Comprehension of an organosolv process for lignin extraction on Festuca arundinacea and monitoring of the cellulose degradation

Industrial Crops and Products 94 (2016) 308–317

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Comprehension of an organosolv process for lignin extraction on Festuca arundinacea and monitoring of the cellulose degradation Quentin Schmetz a,∗ , Guillaume Maniet a , Nicolas Jacquet a , Hiroshi Teramura b , Chiaki Ogino b , Akihiko Kondo b , Aurore Richel a a

Unit of Biological and Industrial Chemistry, Gembloux Agro-Bio Tech—University of Liege, Passage des Déportés, 2-B-5030 Gembloux, Belgium Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodaicho, Nada-ku, Kobe, Hyogo 657-8501, Japan b

a r t i c l e

i n f o

Article history: Received 21 May 2016 Received in revised form 29 August 2016 Accepted 1 September 2016 Keywords: Biorefining Lignin Ethanol Organosolv Tall fescue

a b s t r a c t It is commonly accepted that the current society needs to partially substitute fossil resources by renewable ones. Among many solutions, one approach consists in the development of biorefinery involving lignocellulosic biomass to produce bio-based materials and fuels. This study focuses on the comprehension of an organosolv treatment designed to break the complex lignocellulosic structure for high purity lignin extraction from tall fescue (Festuca arundinacea Schreb.). This grass benefits from an increasing interest in Western Europe and has been suggested as feedstock for biorefinery. However, its use as material for high purity lignin production has not been determined yet. Ethanol/water, 92/8% [v/v] with H2 SO4 0.32 M was investigated at pilot scale under conventional heating (5 ◦ C min−1 during 30 min and stabilized at 148 ◦ C for 5 min). Precipitated lignin were analyzed as well as the composition of sidestream products (recovered cellulosic pulp and the aqueous hydrolysate). Lignin has been recovered at a purity level of 90% with a yield of 60%. The main contaminants were nitrogen containing compounds and degraded hemicelluloses. 2D-HSQC NMR (Two Dimension-Heteronuclear Single Quantum Correlation Nuclear Magnetic Resonance) revealed a co-extraction of ferulates and coumarates function as well as arabinoxylan. Cellulose was recovered at 53% purity with 60% yield. The conditions appear to be too harsh for tall fescue and led to significant amount of cellulose degradation. A process using a lower alcohol concentration will be developed to provide better yields of both cellulose and lignin. © 2016 Published by Elsevier B.V.

1. Introduction Nowadays, the global environmental and economical context incites industries and scientists all over the world to look for cleaner energies and greener chemistry and processes as alternatives to the oil-based economy (Bennett and Pearson, 2009). The global warming, the depletion of fossil fuel and the highenergy consumption by a continuously growing population are many problems to deal with (Kajaste, 2014). One solution is to substitute fossil energies by renewable resources based on lignocellulosic biomass. The main concerned fields are the production of energy (biofuels), molecules (fine chemistry, pharmaceuticals), and materials (i.e. composites) from whole plants. It contributes to the development of biorefineries producing such bio-based products and biofuels using new “greener” processes (Bennett and Pearson,

∗ Corresponding author. E-mail address: [email protected] (Q. Schmetz). http://dx.doi.org/10.1016/j.indcrop.2016.09.003 0926-6690/© 2016 Published by Elsevier B.V.

2009; Octave and Thomas, 2009). The main advantages of lignocellulose as feedstock are its low cost, its abundance, its renewability, and its non-competition with the food industry. The chemical products need low volumes to be produced with a high added-value unlike biofuels such as bioethanol which require larger amounts of biomass and for which the productivity is limited by the availability of feedstock in some geographic areas. (FitzPatrick et al., 2010). The region of Wallonia in Belgium suffers from a limited availability of mobilized lignocellulosic supplies. One solution to deal with the increasing demand for biobased products is to focus more on niche markets relying on relatively low quantity of biomass to produce molecules with high added value (Jacquet et al., 2015). Tall fescue (Festuca arundinacea Schreb.), a cool-season perennial grass widely used as forage, has been investigated as biomass feedstock for biorefinery (Butkute et al., 2014; Godin et al., 2010). This grass beneficiates from a good yield (8.4–14.1 Tons/ha) and reasonable establishment costs (527–600 D /ha) (Danielewickz et al., 2015) in addition to a strong resistance to multiple cutting and grazing. Because of its drought resistance, tall fescue could be used

