Eucalyptus red grandis pretreatment with protic ionic liquids: effect of severity and influence of sub/super-critical CO2 atmosphere on pretreatment performance

Deconstruction of lignocellulosic biomass with low-cost ionic liquids (ILs) has proven to be a promising technology that could be implemented in a biorefinery to obtain renewable materials, fuels and chemicals. This study investigates the pretreatment efficacy of the ionoSolv pretreatment of Eucalyptus red grandis using the low-cost ionic liquid triethylammonium hydrogen sulfate ([N2220][HSO4]) in the presence of 20 wt% water at 10% solids loading. The temperatures investigated were 120 °C and 150 °C. Also, the influence of performing the pretreatment under sub-critical and supercritical CO2 was investigated. The IL used is very effective in deconstructing eucalyptus, producing cellulose-rich pulps resulting in enzymatic saccharification yields of 86% for some pretreatment conditions. It has been found that under a CO2 atmosphere, the ionoSolv process is pressure independent. The good performance of this IL in the pretreatment of eucalyptus is promising for the development of a large-scale ionoSolv pretreatment processes.


Moisture content
For both raw and pretreated biomass the moisture content was determined according to the protocol by the National Renewable Energy Laboratory (NREL) "Determination of Total Solids in Biomass and Total Dissolved Solids in Liquid Process Samples" 1 by weighing out approximately 100 mg of biomass/pulp onto a preweighed piece of aluminium foil and recording the weight using the analytical balance. The foil with the biomass/pulp was folded and oven dried (T = 105 °C) overnight. The hot packets were placed in a desiccator to allow cooling to room temperature. The weight was recorded immediately, and the moisture content calculated.
The moisture content of all Eucalyptus Red Grandis samples (raw and pretreated) ranged from 7% -9%.

Fractionation of Biomass
The ionic liquid/water mixture was prepared by adding 20 wt% of water to the hydrogen sulfate ionic liquid triethylammonium hydrogen sulfate [N2220][HSO4] and mixing until a homogenous solution was obtained. The water content was confirmed by Karl-Fischer titration in triplicate. The required amount of ionic liquid/water mixture was weighed into a 15 ml glass pressure tube with silicone front seal (Ace Glass) and the exact weight recorded. The air-dried biomass (Eucalyptus Red Grandis) was added to obtain a solvent ratio of 1:10 g/g. The containers were capped, and the content mixed with a vortex shaker. The samples were then placed into a preheated convection oven (OMH60 Heratherm Advanced Protocol Oven). After the pretreatment period, they were taken out and left to cool at room temperature. After the Electronic Supplementary Material (ESI) for RSC Advances. This journal is © The Royal Society of Chemistry 2020 pretreatment, 40 mL of ethanol was added to the pretreatment mixture and the suspension transferred into a 50 mL centrifuge tube. The tube was shaken for one minute and the mixture then left at room temperature for at least 1 hour. The tube was mixed again for 30 seconds and then centrifuged at 3500 rpm for 1 hour. The supernatant was decanted carefully into a round bottom flask. The washing step was repeated three more times. The remaining pulp was then transferred into a cellulose thimble and further washed by Soxhlet extraction with refluxing ethanol (150 mL) for 24 hours. The thimbles were then left on the bench overnight to dry. The ethanol used for the Soxhlet extraction was combined with the previous washes and evaporated under reduced pressure at 40 °C, leaving the dried ionic liquid/lignin mixture.
To the dried ionic liquid/lignin mixture, 30 mL of water was added to precipitate the lignin. The suspension was then transferred into a 50 mL falcon tube, shaken for one minute and then left at room temperature for at least 1 hour. The tube was centrifuged and the supernatant decanted and collected in a round bottom flask. This washing step was repeated twice more. The air-dried pulp yield was determined by weighing the recovered biomass from the cellulose thimbles. The oven-dried yield was determined as described in section 1.1.
The lid of the Falcon tube containing the lignin was removed. The tube was covered with a cellulose-paper, to avoid lignin contamination or losses and put into a vacuum oven overnight to dry at 40 °C under vacuum. The dried lignin was weighed the next day to calculate the lignin yields and delignification. • 100% Where, Ligninuntreated is the lignin content in untreated biomass (Eucalyptus Red Grandis), Ligninpulp is the lignin content in the pulp and Yieldpulp is the oven-dried yield of pulp.
The experiments under CO2 atmosphere were carried in a similar way. The biomass and ionic liquid were weighted in a glass liner, which was placed into the Parr reactor described in the experimental section of the main manuscript. The reactor was sealed according to the manufacturer instructions. The agitator of the reactor was used to mix the biomass and the IL, then it was switched off. The reactor was then pressurized with CO2 to the required pressure.
After the experiments, the reactor was let to cooled down to 50 °C and depressurised slowly by opening the depressuring valve. Once the reactor reached ambient temperature and pressure, it was opened, the glass liner removed and the pretreated biomass washed as described previously.
A video describing the experimental protocol is available for consultation. 2

Extractive determination
Ethanol extractives determination was carried out according to a published procedure by NREL "Determination of Extractives in Biomass" 3 in triplicates. Around 2.5 g were weighted and transferred into a cellulose thimble, which was placed into a Soxhlet extraction apparatus with refluxing ethanol (190 + 5 mL) for 24 hours. After this period, the system was allowed to cool down and the thimbles were washed with fresh ethanol and then left overnight to dry at room temperature. The solid were dried and the extractive content calculated:

Compositional Analysis
Compositional analysis was carried out according to a published procedure by NREL "Determination of Structural Carbohydrates and Lignin in Biomass" 4 . 300 mg (on oven dry basis) of air-dry biomass after pretreatment and raw biomass without the extractive was weighed into a 100 mL pressure tube and the weight recorded. 3 mL of 72% sulfuric acid was added, the samples stirred with a Teflon stir rod and the pressure tubes placed into a preheated water bath at 30 °C. The samples were stirred again every 15 min for one hour, they were then diluted with 84 mL distilled water and sealed. The samples were autoclaved (Sanyo Labo Autoclave ML5 3020 U) for one hour at 121 °C and left to cool. The samples were then filtered through filtering ceramic crucibles of a known weight. The filtrate was stored in two plastic tubes and the remaining residue washed with distilled water. The crucibles were placed into a convection oven (VWR Venti-Line 115) at 105 °C for 24±2 hours. They were placed in a desiccator for 15 min and the weight recorded. The crucibles were then placed into a muffle oven (Nabertherm + controller P 330) and ashed to constant weight at 575 °C. The crucible weight after ashing was recorded. The content of acid insoluble lignin (AIL) was determined according to Eq. 9. %AIL = Weight crucible+ AIR -Weight crucible+ ash Weight oven−dry sample • 100 where Weightcrucibles + AIR is the weight of the oven-dried crucibles plus the acid insoluble residue, Weightcrucibles + ash is the weight of the crucibles after ashing to constant temperature at 575 °C. The supernatant was used for the determination of acid soluble lignin content (ASL) by UV analysis at 240 nm (Eq. 10) using a Perkin Elmer Lambda 650 UV/Vis spectrometer.
Where A is the absorbance at 240 nm, l is the path length of the cuvette in cm (1 cm in this case), ε is the extinction coefficient (25 L/g cm), c is the concentration in mg/mL, the ovendried weight of the sample in mg and Vfiltrate is the volume of the filtrate in mL and equal to 86.73 mL. Calcium carbonate was added to the second liquid fraction until pH 5 was reached. The liquid was passed through a 0.2 µm PTFE syringe filter and subsequently submitted to HPLC analysis (Shimadzu, Aminex HPX-97P from Bio-Rad, 300 x 7.8 mm, purified water as mobile phase at 0.6 ml/min, column temperature 85 °C, de-ashing columns were used as pre-filters) for the determination of total sugar content. Calibration standards with concentrations of 0.1, 1, 2 and 4 mg/mL of glucose, xylose, mannose, arabinose and galactose were used. Sugar recovery standards were prepared as 10 mL aqueous solutions close to the expected sugar concentration of the samples and transferred to pressure tubes. 278 µL 72% sulfuric acid was added, the pressure tube closed and autoclaved and 3 the sugar content determined as described above. The sugar recovery coefficient (SRC) was determined according to Eq. 11 and the sugar content of the analysed sample using Eq. 12.
where cHPLC is the sugar concentration detected by HPLC, V is the initial volume of the solution in mL (10.00 mL for the sugar recovery standards and 86.73 mL for the samples), initial weight is the mass of the sugars weighed in, corranhydro is the correction for the mass increase during hydrolysis of polymeric sugars (0.90 for the C6 sugars glucose, galactose and mannose and 0.88 for the C5 sugars xylose and arabinose) and the oven-dried weight of the sample in mg.

Saccharification Assay
Saccharification assays were carried out according to a protocol published by the NREL "Low Solids Enzymatic Saccharification of Lignocellulosic Biomass" 5 in triplicate with blanks (also in triplicate). All reagents were purchased from Sigma Aldrich. The detailed procedure is as follows: 100±10 mg (ODW basis) of air-dried was placed into a Sterilin tube and the weight recorded. Three enzyme only blanks were run with 100 µL of purified water instead of biomass in order to correct for sugar residues present in the enzyme solutions. 9.9 mL solution made from 5 mL 100mM sodium citrate buffer at pH 4.8, 40 µL tetracycline solution (10 mg/mL in 70% ethanol), 30 µL cycloheximide solution (10 mg/mL in purified water), 4.71 mL purified water and 50 µL of Novozymes experimental enzyme mixture NS-22201 was added, the tubes closed and placed into an Stuart Orbital Incubator (S1500) at 50 °C and 250 rpm for 7 days at 50°C and 250 rpm. End point samples were obtained by filtering 1 mL of the saccharification mixture through a PTFE syringe filter. Samples were analysed on Shimadzu HPLC system with RI detector and an Aminex HPX87P column (BioRad, 300 x 7.8 mm) with purified water as mobile phase (0.6 mL/min). The column temperature was 85°C and acquisition time was 40 min. Calibration standards with concentrations of 0.1, 1, 2 and 4 mg/mL of glucose, xylose, mannose, arabinose and galactose and 8 mg/mL of glucose were used. Figure S1. Typical reaction medium temperature inside an unstirred reaction tube (15 ml) at an oven temperature of 150°C for different pretreatments times.