Preparation of wood samples
The biomass samples used in this study were extractive-free wood of hybrid aspen and Norway spruce, and the corresponding torrefied materials, hereafter referred to as T-aspen and T-spruce. The aspenwood and the spruce wood were milled with an A11 Basic Mill (IKA, Staufen, Germany) and sieved with 100–500 μm sieves (Retsch Analytical AS 200, Retsch, Haan, Germany). The milled and sieved aspenwood and spruce wood were freeze-dried to a dry-matter content of 100%. The moisture content was measured using an HG63 Moisture Analyzer (Mettler-Toledo, Greifensee, Switzerland). Using a previously described procedure , portions of 3 g of freeze-dried wood of aspen or spruce were extracted with 200 mL of a 9:1 mixture of petroleum ether (Petroleum Benzene, Merck, Darmstadt, Germany) and acetone by performing 15 extraction cycles with a Soxhlet system (Büchi Extraction System B-811, Büchi, Flawil, Switzerland) followed by air-drying for ~16 h.
Portions of extractive-free wood of aspen and spruce were torrefied in a torrefaction reactor with nitrogen gas at 285 °C for 16.5 min. The weight of the extracts and the losses during the torrefaction were measured. The fraction of extractives amounted to (% dry weight): 0.90 ± 0.16 for aspen and 1.07 ± 0.03 for spruce, whereas the mass remaining after torrefaction (% dry weight) was 57.9 ± 2.7 for aspen and 73.3 ± 4.2 for spruce.
Ionic liquids and Karl-Fischer titration
The three ILs [C4C1im][HSO4], [C4C1im][Cl], and [C4C1im][MeCO2] (all of which are based on the 1-butyl-3-methylimidazolium cation, a.k.a. [Bmim]) were prepared as previously described . The ILs were dried overnight at 65 °C under stirring using an evaporator and a high vacuum pump. In accordance with Brandt et al. , the water content of [C4C1im] [HSO4] was adjusted to 20% by addition of ultra-pure water (Millipore, Billerica, MA). The water content of the other two ILs was <1% as determined by Karl-Fischer titration.
A Karl-Fischer titrator (756 KF coulometer, Metrohm, Herisau, Switzerland) with a diaphragm-free titration cell and Hydranal Coulomat E reagent (Sigma-Aldrich, St. Louis, MO) were used to determine the moisture content of the ILs. After drying, approx. 100 μL of the IL was withdrawn into a desiccator-dried syringe and weighed. After injection of the IL into the Karl-Fischer reactor, the syringe was weighed again to determine the amount of sample injected.
Ionic liquid pretreatment
The IL pretreatment was performed according to Gräsvik et al.  by adding 50 mg wood sample (dry weight) to 950 mg ionic liquid and incubating the reaction at 100 °C for 20 h. The samples were then cooled to room temperature, and the pretreated material was precipitated by addition of 1 g of methanol followed by vigorous mixing. A second precipitation was performed by addition of 10 g of ultra-pure water followed by vigorous mixing. The mixture was centrifuged for 10 min at 14,500 g and the supernatant was decanted and collected. The pellet was washed using 3 × 10 g ultra-pure water. After that, the pellet was freeze-dried as a part of the analytical procedure. The washing liquids (~30 g) were collected and mixed with the supernatant (~11 g), and the chemical composition of the resulting liquid (~41 g) was analyzed. In total, four different woody materials were pretreated with three different ILs, resulting in 12 different pellets/liquids. For mass balance analysis, quadruplicate measurements were performed.