Q. Schmetz et al. / Industrial Crops and Products 94 (2016) 308–317

as feedstock for biorefinery in Western Europe where dry summer are expected to be more frequent as a result of climate change (Cougnon et al., 2013). The present process is an organosolv process of which the main advantage is the capacity to recover both the solvent by distillation and the lignin by precipitation as a solid material. In addition, it allows the recovery of hydrolyzed hemicelluloses (in the aqueous fraction) and a relatively pure cellulose fraction as solid residue. All these available feedstock chemicals make this process promising for biorefinery. Optimizations and reasoned utilizations of these by-products are therefore required (Zhao et al., 2009). The lignin removal relies on dissolution in organic solvent of lignin macromolecules after chemical breakdown. The hydrolysis of ether linkages between carbohydrates and lignin facilitates the extraction (McDonough, 1992). In herbaceous plants, ferulic acid links lignin and polysaccharides via ether and ester bonds respectively. It is therefore almost impossible to extract lignin in pure form. When using dilute acid, ether bonds are cleaved between lignin and ferulic acid (Buranov and Mazza, 2008). The lignin structure suggests an interesting potential as a new feedstock for aromatic chemicals and for the formation of supramolecular materials (Finch et al., 2012). The use of low value lignin such as sulfonated lignin for production of chemicals has been limited by numerous contaminations (salts, carbohydrates) and the higher molecular weights. Lignin is viewed as a mid-long term opportunity as renewable raw material in added value compounds production. (Rochez et al., 2013). The main applications of lignin are divided into three groups. Firstly, small aromatic compounds such as benzene, toluene and xylene (B, T, X) can be produced after fragmentation. As macromolecule, lignin can be used as additive (adhesives) or polymer blend (polyurethane foam). Finally its use as carbon-rich materials for applications such as carbon fibers production has been developed (Hodásová et al., 2015; Van Haveren et al., 2007). These applications depending on the quality and the efficiency of catalytic conversion of extracted lignin, it is therefore important to improve both extraction and catalytic pathways to valorize their whole potential (Zakzeski et al., 2010). The potential of tall fescue as material for high purity lignin production has not yet been determined. In this study an organosolv process, optimized on herbaceous plant to extract lignin, was investigated on tall fescue under conventional heating at pilot scale to assess the industrial viability.

2. Materials and methods 2.1. Raw material A Kora (endophyte free cultivar) tall fescue (Festuca arundinacea Schreb.) was used throughout this study. This cool season grass is a perennial crop. An experimental field from Gembloux (Belgium) was seeded at the end of August 2008. The field was harvested three times a year (at the end of spring, the end of summer and the end of autumn). The three samples were dried during three days at 60 ◦ C, homogenized and conditioned in bags to be stored at room temperature. The grounding was performed prior to analyses using a Fritsch PULVERISETTE 19 equipped with a sieve cassette of 0.75 mm. The biomass was further ground using an IKA WERKE M20 for composition analyses only.

2.2. Methods 2.2.1. Biomass and fractions characterization All analyses were performed in triplicate. Moisture content was quantified based on the NREL method (Ehrman, 1994). 1 g of sample was dried in oven (Memmert SNB 100) during 24 h at 105 ◦ C and moisture was calculated from mass

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loss. Dry matter in liquid fraction was determined by the same method on 20 mL. Ash content was determined by gravimetric method, as the solid remaining after combustion during 6 h (2 h rising to reach a plateau for 4 h) at 575 ◦ C in muffle furnace (Nabertherm controller b180) based on the NREL method (Sluiter et al., 2005). Extractives quantification using Soxhlet apparatus was carried out according to the NREL method (Sluiter et al., 2008). A first extraction was performed on 1.5 g raw biomass during 6 h using water as solvent. A second extraction was then performed on the exhausted solid using ethanol during 12 h. Water-soluble compounds and ethanol-soluble compounds were quantified gravimetrically. Protein content was estimated by measuring the nitrogen content according to Kjeldahl method. Mineralization was performed using a TECATOR 1015 instrument followed by a titration using a Kjeltec 2300 (Foss). The results were multiplied by 6.25, the conversion factor which is used for animal feeds and other materials (Tkachuk, 1997). Total sugar content was determined, including free monosaccharides, non-cellulosic polysaccharides and cellulosic fraction. Free monosaccharides were extracted by stirring the solid fraction in water at 50 ◦ C during 30 min. Non cellulosic polysaccharides were converted into monosaccharides by chemical hydrolysis using H2 SO4 1 M at 100 ◦ C during 3 h. Hydrolysis of cellulose was performed in two steps. A first hydrolysis was carried out at 30 ◦ C during 1 h using H2 SO4 72%. The acid was then diluted to reach a concentration of 1 M to perform the second hydrolysis at 100 ◦ C during 6 h. Monosaccharides were derivatized by 1-methylimidazole and acetic anhydrid into alditol acetates prior to gas chromatography analysis as described in (Blakeney et al., 1983). Analyses were carried out using an Agilent (7890 B series) gas chromatograph. The components were separated using a capillary column, HP1-methylsiloxane (30 m × 0.32 mm ID, 0.25 ␮m film thickness). Helium was used as carrier gas at a flow rate of 1.6 mL min−1 . Injection chamber was set at 290 ◦ C, and the temperature program was 1 min at 120 ◦ C, followed by a linear increase in 4 min to 220 ◦ C and then in 35 min to 290 ◦ C which was maintained for 4 min. Compounds were detected using a flame ionization detector at 320 ◦ C. Data were analyzed using OpenLab ChemStation software. Glucose, xylose, arabinose, mannose, rhamnose, galactose and 2-deoxyglucose (internal standard) were purchased at Sigma-Aldrich (St.-Louis, USA). The anhydro correction factors (0.88 for pentoses and 0.90 for hexoses) were applied to the monomeric sugars concentrations in order to obtain the concentration of the corresponding polymeric sugars. Qualitative determination of derivative sugars was performed using a HP 6890 Series GC system equipped with a HP 7630 series injector and Electronic Impact—Mass Sprectrometry (EI—MS) detector. The separation was performed according to the same temperature program on a VF-5MS (30 m × 0.25 mm ID, 0.25 ␮m film thickness) column. Total Starch content was determined by the method AOAC Method 996.11 using a Megazyme kit: Total starch assay procedure (amyloglucosidase/␣–amylase method). Klason Lignin, the acid-insoluble lignin content, was determined according to the protocol described in (Sluiter et al., 2011). The acid-soluble lignin was measured by spectrophotometry using a UV-1800 Shimadzu spectrophotometer at 205 nm as described in the method (TAPPI UM 250, 1991) in order to minimize interferences from the generated degradation products (Korpinen et al., 2014). 5-Hydroxymethylfurfural (5-HMF) and 2-Furfural, the liquid fractions recovered after precipitation of lignin in the black liquor were analyzed by HPLC using a Waters 2695 Separations module equipped with a UV Detector (Waters 2487 Dual ␭ Absorbance). Separation was carried out on an Agilent Zorbax

