature resulted ingreater delignification. Indeed, in more severe conditions, a largerpart of the lignin was dissolved in the black liquor. The Student ttest and P-value (Table 3), show that cooking temperature had alarger effect on delignification than treatment time.

Figure 1(a) presents a contour plot of the interaction betweencooking time and temperature on delignification. The contourplot indicates that maximum delignification was obtained forthe highest cooking time and temperature. Short time and hightemperature led to better delignification results than long timecombined with low cooking temperature.

The P-value for lack-of-fit (0.916) and the coefficient of multipleregression (97.40%) were evidence for the good fit of the modelto experimental data.

Pulp yield was defined as the ratio (%) between total dry matterin pulp after treatment and total dry matter in the raw material.The pulp yield followed an inverse trend to that of delignification(Table 2), and was inversely affected by temperature and cookingtime. According to the severity of treatment conditions, pulp yieldranged from 55.3 to 88.8%. Increasing temperature and cookingtime resulted in a lower pulp yield. A higher delignification resultedin a smaller pulp yield. The carbohydrates composition of the pulpsis presented in Table 4.

Pulp yield second-order polynomial equation is presented inEquation (3), with cooking time and temperature expressed incoded values.

Y2 = 77.12 − 5.55x1 − 12.24x2 − 3.29x22

(R2 = 94.51%, R2

adj = 92.46%)

(3)

where Y2 corresponds to pulp yield, x1 to the cooking time and x2

to the cooking temperature.The negative regression coefficients signify that cooking

time and temperature inversely affect pulp yield. Lower yieldswere obtained in more severe experimental conditions (highcooking time and temperature). Indeed, during a formic/aceticacid treatment, lignin and hemicelluloses (mainly xylans) werehydrolyzed and dissolved in the black liquor. The more severe thetreatment, the more the lignins are dissolved in the black liquorresulting in low pulp yields. The same conclusion is observed for

Figure 1. Effect of cooking time and temperature on (a) delignification, (b) pulp yield and (c) 2-F concentration.


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Table 4. Carbohydrates composition (% dry matter) of recoveredpulps

Run Pulp yield (%) Glucose (%) Xylose (%) Other sugars (%)

1 67.5 58.7 11.9 2.31

2 87.3 46.9 20.8 3.27

3 76.6 52.2 14.7 2.65

4 88.8 44.3 20.4 3.73

5 86.6 47.3 17.1 2.53

6 68.0 58.5 13.0 2.57

7 84.2 48.6 16.4 3.05

8 79.7 50.6 15.1 2.58

9 74.9 55.2 13.9 2.64

10 80.0 49.6 14.8 2.46

11 74.1 43.6 11.0 2.27

12 * * * *

13 55.3 73.9 9.4 2.00

hemicelluloses and is corroborated by lower xylose contents inthe pulps under severe treatment conditions (Table 4).

The Student t test and the P-value indicated that temperaturehad a stronger effect than cooking time (Table 3). The contourplot of pulp yield (Figure 1(b)) revealed that optimal pulpyield (higher than 90%) was achieved when cooking timeand temperature were below 2h15 and 92 ◦C, respectively.Moreover, higher pulp yields were obtained when the treatmenttime was long and the temperature was low, rather thanconversely. Pulp yield was inversely proportional to delignification:the higher the delignification, the lower the pulp yield.Pulp yield was an interesting dependent variable to proxycellulose enrichment of pulps during treatments in comparisonwith raw material. In severe treatments, pulp yield wasminimized but the solid fraction obtained was richer incellulose as indicated by the total glucose content of about74% (Table 4).

Analysis of the P-value of lack-of-fit (0.507) and the coefficientof multiple regression (94.51%) confirmed that pulp yieldexperimental data were correctly expressed by the second-orderpolynomial model (Table 3). The degradation products of pentoseswere 2-F and 5-hydroxymethylfurfural (5-HMF). However, in thisstudy, only 2-F concentration was taken into account becauseduring acidic treatment 5-HMF was produced but also degradedinto 2-F.27 Table 2 indicates that 2-F concentration ranged from0.1 to 20.0 ppm in the black liquors. The highest amounts of 2-F(20.0 ppm) were obtained during treatment 13 (107 ◦C, 4h30).Experimental data concerning the concentration of 2-F in theblack liquor assayed by HPLC were used for calculation of theregression coefficients of Equation (4) expressing 2-F as a function

of independent variables, both in coded values.

