Sugarcane bagasse (Rocky Point sugar mill, Pimpama, Queensland) comprising long cuticle fibres and core pith particles (mostly ca. 5 mm to 50 mm) was air dried for a week on metal trays then size reduced (to <10 mm) with a knife mill before being mixed and subsampled by the cone and split method and stored at 4°C. Before use the bagasse was ground (to <2 mm) using an electric lab mill (Retsch SM100, Haan, Germany). The material was ground for ca. 1 min per batch to avoid excess heating, placed on top of two brass sieves (0.5 mm and 0.25 mm) and in a sieve shaker for 20 min, and the fraction collected between the two sieves was used as the starting material. Moisture content was measured gravimetrically (convection oven, 105°C, overnight) before every use and was 10 ± 1% mass except where otherwise indicated.
The ionic liquids (1-butyl-3-methylimidazolium chloride [C4mim]Cl (≥ 95%) melting point as per MSDS (m.p.) 73°C, 1-ethyl-3-methylimidazolium chloride [C2mim]Cl (≥ 95%) m.p. 80°C, and 1-ethyl-3-methylimidazolium acetate [C2mim]OAc (≥ 90%) m.p. –20°C, Sigma-Aldrich, NSW) were all dried in a vacuum oven (at 80°C – 90°C, ca. 4 mm Hg, > 12 h) prior to use. Initial moisture content (at the time of weighing the IL for each use) was typically ca. 2% of total mass for [C4mmim]Cl and 1% for [C2mim]Cl and [C2mmim]OAc as measured by Karl Fischer titration. At this point it is worth noting that although the m.p. of neat [C4mim]Cl is 73°C (as per the MSDS provided by the manufacturer), its 2% moisture content was sufficient to maintain it in liquid phase at room temperature. Cellulose (Avicel PH-101), dimethyl sulphoxide (DMSO) (99.9%) and Karl Fischer HYDRANAL titrant 2E and solvent E were purchased from Sigma-Aldrich (Sydney, NSW). Cellulase / β-glucosidase mixture (Accelerase 1000) was purchased from Genencor (Danisco A/S, Denmark). Water was Millipore-filtered and deionised (Milli-Q-plus) to a specific resistivity of 18.2 μS at 25°C. All other solvents and chemicals were analytical grade.
Bagasse soda lignin preparation
Bagasse soda lignin was prepared by soda pulping of bagasse (175°C, 2 h, bagasse 10% mass, NaOH 10% mass) and precipitating the resulting black liquor with acid (2 M H2SO4) add to reduce the pH to 3.0. The precipitate was then redissolved in aqueous NaOH (10% mass) and reprecipitated by addition of acid to reduce the pH to 3.0. The recovered lignin solids were washed and dried (40°C, vacuum oven).
Karl Fischer titration
A Karl Fischer automated titrator (Radiometer Copenhagen TIM 900) with ethanol based HYDRANAL reagents was used to measure moisture content of ILs after drying and prior to use.
Preliminary dissolution experiments
Non-extracted bagasse was treated in [C4mim]Cl, [C2mim]Cl, [C2mim]OAc under conditions used in previously reported work  (150°C, 90 min and 5% mass bagasse in IL). The treated mixture (partially dissolved bagasse in IL) was then diluted with DMSO and the undissolved and the dissolved-then-precipitated (with water) solid fractions were recovered, dried and weighed as described in previous work by the authors . The estimates of standard deviation (absolute) for this technique (based on duplicate dissolution experiments, 5 degrees of freedom, (df)) are 6% starting mass for undissolved solids and 3% starting mass for dissolved-then-precipitated solids.
Mass balance determinations for three IL treatments
Bagasse (0.25 mm – 0.5 mm) was extracted with ethanol and water using a Sohxlet device according to the NREL protocol for biomass extractives . ILs (ca. 30 g of either [C4mim]Cl or [C2mim]Cl or [C2mim]OAc in duplicate) were weighed in sealable pressure glass tubes (ACE glass 50 mL). At this point, IL (ca. 0.5 g) was weighed and set aside for IL recovery analysis (using ion chromatography). Extracted bagasse (3.5% moisture) (ca. 1.5 g for [C4mim]Cl and [C2mim]Cl and 0.75 g for [C2mim]OAc) was added to each pressure tube, sealed with Teflon stoppers and placed in an oil bath which was stabilised at 150°C with magnetic stirring at 200 rpm. Sealing the tubes prevented volatile losses such as acetic acid (b.p. 118.1°C) or furfural (b.p. 161.7°C) from the degradation of xylose. The tubes were left in the oil bath for 60 min (25 min of which at temperature ramp and 35 min at 150°C) and, upon removal, placed in an ice bath with magnetic stirring. After 2 min, the tubes were removed from the ice bath and water was added equal to 0.5 mass fraction of the originally added IL. The tube was sealed again and agitated vigorously until a homogenous solution between water and IL appeared to form. The contents of each tube were quantitatively transferred into a preweighed polypropylene centrifuge tube and centrifuged at 10000 × g for 20 min. The liquid contents of the centrifuge tube were decanted to a new preweighed polypropylene centrifuge tube and weighed (liquid fraction 1 or LF1). The pellet (SF1) was centrifuge washed with distilled water (5 × 30 mL at 10000 × g and 5 min - 10 min cycles), freeze dried overnight (−85°C, 80 mT) and weighed. LF1 1 was precipitated with additional water resulting to a water : IL mass ratio of 2.0. Precipitation and coagulation of solids was aided by storing at 4°C overnight followed by shaker incubating at 55°C - 70°C for 60 min. The resulting precipitate (solid fraction 2 or SF2) was centrifuge washed, freeze dried and weighed. The resulting liquid was acidified to pH ≤1 and mixed with a further 1.5 IL mass equivalents of water to maximise precipitation of lignin in solution. This final precipitate (solid fraction 3 or SF3) was centrifuge washed, freeze dried and weighed.