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Q. Schmetz et al. / Industrial Crops and Products 94 (2016) 308–317 Table 1 Chemical composition of raw tall fescue.

Fig. 1. Recorded internal reactor temperature during experiment 1 and 2.

Componentsa

Weight% dry basis

Lignin Klason lignin Acid soluble lignin Polysaccharides Glucan Xylan Mannan Galactan Arabinan Rhamnan Extractives Water extractives Ethanol extractives Protein Ash Total

19.4 ± 0.3 15.2 ± 0.1 4.2 ± 0.2 46.5 ± 1.3 31.3 ± 0.1 11.2 ± 0.8 0.5 ± 0.0 1.2 ± 0.1 2.1 ± 0.3 0.2 ± 0.0 17.7 ± 1.7 13.8 ± 1.2 3.8 ± 0.5 7.3 ± 0.1 11.5 ± 0.4 102.4 ± 3.8

a

300SB-C18 (4.6 mm ID × 150 mm, 3.5 ␮m particle size) column at 30 ◦ C at a flow rate of 1 mL min−1 . Data were processed using Empower Pro software. 2D HSQC NMR (Two dimensional Heteronuclear Single Quantum Correlation—Nuclear Magnetic Resonance) analysis, 2 g of sample were milled prior to the analysis using a Retsch PM 100 planetary ball milling loaded with 10 zirconium oxide balls (diam. 10 mm). Milling was performed 5 min alternately with 5 min break during 45 min at 600 rpm 30 mg of sample were then soaked in 600 ␮L DMSO/150 ␮L pyridine. Alternation of 10 min vortexing and 10 min sonication was performed to improve lignin solubilization. NMR analysis was conducted using a Bruker AVIIIHD (400 MHz). Molecular weight distribution measurements of lignin were performed by Size Exclusion Chromatography (SEC) on a Waters 2690 separations module equipped with a UV detector (Waters 996 photo diode array detector) and a TSKgel G3000PWXL column 200 Å (7 and 6 ␮m particle size). NaH2 PO4 ·H2 O/NaOH buffer (pH 12) was used as eluent at 0.9 mL min−1 . Sodium polystyrenesulfonates standard samples served for calibration (210 Da, 4.3 kDa, 6.8 kDa, 10 kDa, 17 kDa and 32 kDa). ProtobindTM 1000 lignin (Alm India Pvt ltd) as well as lignin from beech wood and corn stover extracted by the same process were analyzed for comparison. The molecular masses were determined based on the elution time of the UV detector signal at 280 nm at 30 ◦ C. Lignin samples were put in solution at 3 mg mL−1 in eluent by stirring during 48 h and then filtered with 0.45 ␮m filters. 2.2.2. Organosolv treatment Tall fescue was treated according to the protocol described by (Monteil-Rivera et al., 2012) with some modifications. The whole following process was performed in duplicate. 100 g of dried tall fescue were mixed with 2 L of ethanol/water: 92/8% [v/v] with H2 SO4 0.32 M and heated at 5 ◦ C min−1 until reaching a stable temperature of 148 ◦ C as shown on Fig. 1. This set point temperature was maintained during 5 min. It was performed in a stainless steel pressure reactor (7.5 L, Parr Series 4552 HT, Parr Instrument Co), equipped with a 6-blades impeller and a controller system (model 4848 Modular Controller, Parr Instrument Co). The medium was let to cool down at room temperature and then filtered on 100 ␮m nylon filter. The liquid was evaporated to 130 ± 10 mL with a rotary evaporator (Rotavapor® , Büchi Labortecknik). Water acidified at pH 2 was added to reach a total volume of 1.5 L in order to precipitate the lignin. The medium was then centrifuged at 10,000 rpm during 10 min using an Aventi® -JE Centrifuge (Beckman Coulter). Recovered lignin was dried at 60 ◦ C overnight prior to overnight freeze drying using a FreeZone 4.5 (Labconco) system. The solid residue was rinsed with 500 mL warm technical ethanol (96% [v/v]). It was dried and stored at room temperature.