Y3 = 0.67 + 2.25x1 + 5.82x2 + 4.38x22 + 4.48x2

2(

R2 = 87.04%, R2adj = 79.64%

)(4)

where Y3 correspond to 2-F concentration, x1 to the cooking timeand x2 to the cooking temperature.

2-F production was influenced by both cooking time andtemperature. When the temperature ranged between 83 and97 ◦C the 2-F concentrations were very low (0.1–1.8 ppm). Instronger conditions, the hemicelluloses hydrolysis increased andat the same time the amount of 2-F produced was augmented inthe black liquor. The largest concentration of 2-F (20 ppm) wasobtained at 107 ◦C after 4h30. By analysis of the Student t test andthe P-value for each regression coefficient of the 2-F second-orderpolynomial equation, 2-F production depended mainly on thecooking temperature and to less extent on the cooking treatmenttime (Table 3). The contour plot of 2-F (Fig. 1(c)) revealed that overa large part of the surface, 2-F concentration stayed below 10 ppm.At long times and high temperatures such as during treatment 13(107 ◦C, 4h30), 2-F production higher than 10 ppm was detected.However, according to the P-value of lack-of-fit (0.002), i.e. lessthan 0.050, the second-order polynomial model adjusted for 2-Fconcentration would not accurately fit the experimental data.

According to the response surface of delignification, ligninextraction was maximized at the highest cooking time andtemperature. Given that 107 ◦C was the maximum cookingtemperature allowed at atmospheric pressure, lignin extractionwas optimal at 107 ◦C for at least 4h30 (75.2% delignification yield).

In the context of an integrated biorefinery that recycles ligninbut also transforms hemicelluloses and cellulose (notably forthe production of bioethanol through fermentation processes),the optimization of the treatment conditions has to considerdelignification and pulp yield. In this way, multi-criteriaresponse optimization was used for the identification oftreatment conditions (cooking time and temperature) thatmaximized delignification and pulp yield. For the maximization ofdelignification and pulp yield, the target value for optimizationwas 100% but lower boundaries had also to be found and were setrespectively at 70% and 50%. According to these optimizationcriteria, a global solution was found and corresponded to aformic/acetic acid treatment at 104.2 ◦C for 5h07. The predictedresponses for this treatment condition were 70.5% delignificationand 58.7% pulp yield. Experimental values under these conditionswere 66.2% for delignification and 55.9% for pulp yield, close to thepredicted values and confirming the model equations. Under thesetreatment conditions, 2-F production reached an unexpectedvalue of 30.8 ppm, confirming that the second-order polynomialmodel adjusted for 2-F concentration was not satisfactory.

Table 5. Klason lignin, carbohydrates composition and molecular weights (Mp) of selected formic/acetic acid lignins

Lignin sample Experimental run order Klason lignin (%) Glucose (%) Xylose (%) Other sugars (%) Total sugars (%) Mp

87 ◦C 1h30 4 80.5 11.8 0.6 1.5 13.9 1200

87 ◦C 4h30 7 90.5 4.1 1.9 0.5 6.5 800

97 ◦C 3h 3 93.3 0.7 3.1 0.9 4.7 1000

107 ◦C 1h30 6 93.7 3.1 1.0 0.4 4.5 1400

107 ◦C 4h30 13 90.7 0.5 1.6 0.3 2.4 1100


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Figure 2. Expanded aromatic region (a) and expanded oxygenated aliphatic region (b) of HSQC NMR spectra of lignins from treatment 4 (87 ◦C, 1h30)and treatment 13 (107 ◦C, 4h30). By convention, CH and CH3 peaks appeared in blue and CH2 in red. Peak labels refer to Table 5.