Losses of liquid components to washings of pellets were accounted for by weighing pellets prior to washing and after drying (it is assumed that the composition of these lost liquid components is the same as the bulk liquid). Similarly, subsampling for analysis was accounted for by careful attention to mass changes.
SF1 and the starting biomass were characterised using the NREL acid hydrolysis protocol . Solid fractions 2 and 3 were characterised for lignin content with the acetyl bromide protocol described by Iiyama and Wallis . The sample of LF1 was directly injected onto the HPLC and the Ion Chromatograph (IC) for quantification of monosaccharides and IL ions respectively while the soluble oligosaccharides were determined by acid hydrolysis. All methods are described in detail in the following sections. The distribution of cellulose, hemicellulose and lignin between solid fractions, liquid fraction monosaccharides and liquid fraction oligosaccharides was finally reported as% mass of the components in the starting material (Table 3). The estimates of standard deviation (absolute, based on duplicate IL pretreatments, 3 df) of the recovery (and analysis) of these components (as%mass starting component) in SF1 are 2% for glucan, 2% for xylan, 3% for arabinan, 1% for acetyl and 2% for lignin (acid soluble + acid insoluble). In LF1, these estimates of standard deviation for the oligosaccharides are 1% for glucan, 2% for xylan, 18% for arabinan and 3% for acetyl and for the monosaccharides they are 0.2% for glucan, 0.2% for xylan, 15% for arabinan and 0.7% for acetyl. The unacceptably high standard deviation for arabinan is attributed to its very low concentrations in the liquid fractions.
Compositional analysis of “solid fraction 1”
Compositional analysis of SF1 samples was carried out using the standard NREL procedure for determination of structural carbohydrates and lignin in biomass . All samples were freeze dried overnight prior to analysis. Each sample (250 mg) was treated with H2SO4 (72% mass) at 30°C for 1 h. These samples and a sugar recovery standard (SRS, containing known concentrations of glucose, xylose and arabinose) were then exposed to dilute H2SO4 (4%) at 121°C for 1 h. The hydrolysis products were determined by HPLC (Waters) equipped with a RI detector (Waters 410) and a Bio-Rad HPX-87 H column operated at 85°C. The mobile phase consisted of 5 mM H2SO4 with a flow rate of 0.6 mL min-1. The glucose, xylose and arabinose results were corrected for acid decomposition using the% mass recovery from the SRS. The polysaccharide and acetyl mass content were calculated by conversion of the monosaccharide and acetic acid results with appropriate multiplication factors (0.90 for glucose, 0.88 for xylose and arabinose, 0.683 for acetic acid). In bagasse, glucan content is considered equal to cellulose content since there is no glucose in the hemicelluloses of sugarcane and sucrose has been removed previously at the sugar mill. The acid- insoluble lignin (AIL) after acid hydrolysis was measured as the mass loss of insoluble residue at 575°C. The acid-soluble lignin (ASL) was measured by UV–vis spectrophotometer (Cintra 40) at 240 nm with an extinction coefficient value of 25 L g-1 cm-1. Ash was determined by placing separate sample fractions at 575°C.
Compositional analysis of monosaccharides in liquid fraction 1
Each sample of LF1 (0.5 mL) was weighed in 1.5 mL Eppendorf tubes and diluted with water (0.5 mL). The contents were vortexed thoroughly, filtered through a 0.45 μm nylon filter and injected to a Waters HPLC as described earlier. Glucose, xylose, arabinose and acetic acid masses were converted to glucan, xylan, arabinan and acetate masses using appropriate multiplication factors (as listed earlier). In addition, it was assumed that the detected hydroxymethylfurfural (HMF) and furfural were products of cellulose and xylan degradation respectively. Therefore, HMF and furfural masses were converted to cellulose and xylan mass equivalents using multiplication factors of 1.28 and 1.38 respectively .