Each analysis was performed in triplicate.

3. Results and discussion 3.1. Chemical composition of raw material The biomass composition is one of the most important elements to consider regarding the assessment of an extraction process. It provides useful information about the available lignin content for extraction, the potential contaminations and the compounds available as co-products. Table 1 provides the complete characterization of the raw tall fescue according to the protocols described in the previous section. The dry matter was measured equal to 92 ± 1.0% and used as the dry weight basis against which results were compared. The major constituent is glucan, of which 27.6 ± 0.0% was from cellulose, 1.3 ± 0.1% from starch and 2.4 ± 0.1% from hemicelluloses. Hemicelluloses make up 17.6% of the raw biomass and consist mostly constituted by arabinoxylan. A total lignin content of 19.4 ± 0.3%, which includes acid soluble and insoluble fractions, was found. Inorganic matter (ash) remaining after combustion is up to 11.5 ± 0.4%. These results are consistent with the literature (Njoku et al., 2012). The nitrogen content was 1.2 ± 0.0% implying an estimated total protein content of 7.3 ± 0.1%. Protein could slightly increase the lignin content (Norman and Jenkins, 1934) and also be included in extractives. These inaccuracies together with methods imprecisions inherent to the equipment explain why the sum of all the estimated weights of all the fractions is 102.4 ± 3.8% of the original sample mass. 3.2. Composition of the extracted lignin The purity of the precipitated lignin was assessed by the Klason procedure, Kjeldahl (nitrogen) and ash contents (Table 2). A 60% yield of recovered lignin was achieved which is below the 90% yield obtained by (Monteil-Rivera et al., 2012). It was proposed that the use of conventional heating (instead of microwave heating) and scaling up was responsible for the lower performance. A purity over 90% (92.9–90.4%) has been obtained. Composition (%) and yield (%) for both soluble and insoluble lignin were calculated as follow: Composition (%) =

Yield (%) =

g of ligninin precipitate × 100 g of precipitate

g of ligninin precipitate × 100 g of initial lignin

The fraction is constituted by 87% of insoluble lignin which corresponds to 74% of initial insoluble lignin. 13% of acid soluble lignin

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Table 2 Chemical composition of precipitated lignin. Experiment 1 Componentsa Lignin Klason lignin Acid soluble lignin Nitrogen Ash a b

Experiment 2

Compositionb (%) 92.9 ± 1.3 87.8 ± 1.2 5.1 ± 0.1 1.6 ± 0.0 1.0 ± 0.0

Yieldb (%) 59.3 74.2 13.2 16.1 1.1

Compositionb (%) 90.4 ± 1.3 82.4 ± 1.2 8.0 ± 0.1 1.5 ± 0.1 1.3 ± 0.0

Yieldb (%) 63.6 76.7 23.1 16.7 1.6

Each analysis was performed in triplicate. Weight% dry basis.

Table 3 Molecular weight of lignin. Components Tall fescue Corn stover Beech wood P1000

Peak 1 (Da)

Peak 2 (Da)

Peak 3 (Da)