Liquid phase compositional analysis
A portion (0.5 mL) of the liquid phase of each sample was diluted with water (0.5 mL) and the monosaccharide content was analyzed by using high-performance anion-exchange chromatography (HPAEC). Moreover, 14 mL of the liquid phase of each sample were concentrated to 0.25–0.3 mL by freeze-drying. The oligosaccharide content was analyzed by diluting 0.2 mL of the concentrated samples with 0.8 mL of ultra-pure water and acidifying the mixture to a pH of 0.3 with a 72% solution of sulfuric acid (50 μL for samples pretreated with [C4C1im][HSO4] or [C4C1im][Cl], and 100 μL for samples pretreated with [C4C1im][MeCO2]). The samples were vortexed and autoclaved at 121 °C for 60 min. HPAEC was used to analyze the sugar content, and the oligosaccharide content was calculated by subtracting the original monosaccharide content.
Solid fraction compositional analysis
The weight of the dried pellet of each IL-pretreated sample was determined to calculate the recovery. The lignin and carbohydrate contents of the pellets and the four woody materials were determined essentially according to Sluiter et al. . Since the woody materials were already extractive-free, no extraction step was included. Fifty mg (dry weight) of each sample were treated with 1.5 mL 72% sulfuric acid at 30 °C in a water bath for 1 h. Samples were diluted to a sulfuric acid content of 4% followed by autoclaving at 121 °C for 1 h. Monosaccharides were determined using HPAEC (section “HPAEC analysis of monosaccharides”) instead of using HPLC. Compositional analysis of lignocellulosic biomass includes a heating step in the presence of sulfuric acid, intended to generate monosaccharides from hemicelluloses and cellulose. Under such conditions there may be some degradation of monosaccharides to furan aldehydes and aliphatic carboxylic acids. Therefore, to make the compositional analysis as thorough as possible, even monosaccharide degradation products were quantitated. HPLC was used to determine the content of furfural, HMF (5-hydroxymethylfurfural), acetic acid, levulinic acid, and formic acid (section “HPLC analysis of furan aldehydes and carboxylic acids”).
IL pretreatment of 50 mg of the woody materials was performed as previously described (section “Ionic liquid pretreatment”). The enzymatic hydrolysis was performed as described by Gräsvik et al. . The pretreated material was washed with 1 × 10 g 50 mM citrate buffer (pH 5.2) and centrifuged for 10 min at 14,500 g prior to enzymatic hydrolysis. After washing, 900 mg of 50 mM citrate buffer (pH 5.2) and 50 mg of a 1:1 mixture of Celluclast 1.5 L and Novozyme 188 (liquid enzyme preparations obtained from Sigma-Aldrich, St. Louis, MO, USA) were added to each sample. This would correspond to a loading of ~40 mg protein per g non-pretreated substrate. The reaction mixtures were incubated in an orbital shaker set at 170 rpm and 45 °C (Ecotron incubator shaker, Infors, Bottmingen, Switzerland) for 72 h. The four non-pretreated woody materials were digested in the same way. All reactions were performed in triplicate. Samples (10 μL) were withdrawn from the reaction mixtures at the start and after 2 h of incubation, and were used for analysis of the glucose production rate (GPR) using a glucometer (Accu-Chek Aviva, Roche Diagnostics, Rotkreuz, Switzerland). The glucometer was calibrated with a series of glucose solutions (mM): 1.25, 2.5, 5, 7.5, 10, 12.5, 15, and 17.5. Samples for glucometer analysis were diluted with deionized water and mixed (1:1) with glucometer buffer (0.15 mM sodium chloride, 1.2 mM calcium chloride, 0.9 mM magnesium chloride, 2.7 mM potassium chloride, pH 7.4) to reach the calibration range. The yield of monosaccharides after 72 h was determined using HPAEC (section “HPAEC analysis of monosaccharides”). The monosaccharide yields (g g−1 dry woody material) were calculated based on the amount of biomass before pretreatment, which was 50 mg for all samples.