Compositional analysis of oligosaccharides in liquid fraction 1
Each sample of LF1 (0.5 mL) and SRS solution (0.5 mL) were weighed in 2 mL twist-top Eppendorf tubes, diluted with water (1 mL) and acidified (with 72% mass H2SO4) to a pH of 0.3. The contents were vortexed thoroughly and autoclaved (121°C for 60 min; autoclaving did not affect mass). After cooling to room temperature, the autoclaved tube contents were filtered through a 0.45 μm nylon filter and injected onto the HPLC system described earlier. After SRS correction for acid decomposition of sugars and subtraction of the monosaccharide composition results, the difference was converted to polysaccharide mass equivalents (using appropriate multiplication factors as listed earlier) in order to arrive at the composition of the soluble oligosaccharides in LF1.
A small amount of freeze dried fibre, enough to cover the surface of the probe, was placed on the diamond probe of a Thermo Nicolet 870 FTIR (software: OMNIC 7.3). The sample was pressed with an anvil to increase the surface contacting the probe. Sixty-four scans were acquired for each spectrum and the two replicate spectra for each sample were overlayed. No differences in the replicate spectra of this study were observed and thus only the first spectrum of each sample was used for analysis.
Acetyl bromide for lignin quantification in solid fractions 2 and 3
The acetyl bromide method as described by Iiyama and Wallis  was used to determine the% mass lignin content of solid fractions 2 and 3. Freeze dried solids (ca. 10 mg) were weighed in glass tubes and acetyl bromide in acetic acid (25% mass, 10 mL) and then perchloric acid (70% mass, 0.1 mL) were added. The tubes were sealed with Teflon screw caps and placed in temperature controlled rotary shaker (70°C and 100 rpm for 30 min). After cooling to room temperature the tubes were opened and 2 M NaOH (10 mL) and then glacial acetic acid (25 mL) were added. After agitation, absorbance (280 nm, quartz cuvettes, Cintra UV spectrometer) was measured against glacial acetic acid. The resulting solution was analysed with a Cintra-40 UV spectrometer and the absorbance was referenced to a cuvette with glacial acetic acid. Dilutions with glacial acetic acid were necessary for some samples so that the absorbance was <1.0. The absorbance was converted to percent mass concentration of lignin using an extinction coefficient of 25 L·g-1·cm-1. The extinction coefficient was determined with the use of a calibration curve based on bagasse of known lignin content. The estimate of standard deviation (absolute) of this technique (duplicate samples of untreated bagasse and soda lignin, 4 df) (as% dry mass of solid analysed) is 3.
Enzymatic saccharification of solids from 3 IL treatments
Enzymatic hydrolysis experiments (SF1 fraction) were performed in 20 mL scintillation vials on a rotary shaker (150 rpm, 50°C) in volumes of 5 mL with a biomass load of 50 mg cellulose equivalent and Accelerase 1000 (Genencor) activity of 15 FPU g-1 (25 μL of Accelerase as received) in 50 mM citrate buffer (pH 4.7). Samples (0.2 mL) were periodically removed, placed in ice, then in boiling water (2 min) and centrifuged. Cellobiose, glucose and xylose concentrations were measured by HPLC (HPLC system as described earlier except a Shodex SPO-810 HPLC column at 85°C with a mobile phase of ultrapure water at 0.6 L min-1 were used.). The glucose and cellobiose results were converted to glucan mass equivalents and xylose was converted to xylan mass equivalents using appropriate multiplication factors.
The estimates of standard deviation (absolute) of this analysis (based on duplicate IL pretreatments’ saccharification extents at different time points, 18 df) are 2% mass of glucan and 0.9% mass of xylan.
Recovery of IL
IL set aside at the start of mass balance experiments (starting IL) was brought to a volume of 50 mL with deionised water. Similarly, known masses of LF1 were diluted with deionised water and injected onto the ion chromatograph (Metrohm 761 with a conductivity detector). For cation analysis, samples were injected onto a Metrosep C 2 150 (150 mm × 4 mm) column with an aqueous mobile phase (25% volume acetone, 6 mM tartaric acid and 0.75 mM dipicolinic acid) at 1 mL min-1. For anion analysis samples were injected onto a Metrosep ASupp5 (150 mm × 4 mm) column with an aqueous mobile phase (1 mM NaHCO3 and 3.2 mM Na2CO3) at 0.7 mL min-1 and suppressed by post-column addition of H2SO4 (50 mM). IL mass balance was determined from the results of these analyses. The estimate of standard deviation (absolute) of this technique (as% mass of ions in starting IL) for both cations and anions and for all 3 ILs is 2 (based on duplicate IL pretreatments, 6 df).