2600 2550 2300 2600

500 750 600 600

150 200 170 /

are recovered. This suggests that a major part of soluble lignin is lost during the process and did not precipitate together with insoluble lignin. A great part could remain in the aqueous phase. About 1.5% of nitrogen was measured. It accounts for 16% of the initial nitrogen in raw biomass and suggests that nitrogenous compound co-precipitated with the lignin. Due to the harsh conditions, proteins probably degraded. Some of these protein fission products could probably be resistant causing a slight overestimation of the percentage of Klason lignin. However, it was considered as negligible since applying a correction on the measured nitrogen in lignin to calculate the protein amount and subtracting it would likely introduce an error greater than that caused by the presence of nitrogenous material (Norman and Jenkins, 1934). 3.3. Molecular weight of extracted lignin The molecular weight (MW) distribution of lignin separated from tall fescue has been assessed by Size Exclusion Chromatography (SEC). Results were compared with lignin extracted by the same process from corn stover and beech wood and to lignin extracted from herbaceous plant by Alm India Pvt and sold as ProtobindTM 1000 (P1000). The overall patterns of MW distribution were similar for experiment 1 and 2. The chromatograms obtained are provided in Fig. 2. The corresponding MW average are given in Table 3. The major peaks labeled 1 on Fig. 2 correspond to molecular weights of about 2600 Da for tall fescue (Fig. 2A), P1000 (Fig. 2B) and corn stover (Fig. 2C), shorter lignin polymers (2300 Da) were found in the beech wood sample (Fig. 2D). The present process seems to produce lower molecular masses, corresponding to peaks labeled 3 on Fig. 2A, C and D, than the Alm process used to extract P1000 lignin (absence of peak 3 on Fig. 2B). Tall fescue appears to be an interesting choice to produce short polymers (500 Da, 150 Da) in comparison to corn stover. Finally, a small peak at 14.08 min in the chromatogram of tall fescue corresponds to high MW of about 62400 Da. It could probably come from high molecular weight lignin or co-precipitated protein degradation products. 3.4. 2D NMR analysis of solid fractions and raw material 2D-Heteronuclear single quantum coherence Nuclear Magnetic resonance (2D-HSQC NMR) gives an overview of different lignin fractions such as aromatic rings, side-chain/inter bonds and contamination by polysaccharides. Fig. 3 shows HSQC NMR spectra of lignin from raw fescue (1), treated solid (2) and precipitated lignin (3). The spectra are divided into three different regions; aromatic region (␦1 H/␦13 C 100–150/6.4–7.9) (A), oxygenated aliphatic

side chain region (␦13 C/␦1 H 50–90/3.1–5.1) (B) and carbohydrates region (␦13 C/␦1 H 91–150/4.0–5.3) (C). Peak assignments from literature and their ı13 C/ı1 H cross-signals are summarized in Table 4 (Del Río et al., 2012; Kim and Ralph, 2010; Samuel et al., 2011; Villaverde et al., 2009; Wen et al., 2013; Yuan et al., 2011). The aromatic carbons were numbered from 1 to 6 and the aliphatic carbons from ␣ to ␥ by convention. The aromatic region of raw tall fescue lignin spectrum (Fig. 3A.1) displays Guaiacyl (G) ring cross-signals at ı13C/ı1H 114.8/6.83 and 110.85/7.08 ppm attributed respectively to C5,6 –H5,6 and C2 –H2 correlations (Del Río et al., 2012; Villaverde et al., 2009). The crosssignals at 103.65/6.76 and 128.7/7.26 ppm corresponds to C2,6 –H2,6 correlations in Syringyl (S) (Del Río et al., 2012; Villaverde et al., 2009) and p-Hydroxyphenyl (H) (Del Río et al., 2012; Wen et al., 2013) respectively. These results agreed with literature reporting that grasses lignin contains the three different units (Samuel et al., 2011). Ferulic acid (FA) signals were correlated at 111.0/7.37 and 123.2/7.13 ppm for C2 –H2 and C2,6 –H2,6 respectively. p-coumaric acid (PCA) C2,6 –H2,6 correlation was found at 130.0/7.46 ppm (Del Río et al., 2012). A comparison with the cellulosic pulp spectrum (Fig. 3A.2) shows the evidence of a successful delignification. H and G units cross-signals are weakly present while S units, FA and PCA appear to be completely removed from the pulp. S units seems to be depolymerized more likely. It can be due to the no vacant 5-position as stated by (Ziebell et al., 2010). The spectrum acquired from precipitated lignin (Fig. 3A.3) shows cross-signals similar to the raw material spectrum. The three types of subunit were recovered. The appearance of an additional cross-signal corresponding to oxidized guaiacyl unit (G’) at 111.5/7.50 ppm (Yuan et al., 2011) attests to the occurrence of depolymerization. The strong cross-signal of FA and PCA revealed that these acids were concentrated from the raw material. It suggests a good recovery of these compounds, and in the case of FA, a possible contamination by hemicelluloses since this acid acts as a bridge between hemicelluloses and lignin units (Buranov and Mazza, 2008). The oxygenated aliphatic side-chain region of raw fescue spectrum (Fig. 3B.1) was analyzed to determine the type of linkages in the native lignin. The strongest signal was observed at 55.4/3.73 ppm and was attributed to methoxyls groups ( OCH3) substituted to lignin subunits (Del Río et al., 2012; Samuel et al., 2011; Wen et al., 2013). C␥ –H␥ of ␥ acetylated ␤-O-4 substructures was attributed to the signal at 62.9/3.88 ppm (Del Río et al., 2012). ␤-O-4 linkages were present only in raw material (Table 4). The cellulosic pulp (Fig. 3B.2) spectrum displays ␤-d-Xylopyranoside ring cross-signals at 72.8/3.22, 74.0/3.42 and 75.3/3.63 ppm attributed respectively to C2 –H2 , C3 –H3 and C4 –H4 correlations (Del Río et al., 2012; Wen et al., 2013) similar to signal found in the raw fescue spectrum (72.7/3.20, 73.9/3.41 and 75.3/3.64 ppm). It attests of residual hemicelluloses in pulp. A weak signal from methoxyl reveals the presence of lignin contamination. Methoxyl group signals are even stronger in extracted lignin (Fig. 3B.3) than in raw material due to a concentration of this function. The study of the carbohydrates region reveals a great variety of compounds in raw material (Fig. 3C.1) including cellulose, cellobiose, xylan, arabi-

