Value Addition of Agricultural Wastes for Improved Production …
267
flasks were placed at 30 °C for 48 h to activate the fungi. All the media components
were purchased from Himedia Laboratories Pvt. Ltd., India (Mumbai, India). All the
chemicals used were of analytical grade and were procured from Merck, India.
2.2 Lignocellulosic Biomass Collection and Pretreatment
Cauliflower wastes (stalk and leaf) were collected from local market of Shibpur,
Howrah, West Bengal, India. They were first washed with deionized water to remove
dirt and impurities. Finely crushed cauliflower wastes were pretreated separately with
1% (v/v) 0.1 N dilute HCl and 1% (w/v) 0.1 N alkali (NaOH). For organosolvent
pretreatment, 10 gm of cauliflower stalk and leaf were suspended separately in 100 ml
of methanol with 0.1 M sodium acetate as catalyst. Pretreatment was carried out in
autoclave at 121 °C for 15 min. The solution was centrifuged and the solid portions
were collected and washed with distilled water and the solution was adjusted to pH
7 with 0.1 M HCl. The pretreated cauliflower wastes were dried in oven at 50 ± 5 °C
and stored in airtight container for further use.
2.3 Inoculum Preparation
The inoculum was prepared by growing the fungal strains under submerged fermentation in 150 ml Erlenmeyer flasks containing 50 ml of sterile Czapek Yeast extract
(for A. oryzae) and Malt extract and Malt Yeast extract media (for A. niger and
C. intermedia), respectively. Fungi were co-cultivated by inoculating 2 μl of spore
suspension on PDA plate with a distance of 3 cm by mixing the spores and inoculating
it in the centre. The plates were incubated at 29 °C for 7 days.
2.4 Evaluation of Cauliflower Wastes for Extracellular
Enzyme Production by Submerged Fermentation
In the first set of experiment, fermentation was carried out in 250 ml of Erlenmeyer
flasks containing 5 gm (dry weight) of each above-mentioned pretreated wastes as
substrate was dissolved in distilled water and autoclaved at 121 °C for 15 min,
cooled and then inoculated with spore suspension containing 1 × 10
7 spores/ml of
actively growing mycelia of A. oryzae, A. niger and C. intermedia. In another set of
experiment, fermentation was carried out using spore suspension of co-cultures of
the above fungi (in the ratio of 1:1:1) under the same conditions as described above.
All the flasks were incubated under static condition at 30 °C and enzymatic extraction
267
flasks were placed at 30 °C for 48 h to activate the fungi. All the media components
were purchased from Himedia Laboratories Pvt. Ltd., India (Mumbai, India). All the
chemicals used were of analytical grade and were procured from Merck, India.
2.2 Lignocellulosic Biomass Collection and Pretreatment
Cauliflower wastes (stalk and leaf) were collected from local market of Shibpur,
Howrah, West Bengal, India. They were first washed with deionized water to remove
dirt and impurities. Finely crushed cauliflower wastes were pretreated separately with
1% (v/v) 0.1 N dilute HCl and 1% (w/v) 0.1 N alkali (NaOH). For organosolvent
pretreatment, 10 gm of cauliflower stalk and leaf were suspended separately in 100 ml
of methanol with 0.1 M sodium acetate as catalyst. Pretreatment was carried out in
autoclave at 121 °C for 15 min. The solution was centrifuged and the solid portions
were collected and washed with distilled water and the solution was adjusted to pH
7 with 0.1 M HCl. The pretreated cauliflower wastes were dried in oven at 50 ± 5 °C
and stored in airtight container for further use.
2.3 Inoculum Preparation
The inoculum was prepared by growing the fungal strains under submerged fermentation in 150 ml Erlenmeyer flasks containing 50 ml of sterile Czapek Yeast extract
(for A. oryzae) and Malt extract and Malt Yeast extract media (for A. niger and
C. intermedia), respectively. Fungi were co-cultivated by inoculating 2 μl of spore
suspension on PDA plate with a distance of 3 cm by mixing the spores and inoculating
it in the centre. The plates were incubated at 29 °C for 7 days.
2.4 Evaluation of Cauliflower Wastes for Extracellular
Enzyme Production by Submerged Fermentation
In the first set of experiment, fermentation was carried out in 250 ml of Erlenmeyer
flasks containing 5 gm (dry weight) of each above-mentioned pretreated wastes as
substrate was dissolved in distilled water and autoclaved at 121 °C for 15 min,
cooled and then inoculated with spore suspension containing 1 × 10
7 spores/ml of
actively growing mycelia of A. oryzae, A. niger and C. intermedia. In another set of
experiment, fermentation was carried out using spore suspension of co-cultures of
the above fungi (in the ratio of 1:1:1) under the same conditions as described above.
All the flasks were incubated under static condition at 30 °C and enzymatic extraction