Chemical and structural characterization of formic/acetic acidligninsFor physicochemical and structural characterization offormic/acetic acid lignins, five representative lignins of theexperimental design were chosen: lignins from treatment 4 (87 ◦C,1h30), treatment 7 (87 ◦C, 4h30), treatment 3 (97 ◦C, 3h), treatment6 (107 ◦C, 1h30) and treatment 13 (107 ◦C, 4h30) (Table 2). The aimof this characterization was to study the influence of treatmentconditions on the physicochemical and chemical structure ofextracted lignins and not only to elucidate the structure of aformic/acetic acid wood lignin.

The purity of the extracted lignins was assessed by the Klasonprocedure and the monosaccharides contents (Table 5). The Klasonlignin trends were similar for the four lignins obtained with theharsher treatments whereas the lignin recovered from treatment4 (87 ◦C, 1h30) had lower lignin purity (80.5%). Monosaccharidescomposition revealed that the amount of carbohydrates in thislignin was the highest (13.9%) with glucose as the main constituent.These results suggest that cellulose was linked to lignins by bonds

resistant to formic/acetic acid treatment under soft experimentalconditions.28 Another explanation is imprisonment of cellulosechains during lignin precipitation that led to their aggregationwith lignin molecules. Xylose content ranged from 0.6 to 3.1% andindicated that part of the hemicelluloses (glucuronoxylans)-ligninlinkages seemed to be resistant to formic/acetic acid treatment.Other monosaccharides (mannose, galactose and arabinose) werepresent in small amounts.

Heteronuclear single quantum coherence (HSQC) NMR gives anaccurate description of the composition of the different lignin frac-tions such as aromatic rings, inter-unit bonds and polysaccharidescontamination. Figure 2 shows HSQC NMR spectra of lignins fromtreatment 4 (87 ◦C, 1h30) and treatment 13 (107 ◦C, 4h30). HSQCNMR spectra of lignins recovered from treatment 7 (87 ◦C, 4h30),treatment 3 (97 ◦C, 3h) and treatment 6 (107 ◦C, 1h30) were notcompiled in Fig. 2 but presented a similar profile to lignin fromtreatment 13 (107 ◦C, 4h30). Three different regions could be distin-guished in these HSQC NMR spectra: the aromatic region (δ13C/δ1H


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Figure 3. Main structural patterns elucidated by HSQC NMR experiments: gauaicyl unit (G), syringyl unit (S), oxidized (Cα=O) phenolic syringyl unit (S′),β-O-4’ substructure (A), γ -acetylated β-O-4’ substructure (A′) and resinol substructure (R).

Table 6. General signal assignment for selected formic/acetic acid lignins HSQC NMR spectra

Labels δ13C (ppm) δ1H (ppm) Assignment*

1 119.3 6.73 C6-H6 in guaiacyl units (G)




5 107.4 6.44 n.a**

6 107.1 7.29 C2-H2 and C6-H6 in oxidized (Cα=O) phenolic syringyl units (S’)

7 106.6 6.39 n.a

8 104.1 6.63 C2-H2 and C6-H6 in etherified syringyl units (S)

9 86.5 4.09 Cβ -Hβ in β-O-4’ linked to a syringyl unit (A)

10 85.5 4.63 Cα-Hα in resinol substructures (R)

11 83.7 4.28 Cβ -Hβ in β-O-4’ linked to a guaiacyl unit (A)Cβ -Hβ in γ -acetylated β-O-4’ linked to a syringyl unit (A’)

73.6 3.62 Carbohydrate contamination





12 72.3 4.82 Cα-Hα in β-O-4’ linked to a syringyl unit (A)Cα-Hα in γ -acetylated β-O-4’ substructures (A’)

13 71.7 4.71 Cα-Hα in β-O-4’ linked to a guaiacyl unit (A)

14 71.7 4.15 Cγ -Hγ in resinol substructures (R)

15 71.6 3.77 Cγ -Hγ in resinol substructures (R)

16 67.6 4.12 n.a

17 66.2 4.01 n.a

18 63.3 4.27 Cγ -Hγ in γ -acetylated β-O-4’ substructures (A’)

19 61.3 3.42 Cγ -Hγ in β-O-4’ substructures (A)

20 60.7 3.59 Cγ -Hγ in β-O-4’ substructures (A)

21 61.1-59.2 3.85-3.04 n.a

22 56.2 3.69 C-H in methoxyls

23 54.1 3.02 Cβ -Hβ in resinol substrucures (R)

*Assignment according to Refs 17,29,30**n.a denotes not assigned.


