268
S. Majumdar and J. Bhowal
was carried out after 7 days of fermentation. All the submerged fermentations were
carried out in triplicate.
2.5 Extraction of Enzyme
After 7 days of incubation, enzymes secreted during submerged fermentation were
extracted with sodium acetate buffer (0.1 M, pH 5) under shaking at 150 rpm for
1 h at room temperature. Solids were separated through centrifugation at 3000 rpm
for 20 min. Supernatants were collected, freeze-dried and stored at −30 °C for
subsequent enzymatic [cellulase (endoglucanase and exoglucanase), β-glucosidase]
and xylanase activities.
2.6 Enzyme Assay
Endo-β1, 4 D-glucanase (carboxymethylcellulase, CMCase) and Exo-β1, 4 Dglucanase (cellobiohydrolase) in the culture supernatants were analysed according to
Ghosh (1987) using 2% carboxymethyl cellulose and 1% microcrystalline cellulose
(Merck Pvt. Ltd. Darmstadt, Germany) as substrate. The absorbance was recorded
at 540 nm using Spectrophotometer (JASCO V620 UV Vis spectrophotometer,
Maryland, USA) (Miller 1959). β-glucosidase (cellobiase) activity was evaluated
according to Herr (1979) using 2 mM p-nitrophenyl-β-D glucopyranoside (pNPG)
(Merck Pvt. Ltd. Darmstadt, Germany) as substrate. Xylanase activity was assayed
according to Bailey et al. (1992) using 1% corncob xylan as substrate.
All the assays were carried out in triplicates.
2.7 Enzymatic Saccharification with Crude Fungal Enzymes
and Commercially Available Enzymes
Enzymatic hydrolysis of organosolvent pretreated cauliflower stalk and leaf was
carried out according to Sindhu et al. (2012). The commercially available enzymes
and crude enzymatic extracts obtained from mono-culture of A. niger and co-culture
cultivations as described previously were used for carrying out the biomass saccharification of cauliflower waste biomasses. In the first substrate batch, a suspension
of organosolvent pretreated (5 gm) waste biomass in 50 mM citrate buffer was incubated separately with 1 ml of commercial cellulases [Celluclast
® from Trichoderma
reesei (700 EGU/g)] and xylanase, [recombinant A. oryzae (≥2500 U/g)] in screwcapped tubes. In another set of experiment, crude fungal cellulases obtained from
mono-culture of A. niger [Endoglucanase (160 ± 0.69 IU ml
−1 ) Exoglucananase
S. Majumdar and J. Bhowal
was carried out after 7 days of fermentation. All the submerged fermentations were
carried out in triplicate.
2.5 Extraction of Enzyme
After 7 days of incubation, enzymes secreted during submerged fermentation were
extracted with sodium acetate buffer (0.1 M, pH 5) under shaking at 150 rpm for
1 h at room temperature. Solids were separated through centrifugation at 3000 rpm
for 20 min. Supernatants were collected, freeze-dried and stored at −30 °C for
subsequent enzymatic [cellulase (endoglucanase and exoglucanase), β-glucosidase]
and xylanase activities.
2.6 Enzyme Assay
Endo-β1, 4 D-glucanase (carboxymethylcellulase, CMCase) and Exo-β1, 4 Dglucanase (cellobiohydrolase) in the culture supernatants were analysed according to
Ghosh (1987) using 2% carboxymethyl cellulose and 1% microcrystalline cellulose
(Merck Pvt. Ltd. Darmstadt, Germany) as substrate. The absorbance was recorded
at 540 nm using Spectrophotometer (JASCO V620 UV Vis spectrophotometer,
Maryland, USA) (Miller 1959). β-glucosidase (cellobiase) activity was evaluated
according to Herr (1979) using 2 mM p-nitrophenyl-β-D glucopyranoside (pNPG)
(Merck Pvt. Ltd. Darmstadt, Germany) as substrate. Xylanase activity was assayed
according to Bailey et al. (1992) using 1% corncob xylan as substrate.
All the assays were carried out in triplicates.
2.7 Enzymatic Saccharification with Crude Fungal Enzymes
and Commercially Available Enzymes
Enzymatic hydrolysis of organosolvent pretreated cauliflower stalk and leaf was
carried out according to Sindhu et al. (2012). The commercially available enzymes
and crude enzymatic extracts obtained from mono-culture of A. niger and co-culture
cultivations as described previously were used for carrying out the biomass saccharification of cauliflower waste biomasses. In the first substrate batch, a suspension
of organosolvent pretreated (5 gm) waste biomass in 50 mM citrate buffer was incubated separately with 1 ml of commercial cellulases [Celluclast
® from Trichoderma
reesei (700 EGU/g)] and xylanase, [recombinant A. oryzae (≥2500 U/g)] in screwcapped tubes. In another set of experiment, crude fungal cellulases obtained from
mono-culture of A. niger [Endoglucanase (160 ± 0.69 IU ml
−1 ) Exoglucananase