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Fig. 2. Distribution of lignin molecular mass. Chromatograms obtained by Size Exclusion Chromatography (SEC) from A) tall fescue B ProtobindTM 1000 C) corn stover D) beech wood. Table 4 Coordinates (␦ 13 C/␦ 1 H) and their respective assignments in the three HSQC NMR spectra main regions of raw fescue, cellulosic pulp and precipitated lignin. Assignmentsa

␦ 13 C/␦ 1 H (ppm) Raw fescue

Aromatic region C2,6 –H2,6 p-coumarate (PCA) C2,6 –H2,6 p-hydroxyphenyl units (H) C6 –H6 ferulate (FA) C 5,6 –H5,6 guaiacyl units (G) C2 –H2 ferulic units (FA) C2 –H2 guaiacyl units (G) C2 –H2 oxidized guaiacyl units (G’) C2,6 –H2,6 syringyl units (S) Carbohydrates region ␣-l-arabinose f

130.0/7.46 128.7/7.26 127.9/7.21 123.2/7.13 118.9/6.85 114.8/6.83 115.7/6.86 111.0/7.37 110.85/7.08

128.3/7.23 127.9/7.21

114.7/6.72

103.65/6.76

(1 → 6)-␤-d-Glu p (hemicellulose) (1 → 4)-␤-d-Xyl (xylan) & (1 → 4)-␣-d-Glu (starch) 2-O-Ac-␤-d-Xyl p 2-O-Ac-Man p (1 → 4)-␤-d-Glu p (reducing end cellobiose) (1 → 4)-␣-d-Xyl (reducing end xylose) & 1 → 4)-␤-d-Glu (reducing end glucose) Cellulose peaks

107.9/4.91 106.9/5.53 103.3/4.27 101.7/4.38 99.3/4.60 99.2/4.81 96.8/4.45 92.4/5.07 102.1–102.9/4.40–4.53 & 103.1/4.39

Side chain region C4 –H4 ␤-d-Xylopyranoside C3 –H3 ␤-d-Xylopyranoside C2 –H2 ␤-d-Xylopyranoside C␤–H␤ ␤-O-4 substructure linked to S C␤–H␤ in ␥ acetylated ␤-O-4 linked to S or in ␤-O-4 linked to G/H C␥ –H␥ ␥ acetylated ␤-O-4 substructures O–CH3 (methoxyl)

75.3/3.64 73.9/3.41 72.7/3.20 86.2/4.15 (weak) 83.9/4.44 62.9/3.88 55.4/3.73

a

Cellulosic pulp

Lignin fraction 130.0/7.56 128.6/7.24 128.0/7.22 122.8/7.15 118.7/6.84 114.9/6.75 115.5/6.87 110.9/7.37 110.3/6.97 111.5/7.50 103.39/6.70 107.7/4.86

101.7/4.38

96.68/4.48 92.0/5.05 102.7/4.47

92.4/5.03 103/4.22–4.45 (weak)

75.3/3.63 74.0/3.42 72.8/3.22

73.2/3.12

55.4/3.74 (weak)

55.4/3.75

Assignments according to Refs: Del Río et al., 2012; Kim and Ralph, 2010; Samuel et al., 2011; Villaverde et al., 2009; Wen et al., 2013; Yuan et al., 2011.

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Fig. 3. 2D HSQC NMR spectra in A. aromatic region (␦13 C/␦1 H 100–150/6.4–7.9), B. side chain region (␦13 C/␦1 H 50–90/3.1–5.1), C. carbohydrates region (␦13 C/␦1 H 91–150/4.0–5.3) from (1) raw fescue, (2) cellulosic pulp and (3) precipitated lignin.

nose and acetylated compounds (Table 4) (Kim and Ralph, 2010). A signal of (1 → 4)-␤-d-Xylan was found in the cellulosic pulp spectrum (Fig. 3C.2) at 101.7/4.38 ppm and increases evidence of hemicelluloses contamination. Presence of ␣-l-arabinose correlation at 107.7/4.86 ppm in extracted lignin spectrum (Fig. 3C.3) further substantiates the apparent co-extraction of arabinoxylan from hemicelluloses together with ferulates. Acetylations are no longer found in pulp and lignin suggesting a de-acetylation of xylose aor removal with the associated carbohydrate. 3.5. Composition of the recovered cellulosic pulp The composition of the residual pulp after treatment was assessed using the Klason procedure (lignin), hydrolysis followed by GC-FID (carbohydrates), Kjeldahl method (protein) and calcination (ash) (Table 5) and was calculated as follow: g recovered in pulp × 100 g of dried pulp In order to estimate the recovery of compounds from raw matein pulp rial the yields (%) were calculated as follow: g gofrecovered × 100. initial compound