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90-125/5.5-8.5) (Fig. 3(a)), the side-chain region (δ13C/δ1H 50-90/2.5-5.5) (Fig. 2(b)) and the aliphatic region (δ13C/δ1H 10-50/0.5-2.5).29 The aromatic region and the oxygenated aliphatic side-chainregion of HSQC spectra provided accurate information about thestructural composition (constitutive units and linkages) of ligninsamples by the correlation between 1H and 13C chemical shifts. Theassignment of δ13C/δ1H cross-signals is presented in Table 6.29,30

The main structural patterns observed in HSQC experiments areproposed in Fig. 3. By convention, the aromatic carbons werenumbered from 1 to 6 and the aliphatic carbons from α to γ . Inthe aromatic region of lignin HSQC spectra (Fig. 2(a)), typical gua-iacyl ring cross-signals (G) were observed at δ13C/δ1H 119.3/6.73,115.5/6.68-6.94 and 111.5/6.93 ppm and were attributed to C6 –H6,C5 –H5 and C2 –H2 correlations. The double C5 –H5 signal indicateda certain heterogeneity of guaiacyl units, probably due to the dif-ferent possibilities of substitutions at C4.30 The strong cross-signalat 104.1/6.63 ppm corresponded to C2 –H2 and C6 –H6 correla-tions in etherified syringyl units (S). The appearance of a signalat 107.1/7.29 ppm corresponding to C2 –H2 and C6 –H6 in oxi-dized (Cα=O) phenolic syringyl units (S’) was observed whencooking time and temperature were increased. The advent ofthe oxidized syringyl unit constituted a depolymerization signof lignin in smaller residues by the action of the formic/aceticacid mixture. No signals corresponding to p-hydroxyphenyl unitswere reported. These results agreed with literature reporting thatbeech wood lignin contained mainly guaiacyl and syringyl unitsin equal proportions.2 The oxygenated aliphatic side-chain region(Fig. 2(b)) was used for the determination of the different linkagesimplicated between the constitutive units. Two different linkageswere identified: the β-O-4’ (A) and the resinol (R) substructureswith typical cross-signals listed in Table 5. The stronger signal of theoxygenated aliphatic side-chain region observed at 56.2/3.69 ppmwas attributed to C–H in methoxyls groups (−OCH3) substitutedto the aromatic group of guaiacyl and syringyl units.19 Finally, thelignin from treatment 4 (87 ◦C, 1h30) (Fig. 2(b)) presented originalcross-signals in the region of 70.6–73.6/3.02–3.62 ppm assignedto carbohydrates.

GPC performed on acetylated lignins dissolved in THF alloweddetermination of the molecular weight of the lignin fragmentsafter isolation processes. The molecular weight is an attributefor the development of lignin-based products. Lignin molecularweights (Mp), corresponding to the molecular weight of thehighest peak of gel permeation chromatograms, were expressedin polystyrene equivalents and ranged from 800 to 1400(Table 5). Cooking time and cooking temperature influencedthe molecular weight differently. At equal cooking temperatures,lignin molecular weight decreased with increasing treatmenttime whereas at equal cooking times, the molecular weightincreased with increasing temperature. Cooking time could actas a depolymerizator while cooking temperature increased thelignin molecular weight by lignin repolymerization occurringduring the formic/acetic acid treatment.31 Indeed, under acidicconditions, two antagonist reactions occurred. The first possesseda depolymerization character due to the acidic cleavage of theβ-O-4′ linkages. The second reaction presented a repolymerizarionaction by the acid-catalyzed condensation of an aromatic C5 orC6 and a carbonium ion.19,31 Gel permeation chromatographywas an interesting tool to investigate the molecular weights ofselected formic/acetic acid lignins. However, acetylation could beinvasive and chromatographic analyses were done only on thesolubilized fractions and gave no information about the molecularconformation.

