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3.2 Materials and Methods .1 Reagents and chemicals
3.2.5 Pretreatment of substrates
Lignocellulosic biomass comprise of a complex matrix made up of cellulose and lignin bound by chains of hemicellulose. Pretreatment degrades this matrix reducing the degree of crystallinity of the cellulose with subsequent rise in the amorphous cellulose and hemicellulose fraction for effective enzymatic action (Sanchez and Cardona, 2008). The finely powdered wild grass and water hyacinth were subjected independently to nine pretreatment strategies and one mixed pretreatment strategy.
3.2.5.1 Steam explosion
One gram each of the dry ground wild grass and water hyacinth was taken separately in 100 mL Erlenmeyer flask. In an autoclave, the flasks were kept at 121°C and 15 psi for 1 h. The autoclave was exposed to sudden steam depressurization by completely opening the steam exhaust valve, intending to gain maximum quantity of fermentable sugars in least treatment time (Sharma et al., 2007).
3.2.5.2 Alkali treatment
One gram each of the powdered wild grass and water hyacinth was retained in
autoclaving of the mixture was done at 115°C and 15 psi for 10 min (Okeke and Obi, 1995). Subsequently, the mixture was cooled to room temperature and washed with distilled water. Each washing was followed by centrifugation (5,876g, 10 min, 25ºC).
The residues were washed with distilled water until a pH of 7 was reached.
Subsequently, the residues were dried at 50°C in an oven for 12 h.
3.2.5.3 Wet oxidation
One gram each of the powdered wild grass and water hyacinth was mixed with 17 mL of water. The pH of the mixture was adjusted to 3.5 with the addition of 0.03 mL of concentrated sulphuric acid (36.5%, w w-1). The wet oxidation of each mixture was carried out independently in a 0.5 L high pressure reactor (Amar Equipments, Mumbai, India) by continuous circulation of oxygen gas (at 12 bar) following Palonen method (Palonen et al., 2003) with modification in residence time (1 h) and temperature (120ºC). Subsequently, cooling of each substrate was done to room temperature (25ºC). Finally, vacuum filtration of each substrate was done using a vacuum filtration unit (Millipore, Massachusetts, USA) with nylon membrane having pore size of 0.45 µm. After filtration, the left over residue of each substrate was collected and dried at 50°C in an oven for 12 h.
3.2.5.4 Phosphoric acid (H3PO4) - acetone
One gram each of the powdered wild grass and water hyacinth was mixed with 8 mL of concentrated phosphoric acid in two separate 250 mL Erlenmeyer flask (Li et al., 2009). Each of the mixture was then incubated at 50°C and 120 rpm for 1 h. The slurry was then poured into 24 mL chilled acetone and the mixture was centrifuged at
5,876g for 10 min and 25ºC. The supernatant was discarded and the pellet collected was washed three times with distilled water. Before third wash, the pH of the resuspended pellet in water was adjusted to 5-6 using 1 N NaOH. Then, each pellet was dried at 50°C in an oven for 12 h.
3.2.5.5 Ammonia fibre expansion (AFEX)
The ammonia fibre explosion (AFEX) treatment of each of the powdered wild grass and water hyacinth was carried out by following the method of Holtzapple (Holtzapple et al., 1991) with modification in reaction vessel, residence time and temperature. 1 g each of the powdered substrate was treated with 2.5 mL of ammonia solution in crucible. The crucibles were covered with aluminium foil restricting the entry of air and kept at 100°C for 1 h in a hot air oven. Then, the aluminium foil was removed and the samples were kept in a fumigating hood overnight to remove the residual excess ammonia.
3.2.5.6 Organosolv pretreatment
One gram each of the powdered wild grass and water hyacinth was treated with ethanol: water mixture (70:30 v v-1) containing 1% of concentrated sulphuric acid, hydrochloric acid, acetic acid and phosphoric acid at 70⁰C for 1 h. The pretreated substrates were then washed initially with 95% (v v-1) ethanol at 60⁰C for 4 h and then 70% (v v-1) ethanol at 30⁰C for 1 h. The residues were then treated with hydrogen peroxide for 16 h and again washed with 70% ethanol at 30⁰C for 1 h (Geng et al., 2012). Then, each residue was dried at 50°C in an oven for 12 h.
3.2.5.7 pH controlled hot water pretreatment
One gram each of the powdered wild grass and water hyacinth was mixed with 20 mL of water at pH 4.0 (adjusted by 1 mL of concentrated sulphuric acid,
36.5%, w w-1). The pH controlled hot water pretreatment of each mixture was carried out in a 0.5 L high pressure reactor (Amar Equipments, Mumbai, India) by continuous circulation of steam following the method described by Mosier et al., 2005 with modification in pressure (8 bar), residence time (40 min) and temperature (90°C).
Subsequently, each mixture was cooled down to room temperature (25ºC). Thereafter, vacuum filtration of each substrate was done using a vacuum filtration unit (Millipore, Massachusetts, USA). A nylon membrane of pore size 0.45 µm was used for filtration. Then, the left over residue of each substrate was collected and dried in an oven (50°C) for 12 h.
3.2.5.8 Dual step dual temperature (DSDT) mild acid hydrolysis
One gram each of the powdered wild grass and water hyacinth was mixed separately with 1% (v v-1) dilute sulphuric acid adjusted at pH 3.5. The dual step dual temperature (DSDT) mild acid hydrolysis of each mixture was carried out independently in a 0.5 L high pressure reactor (Amar Equipments, Mumbai, India) by continuous circulation of steam (at 10 bar) following the method of Bosch et al., (2010) with modification in residence time and temperature (8 min at 90°C and 6 min at 99°C). Finally, each substrate was filtered using a vacuum filtration unit (Millipore, Massachusetts, USA) with a nylon membrane of pore size (0.45 µm). The residue of each substrate was collected and dried in an oven at 50°C for 12 h.
3.2.5.9 Microwave assisted alkali (MAA) pretreatment
Twenty gram each of the powdered wild grass and water hyacinth was suspended separately in 160 mL of 1% (v v-1) sodium hydroxide solution in two 500 mL beakers. Each beaker was positioned at the centre of a rotating circular glass plate in a domestic microwave oven for microwave treatment. The applied microwave power was 900 W for 25 min (Zhu et al., 2006). Then, each substrate was filtered by a vacuum filtration unit (Millipore, Massachusetts, USA) with a nylon membrane of pore size (0.45 µm). Then, the residue of each substrate was collected and dried in an oven at 50°C for 12 h. Finally, 1 g of each substrate was subjected for further studies.
3.2.6 Mixed microwave-assisted alkali (MAA) and organosolv pretreatment