Both experiments show the same composition trend. The pulps contain 10–12% of the lignin initially present in the raw tall fescue which attests of a successful delignification. The composition of the pulp is 6–7% of lignin and less than 55% of cellulose. About 40% of the initial amount of cellulose have been removed. These results suggest severe degradations of the cellulose fibers (Monteil-Rivera et al., 2013). Hemicelluloses have been almost entirely removed. In addition, this fraction is probably overestimated by the glucose content. It could probably come from the cellulosic fraction of the pulp since the cellulose from the treated pulp is more accessible and thus more easily hydrolyzed. Therefore, as the same standard analysis method as applied on the raw biomass was performed, the hydrolysis is probably less suitable. Regarding the protein content, only 14–15% of the initial amount is still present and the major part was removed. Finally, about 70% of the inorganic fraction was insoluble and therefore accumulated in the pulp. The two pulps obtained account for 33.75% and 35.02% of the initial biomass respectively. Experiment 2 seems to have been less efficient to remove some resistant constituents such as protein and lignin which increases the mass balance. The residual solid contains about 2% more of initial lignin and proteins compared with experiment 1. This is probably due to re-polymerization and condensation

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Table 5 Chemical composition of cellulosic pulp after experiment 1 and experiment 2. Experiment 1 Componentsa Lignin Klason lignin Acid soluble lignin Cellulose Hemicellulose Glucose Xylose Mannose Galactose Arabinose Rhamnose Protein Ash a b c

Compositionb (%) 6.0 ± 0.2 4.4 ± 0.1 1.5 ± 0.1 53.6 ± 0.1 3.6 ± 0.4 2.6 ± 0.3 0.9 ± 0.0 0.0 ± 0.0 0.0 ± 0.1 0.0 ± 0.0 0.0 ± 0.0 2.9 ± 0.0 23.1 ± 0.0

Experiment 2 Yieldc (%) 10.0 9.8 10.6 60.5 6.4 24.3 2.7 2.0 0.0 0.6 0.0 13.5 68.1

Compositionb (%) 7.1 ± 0.4 5.3 ± 0.2 1.8 ± 0.2 50.5 ± 0.3 3.5 ± 0.2 2.4 ± 0.1 0.9 ± 0.1 0.0 ± 0.0 0.0 ± 0.0 0.1 ± 0.0 0.0 ± 0.0 3.2 ± 0.1 24.4 ± 0.2

Yieldc (%) 12.4 12.3 12.9 59.1 6.4 23.2 2.9 2.1 0.0 1.0 0.0 15.4 74.7

Each analysis was performed in triplicate. g (recovered in pulp) × 100/g (total pulp). g (recovered in pulp) × 100/g (initial compound).

Fig. 4. Total Ion Chromatogram (TIC) of acetylated sugars in the liquid fraction recovered after lignin precipitation.

Table 6 Compounds obtained in liquid fraction after experiment 1 and experiment 2. Experiment 1 Componentsa Monosaccharides Glucose Xylose Mannose Galactose Arabinose Unknown 5c Total saccharides Glucose Xylose Mannose Galactose Arabinose Rhamnose Ashd Degradation products 5-HMF Furfural a b c d

Concentration (mg mL−1 ) 3.9 ± 0.1 0.9 ± 0.0 2.2 ± 0.1 0.1 ± 0.0 0.3 ± 0.0 0.4 ± 0.0 1.9 ± 0.1 5.5 ± 0.2 1.2 ± 0.0 3.1 ± 0.1 0.1 ± 0.0 0.3 ± 0.0 0.6 ± 0.1 0.1 ± 0.0 1.0 ± 0.0 Concentration (mg L−1 ) 33.1 ± 0.4 99.3 ± 0.6

Each analysis was performed in triplicate. Weight% dry basis of hemicellulose content. Not derivated from hemicellulose No Data Available. Weight% dry basis of initial ash in the input raw biomass.

Experiment 2 Yieldb (%) 30.7 37.0 29.5 20.0 39.3 26.9 NDA 61.0 70.1 58.8 34.0 58.2 63.5 73.9 12.4 Conversion (%) 1.2 1.5

Concentration (mg mL−1 ) 6.0 ± 0.0 1.4 ± 0.0 3.5 ± 0.0 0.1 ±0.0 0.4 ± 0.0 0.6 ± 0.0 1.7 ± 0.0 7.3 ± 0.1 1.5 ± 0.0 4.3 ± 0.0 0.1 ± 0.0 0.4 ± 0.0 0.9 ± 0.1 0.1 ± 0.0 1.2 ± 0.1 Concentration (mg L−1 ) 43.5 ± 0.2 213.4 ± 0.0

Yieldb (%) 47.5 55.9 47.1 24.0 50.8 43.3 NDA 69.9 77.3 68.5 34.0 63.9 75.5 82.6 15.7 Conversion (%) 1.6 3.3

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Fig. 5. Proposed general structure of unknown compound 5.