CONCLUSIONSThe adapted formic/acetic acid treatment of beech wood particlespresented good potential for lignin extraction. Response surfacemethodology indicated that cooking temperature had the majoreffect on delignification, pulp yield and 2-F concentration. Thebest delignification was obtained at the highest cooking times andtemperatures. However, allowing that an integrated biorefineryalso recycled all the by-products, a formic/acetic acid treatmentat 104.2 ◦C for 5h07 was found by multi-criteria optimization ofdelignification. Under these conditions, delignification reached70.5%, pulp yield 58.7% and 2-F concentration 14.9 ppm.

Concerning the chemical composition and the structure ofextracted lignins, low cooking time and temperature decreasedthe purity of lignin because of the resistance of cellulose–ligninlinkages or because of the imprisonment of cellulose duringlignin precipitation. Lignin depolymerization in acidic conditionswas structurally identified by the appearance of an oxidized(Cα=O) phenolic syringyl unit when cooking time and temperatureincreased. Finally, molecular weights revealed the existenceof lignin depolymerization and repolymerization during theformic/acetic acid treatment by the respective actions of cookingtime and temperature.

ACKNOWLEDGEMENTSThe authors thank the ‘Fonds pour la Recherche scientifique dansl’Industrie et l’Agriculture’ (FRIA) for a fellowship to MathildeSimon, and the Walloon Region (Technose excellence researchProgram, project number 716757). Professor Jacques Hebert andMrs N. Lemoine, Department of Forestry and Nature, are thankedfor providing the woody material. Mr Mario Aguedo is thankedfor his guidance in lignin molecular weight determinations. MsVirginie Byttebier and Mrs Lynn Doran are thanked for technicalassistance.

REFERENCES1 Boerjan W, Ralph J and Baucher M, Lignin biosynthesis. Ann Rev Plant

Biol 54:519–546 (2003).2 Zakzeski J, Bruijnincx PCA, Jongerius AL and Weckhuysen BM, The

catalytic valorization of lignin for the production of renewablechemicals. Chem Rev 110:3552–3599 (2010).

3 Boeriu CG, Bravo D, Gosselink RJA and van Dam JEG, Characterizationof structure-dependent functional properties of lignin with infraredspectroscopy. Ind Crops Prod 20:205–218 (2004).

4 Gosselink RJA, Snijder MHB, Kranenbarg A, Keijsers ERP, de Jong E andStigsson LL, Characterization and application of NovaFiber lignin.Ind Crops Prod 20:191–203 (2004).

5 Vinardell MP, Ugartondo V and Mitjans M, Potential applicationsof antioxidant lignins from different sources. Ind Crops Prod27:220–223 (2008).

6 Kosıkova B, Demianova V and Kacurakova M, Sulfur-free lignins ascomposites of polypropylene films. J Appl Polym Sci 47:1065–1073(1993).

7 Lam HQ, Le Bigot Y, Delmas M and Avignon G, A new procedurefor the destructuring of vegetable matte rat atmospheric pressureby a catalyst/solvent system of formic/acetic acid. Applied to thepulping of triticale straw. Ind Crops Prod 14:139–144 (2001).

8 Delmas GH, La biolignineTM: structure et application a l’elaboration deresines epoxy. PhD Thesis, University of Toulouse, Toulouse (2011).

9 Delmas GH, Benjelloun-Mlayah B, Le Bigot Y and Delmas M,Functionality of wheat straw lignin extracted in organic acid media.J Appl Polym Sci 121:491–501 (2011).

10 Vanderghem C, Brostaux Y, Jacquet N, Blecker C and Paquot M,Optimization of formic/acetic acid delignification of miscanthus× giganteus for enzymatic hydrolysis using response surfacemethodology. Ind Crops Prod 35:280–286 (2012).


13

6


11 Sluiter A, Hames B, Ruiz R, Scarlata C, Sluiter J and TempletonD, Determination of ash in biomass. NREL Laboratory AnalyticalProcedure (LAP) (2005).

12 Sluiter A, Hames B, Hyman D, Payne C, Ruiz R, Scarlata C, Sluiter J,Templeton D and Wolfe J, Determination of total solids in biomassand total dissolved solids in liquid process samples. NREL LaboratoryAnalytical Procedure (LAP) (2008).

