Fungal Production of Single Cell Oil Using Defatted Oilseed …
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2.3 Screening and Isolation of Oleaginous Fungi
Oleaginous fungi were isolated from the collected soil sample by serial dilution
using 0.9% sterile saline water and plating in potato dextrose agar (PDA) medium
and incubated at 28 °C for 2–4 days. The isolated colonies were further screened for
their lipid producing abilities by qualitative analysis with the Sudan Black B staining
technique (Thakur et al. 1988).
2.4 Identification of Fungi
Fungal strains were identified by 18s rDNA amplification followed by denaturation,
annealing, and extension by Bio Axis DNA Research Centre (P) Ltd., Hyderabad.
2.5 Preparation of Oilseed Meals
About 25 g of each flaxseed, black mustard seed, and rice bran were collected from
the market and dried to reduce the moisture content and subjected to oil extraction
by Soxhlet after grinding.
2.6 Proximate Composition of the Meals
Proximate composition of the deoiled meals, such as moisture, carbohydrate, protein,
ash, and fiber, was determined by standard AOAC methods (2000).
2.7 Inoculation of Filamentous Fungi in Fermentation
Media for Oil Production
The activated seed cultures of oleaginous fungi were transferred into preculture
medium consisted of (in g/L): glucose 30 g, peptone 5 g, yeast extract 5 g with initial
pH of 5.0 and grown at 28 °C for 2–3 days. Ten percent of precultures were added
to two different fermentation media for determination of highest growth and lipid
production. Medium I contained (in g/L): glucose 50 g, (NH 4 ) 2 SO 4 3 g, KH 2 PO 4
0.8 g, K 2 HPO 4 0.2 g, MgSO 4 , 7H 2 O 0.5 g and Medium II contained 100 mL of
potato dextrose broth. The fermentation media were incubated at 28 °C and biomass
growth and lipid production were measured for 10 days. In another experiment, 1, 2,
53
2.3 Screening and Isolation of Oleaginous Fungi
Oleaginous fungi were isolated from the collected soil sample by serial dilution
using 0.9% sterile saline water and plating in potato dextrose agar (PDA) medium
and incubated at 28 °C for 2–4 days. The isolated colonies were further screened for
their lipid producing abilities by qualitative analysis with the Sudan Black B staining
technique (Thakur et al. 1988).
2.4 Identification of Fungi
Fungal strains were identified by 18s rDNA amplification followed by denaturation,
annealing, and extension by Bio Axis DNA Research Centre (P) Ltd., Hyderabad.
2.5 Preparation of Oilseed Meals
About 25 g of each flaxseed, black mustard seed, and rice bran were collected from
the market and dried to reduce the moisture content and subjected to oil extraction
by Soxhlet after grinding.
2.6 Proximate Composition of the Meals
Proximate composition of the deoiled meals, such as moisture, carbohydrate, protein,
ash, and fiber, was determined by standard AOAC methods (2000).
2.7 Inoculation of Filamentous Fungi in Fermentation
Media for Oil Production
The activated seed cultures of oleaginous fungi were transferred into preculture
medium consisted of (in g/L): glucose 30 g, peptone 5 g, yeast extract 5 g with initial
pH of 5.0 and grown at 28 °C for 2–3 days. Ten percent of precultures were added
to two different fermentation media for determination of highest growth and lipid
production. Medium I contained (in g/L): glucose 50 g, (NH 4 ) 2 SO 4 3 g, KH 2 PO 4
0.8 g, K 2 HPO 4 0.2 g, MgSO 4 , 7H 2 O 0.5 g and Medium II contained 100 mL of
potato dextrose broth. The fermentation media were incubated at 28 °C and biomass
growth and lipid production were measured for 10 days. In another experiment, 1, 2,