of pseudo-lignin as described by (Hu et al., 2012), together with ash and proteins, which deposits on the pulp during the treatment. The higher yield of proteins (15.4%) and ash (74.7%) compared with experiment 1 (13.5% and 68.1% respectively) supports this coprecipitation hypothesis. Lignin precipitation can also occur after filtration without being removed during the washing step. Indeed, these phenomena are hard to control and hardly repeatable. However, it does not affect significantly the polysaccharides removal; 0.6% less residual cellulose and no difference in residual hemicelluloses were assessed. 3.6. Composition of the liquid fraction Derivatization by acetylation has been performed on the recovered liquid phase in order to analyze the saccharides from degradation of hemicelluloses and cellulose gas chromatography. The qualitative analysis by GC–MS of the liquid fraction surprisingly reveals an incomplete degradation of both C5- and C6sugars containing polymers. The Total Ion Chromatogram (TIC) (Fig. 4) indicates six major peaks not attributed to conventional sugar monomers. The first three peaks labeled 1 (18.83 min), 2 (19.07 min) and 3 (19.33 min) were related to C5 derivatives. The peaks labeled 4 (25.85 min), 5 (26.06 min) and 6 (26.23 min) are related to C6 derivatives. The mass spectra strongly suggest a partial degradation of hemicelluloses and cellulose. The degradation of cellulose into nonmonomer or degradation products (5-HMF) could explain the low percentage of glucose in both solid and liquid fractions. The mass spectrum of the compound labeled 5 has been chosen as example. The spectrum of the unknown compound (Fig. A1) corresponds at 87% to the spectrum of tretraacetate (from derivatization) glucose propylglycoside from Wiley 275 database (Fig. A2). Therefore, the hypothesis of a carbohydrate structure grafted by a three carbon chain has been proposed (Fig. 5). It probably comes from the adjacent glucose on the cellulose structure. Table 6 displays the composition of the liquid fractions. The partial degradation trend is similar for both duplicates. However, in the case of experiment 2, about 17% more of hemicelluloses have been converted into monomers. This higher availability of soluble monosaccharides leads to a higher degradation. Twice of initial C5 monosaccharides were converted into furfural. The conditions during the second treatment were probably more severe. The variability could be introduced during the evaporation of the liquid phase. The final volume of concentrated liquid phase prior to lignin

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precipitation was hardly controllable and additional degradation could occur. These compounds were produced in small amounts. 1.6% of C6-sugars were converted into 5-HMF (max 43 mg L−1 ) and 3.3% of C5-sugars were converted into furfural (max 213 mg mL−1 ). The dilution during the precipitation step is partially responsible of the very low concentration. Moreover, even with an additional 10 times concentration of the liquid stream the furfural concentration only reaches 22.2 mmol L−1 . In the view of a valorization of saccharides by a fermentation process, this concentration remains below the concentration of 29 mmol L−1 reported as beneficial for yeast due to regeneration of NAD+ (Palmqvist et al., 1999). Counting the total saccharides content, including oligomers, the difference between the two experiments is reduced. The yield of xylose from the second experiment was 10% higher than the first. This variability remains unexplained. The non-precipitated acid soluble lignin present in the liquid phase could not be measured by UV analysis since other compounds from the matrix such as proteins degradation products may interfere at ␭ 205 nm. An estimated concentration of 2.4 g L−1 of acid soluble lignin has been proposed based on the unrecovered soluble lignin in both cellulosic residue and precipitated lignin. 4. Conclusion Lignin has been recovered at a purity level of 90% with a yield of 60% on tall fescue using a dilute sulfuric acid/ethanol organosolv treatment. Over 75% of the initial Klason lignin was recovered. Soluble lignin is, in major part, lost during the process. The value is slightly decreased by nitrogen containing compounds probably coming from degraded proteins which co-precipitate with the lignin. Higher molecular mass (2600 Da) counts for the major part of the lignin fraction whereas production of short lignin polymers (500 Da, 150 Da) was observed. An additional contamination by arabinose, probably linked to ferulate, was observed by 2D-HSQC NMR. G, S and H lignin subunits, ferulate and coumarate were observed in both raw and extracted lignin material. Cellulose was recovered with a yield of 60% at a purity level of 53%. The purity was decreased by the accumulation of inorganic matter in the pulp (23%). The conditions seem to be too harsh for tall fescue and led to significant amount of cellulose degradation. Optimization of conditions for both extraction treatment and precipitation would be required. A process using alcohol in less quantity providing better yields of both cellulose and lignin will be developed to fully valorize the plant material. Acknowledgements The authors are grateful to Caroline Vanderghem and Mario Aguedo for their guidance in lignin analyses. Virginie Byttebier is thanked for technical assistance and Glenn Bousfield for his help in English revision. Appendix A.

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Fig. A1. Mass spectrum of unknown compound 5.

Fig. A2. Mass spectrum of glucose propylglycoside from Wiley 275 database.

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