3.2.1. Direct delignification
Delignification of MxG with no prior treatment, in this work called direct delignification, was performed applying a modified organosolv method. In his work, Roque (2013) optimised lignin extraction from MxG using water/ethanol under subcritical conditions and applying CO2 as modifier to acidify the media (Roque, 2013). The process extracts lignin as well as hemicellulose and extractives from the lignocellulose matrix, leaving a solid cellulose-enriched fibre.
250mL of 50% (v/v) ethanol and distilled water was prepared and pre-heated to 50oC. 5g of MxG was then soaked in the solution for 20min. As biomass is not soluble in water, the soaking stage prior to the reaction is performed in order to increase the wettability, i.e., facilitate the interaction between water and biomass (Matsunaga et al., 2008). The biomass and solution mixture was ground in a blender (Philips, 400W, 1.5L) for 3min.
The rig used for reaction (Figure 3-1) was composed of a 500mL high-pressure reactor (Parr, alloy C276), a heating jacket, an automatic stirrer, inlet and outlet valves, a cooling loop, and a controller (Parr, model 4836). The same rig was used for sequential extraction described in section 3.2.2.
Ground biomass suspension was placed inside the reactor and the reactor was sealed. Vapour withdrawal CO2 (BOC UK, 99.8%) was used to purge the air inside the reactor and pressurise the reactor to ~50bar. The reactor heating jacket was then turned on and temperature
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was set to 200oC. The reaction lasted for 1h after the temperature achieved the set point. Temperature was kept constant during the reaction time. After that, the reactor temperature was decreased by inserting the reactor in an ice bath and turning on the cooling line.
Figure 3-1 - Scheme of the rig used for direct delignification and for the three steps of sequential extraction.
When the temperature cooled to ~60oC, the reactor was depressurised and opened. Cooling time was usually between 5-10min. The solid fraction (fibres) were vacuum filtered with a porcelain sintered disc (porosity 1) and rinsed three times with 50mL (each time) of water/ethanol solution (1:1) heated to about 50oC. The liquid fraction was stored in a freezer for analysis in High Performance Anion Exchange Chromatography (HPAEC). Fibres were completely dried at 65oC and then placed into a desiccator until room temperature. Dried fibres were weighed and recovery of solid fraction was calculated by Equation 3-1:
π ππππ£πππ¦ (%) = ππ
ππ β 100 Equation 3-1 In which:
ππ = initial mass of Miscanthus used for delignification (~ 5g) ππ = final solid fibres after delignification.
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After the delignification, percentage of lignin remaining in the recovered fibres was quantified by Klason lignin procedure. Delignification percentage was calculated by Equation 3-2.
π·πππππππππππ‘πππ (%) =πππππππ β πππππππ
πππππππ β 100 Equation 3-2
In which lignini/f are the amount of initial/final lignin in grams calculated by multiplying mi and mf by the percentage of Klason lignin.
The fibres remaining from direct delignification were named βDELβ fibres in this work. It is possible to recover lignin from the liquid fraction after modified organosolv delignification method by decreasing the ethanol concentration of the liquid fraction followed by centrifugation. However, this step was not performed in this work.
3.2.2. Sequential extraction
Sequential MxG extraction was performed in three steps described below. 1. Extraction of non-bounded compounds
200mL of distilled water was pre-heated at 50oC and used to soak 10g of MxG for 20min. The suspension was then ground in a blender for 3min. The same reactor rig used for direct delignification was used for all 3 steps of sequential extraction. Ground biomass was transferred to the reactor. After sealing the reactor, the air was purged and the reactor was pressurised to ~50bar using nitrogen. Temperature was set to 120oC. Once this temperature was achieved, the reaction lasted for 30min at constant temperature.
At the end of 30min, the reactor was inserted into an ice bath and the cooling line was opened. When temperature was ~60oC, the reactor was depressurised and opened. The solid fraction (fibres) was sieved in a 45-mesh sieve and dried at room temperature for 48h. These
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fibres were named β120oCβ fibres throughout this work and the liquid fraction was stored at - 20oC until analysis by HPAEC.
2. Extraction of hemicellulose
Hemicellulose extraction was performed after removal of biomass extractives in the first step. 10g of 120oC fibres were soaked in 200mL of distilled water (50oC) for 20min and then transferred to the reactor.
After sealing and purging the reactor, pressure was increased to ~50bar using nitrogen. Temperature was set to 180oC and the reaction lasted for 30min at constant temperature. After the reaction time, the reactor was cooled to ~60oC using an ice bath and the cooling line, and then opened. Fibres were once again sieved using the 45-mesh sieve and dried at room temperature for 48h. These fibres were named β180oCβ fibres and liquid fraction was kept in - 20oC until analysis by HPAEC.
Xylan, arabinan and galactan extraction were calculated by Equation 3-3: ππππ ππ₯π‘ππππ‘πππ (%) =(πππππ β πππππ)
πππππ β 100% Equation 3-3
In which carbi/f is the initial/final mass (in grams) of the carbohydrate (xylan, arabinan and galactan). Carbohydrates initial/final mass was calculated by using their percentage in initial/final fibres obtained using HPAEC and multiplying it by initial/final mass.
3. Extraction of lignin
The exactly same procedure for direct extraction of lignin was used in this step. However, instead of using raw MxG, 180oC fibres were used in this third step and no additional grinding was performed (because these fibres were ground in the 1st-step extraction). After delignification, the fibres were named βSEQβ fibres. The liquid fraction was kept at -20oC until analysis by HPAEC.
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3.2.3. Qualitative analysis of liquid fractions
Liquid fractions resulting from each step of sequential extraction and also after direct delignification were analysed using HPAEC for carbohydrates quantification. HPAEC method is described in section 2.6. Prior to analysis, samples were hydrolysed using the 2-step acid hydrolysis of Klason lignin procedure (section 2.3) in order to obtain monosaccharides, which are more easily quantified by HPAEC.
3.2.4. Qualitative analysis of solid fractions (fibres)
After the extractions performed (sequential and direct), resulting fibres (120oC, 180oC, SEQ and DEL) were analysed using SEM and FTIR as well as evaluated by PCA. The methods used are described in sections 2.4 and 2.5. For the FTIR and PCA analysis, commercial cellulose (Avicel PH 101, Sigma, UK) was also evaluated for comparison with fibres obtained from MxG.
Klason lignin contents of MxG and each fibre obtained after biomass processing (120oC, 180oC, SEQ and DEL) was quantified according to Klason lignin procedure. Hemicellulose fraction (xylan, arabinan and galactan), was quantified by HPAEC after 2-step acid hydrolysis.