HPAEC analysis of monosaccharides
HPAEC was used for determination of the contents of arabinose (Ara), galactose (Gal), glucose (Glc), mannose (Man), and xylose (Xyl) in the liquid phase after pretreatment, in compositional analysis of the solid fraction after pretreatment, in compositional analysis of untreated wood, and in enzymatic hydrolysates. All samples were diluted with ultra-pure water and filtered through 0.2 μm nylon membranes (Millipore). The analysis was performed using an ICS-5000 instrument equipped with an electrochemical detector, a CarboPac PA1 (4 × 250 mm) separation column, and a CarboPac PA1 (4 × 50 mm) guard column (all from Dionex, Sunnyvale, CA). The temperature of the column oven was 30 °C. Prior to injection, the column was regenerated with a solution of 260 mM sodium hydroxide (Sodium Hydroxide Solution for IC, Sigma-Aldrich) and 68 mM sodium acetate (Anhydrous Sodium Acetate for IC, Sigma-Aldrich) for 12 min followed by ultra-pure water for 2 min. Each sample was injected once, and elution was performed with ultra-pure water during 25 min. The flow rate was 1 mL min−1. External calibration curves were established by using 0.5, 1, 5, 10, 20, and 40 ppm (mg L−1) solutions of Ara, Gal, Glc, Man, Xyl [prepared from monosaccharide preparations (>99%) obtained from Sigma-Aldrich].
HPLC analysis of furan aldehydes and carboxylic acids
Reaction mixtures from the compositional analysis (section “Liquid phase compositional analysis”) were analyzed with regard to carbohydrate by-product formation. Determination of the concentrations of the furan aldehydes furfural and HMF, and the carboxylic acids formic acid, acetic acid, and levulinic acid was performed with an Agilent Technologies 1200 series HPLC system (Agilent, Santa Clara, CA). For analysis of the furan aldehydes, the device was equipped with an autosampler, a diode-array-detector (DAD), a binary pump, and a degasser (all from Agilent), and the chromatographic separation was performed using a 3.0 × 50 mm, 1.8 μm Zorbax RRHT SB-C18 column (Agilent) with aqueous 0.1% (volume fraction) formic acid as Eluent A, and acetonitrile with 0.1% (volume fraction) formic acid as Eluent B. The separation program was: 3 min 3% B, 4 min 10% B, and 2 min post run-time for re-equilibration of the column. The flow rate was set to 0.5 mL min−1, the absorption at 282 nm was recorded, and the temperature of the column oven was 40 °C. Quantitation was performed using an external calibration curve covering the interval 5 μM - 250 μM.
For carboxylic acids the device was equipped with an autosampler, a refractive index detector (RID), a binary pump, and a degasser (all from Agilent). The chromatographic separation was performed using a Bio-Rad Aminex HPX-87H column (Bio-Rad Laboratories AB, Solna, Sweden). Separation was achieved with an eluent consisting of an aqueous solution of 0.01 M H2SO4 applied with a flow rate of 0.6 mL min−1. The temperature of the column oven was 60 °C and the temperature of the detector cell of the RID was 55 °C. An external calibration curve covering the interval 5 mM - 250 mM was used for quantitation.
In the mass balance analysis, the masses of formic acid, levulinic acid, and HMF (Additional file 1: Table S1) were converted to glucan by using division factors of 0.28, 0.72, and 0.78, respectively. The mass of furfural (Additional file 1: Table S1) was converted to xylan by using a division factor of 0.73. This was based on the assumption that HMF, levulinic acid, and formic acid were derived mainly from glucan, whereas furfural was derived mainly from xylan.
Fluorescence microscopy was carried out at the Biochemical Imaging Centre of the Chemical-Biological Centre (KBC) (Umeå, Sweden). Following the sample-preparation procedure of Rahikainen et al. , the solid residues obtained after the IL treatments were embedded in London Resin White (TAAB Laboratories Equipment Ltd., Aldermaston, England). The specimens were then sectioned (2 μm) with a rotary microtome (Leica RM2245, Germany) and the autofluorescence of lignin was visualized by excitation at 470 nm and emission at 525 nm using a fluorescence microscope (Axioimager Z1, Carl Zeiss MicroImaging GmbH, Göttingen, Germany).