13 Hames B, Scarlata C and Sluiter A, Determination of protein content inbiomass. NREL Laboratory Analytical Procedure (LAP) (2008).

14 Sluiter A, Ruiz R, Scarlata C, Sluiter J and Templeton D, Determinationof extractives in biomass. NREL Laboratory Analytical Procedure(LAP) (2005).

15 Voragen AGJ, Schols HA and Pilnik W, Determination of the degree ofmethylation and acetylation by h.p.l.c. Food Hydrocolloid 1:65–70(1986).

16 Blakeney AB, Harris PJ, Henry RJ and Stone BA, A simple andrapid preparation of alditol acetates for monosaccharides analysis.Carbohydr Res 113:291–299 (1983).

17 Sluiter A, Hames B, Ruiz R, Scarlata C, Sluiter J, Templeton D andCrocker D, Determination of structural carbohydrates and lignin inbiomass. NREL Laboratory Analytical Procedure (LAP) (2008).

18 Zappala M, Fallico B, Arena E and Verzera A, Methods for thedetermination of HMF in honey: a comparison. Food Control16:273–277 (2005).

19 Vanderghem C, Richel A, Jacquet N, Blecker C and Paquot M,Impact of a formic/acetic acid and ammonia pre-treatments onchemical structure and physico-chemical properties of Miscanthus× giganteus lignins. Polym Degrad Stabil 96:1761–1770 (2011).

20 Huang F, Singh PM and Ragauskas AJ, Characterization of milled woodlignin (MWL) in loblolly pine stem wood, residue and bark. J AgricFood Chem 59:12910–12916 (2011).

21 Dapia S, Santos V and Parajo JC, Study of formic acid as an agent forbiomass fractionation. Biomass Bioenergy 22:213–221 (2002).

22 Kosikova B, Hricovini M and Cosentino C, Interaction of lignin andpolysaccharides in beech wood (Fagus sylvatica) during dryingprocesses. Wood Sci Technol 33:373–380 (1999).

23 Demirbas A, Relationships between lignin contents and fixedcarbon contents of biomass samples. Energy Convers Manage44:1481–1486 (2003).

24 Popescu MC, Popescu CM, Lisa G and Sakata Y, Evaluation ofmorphological and chemical aspects of different wood speciesby spectroscopy and thermal methods. J Mol Struct 988:65–72(2011).

25 Teleman A, Tenkanen M, Jacobs A and Dahlman O, Characterizationof O-acetyl-(4-O-methylglucurono)xylan isolated from birch andbeech. Carbohydr Res 337:373–377 (2002).

26 Girio FM, Fonseca C, Carvalheiro F, Duarte LC, Marques S and Bogel-Lukasik R, Hemicelluloses for fuel ethanol: a review. BioresourceTechnol 101:4775–4800 (2010).

27 Shen DK and Gu S, The mechanism for thermal decomposition ofcellulose and its main products. Bioresource Technol 100:6496–6504(2009).

28 Jin Z, Katsumata KS, Lam TBT and Iiyama K, Covalent linkages betweencellulose and lignin in cell walls of coniferous and nonconiferouswoods. Biopolymers 83:103–110 (2006).

29 Rencoret J, Marques G, Gutierrez A, Ibarra D, Li J, Gellerstedt G,Santos I, Jimenez-Barbero J, Martinez AT and del Rio JC, Structuralcharacterization of milled wood lignins from different eucalyptspecies. Holzforschung 62:514–526 (2008).

30 Yuan TQ, Sun SN, Xu F and Sun RC, Structural characterization oflignin from triploid of Populus tomentosa Carr. J Agric Food Chem59:6605–6615 (2011).

31 Li J, Henriksson G and Gellerstedt G, Lignin depolymerisa-tion/repolymerisation and its critical role for delignification ofaspen wood by steam explosion. Bioresource Technol 98:3061–3068(2007).


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Optimization of a formic/acetic acid delignification treatment on beech wood and its influence on the structural characteristics of the extracted lignins