Inoculum preparation

Aqueous spores suspension of Aspergillus niger was prepared in the Erlenmeyer flask (500ml) by growing the fungus in inoculum medium for 3 days at pH 4.5 and 37oC temperature. The inoculum medium was the 50X Vogel’s medium (Vogal 1956).


Composition of Vogal’s medium used for preparation of inoculum of Aspergillus niger.

s.no


Components Quantity (g/100ml)

1 KH2PO4 0.5

2 NH4NO3 0.2

3 (NH4)2SO4 0.4

4 MgSO4.7H2o 0.02

5 Peptone 0.1

6 Trisodium citrate 0.5

7 Yeast Extract 0.2

8 Glucose 50 % w/v

9 Distilled water 100ml


pH, 5.5; temperature, 37oC.


The inoculum was prepared as described below;

a. 100 ml Vogal’s medium was taken in 550ml conical flask and marble gravels (2-3) were added in the flask.

b. The medium was adjusted at pH 5.5 using 1M NaOH/1M HCl and autoclaved (121oC) for 15minutes under 15 lb/in2 pressure.

c. 50 % W/V sterilized solution of glucose was prepared. Glucose with a concentration of 2% (v/v) was aseptically added in laminar flow in the autoclaved Vogal’s medium flask as a carbon source.

d. A loopful spore of Aspergillus niger were transferred to the sterilized medium with the help of sterilized pipette and the flask was incubated at 30oC for 3 days in rotary shaker operating at 20 rpm. The spore suspension was adjusted to get 106--108 spores/ml.

MATERIALS AND METHODS

The present project was done in the Industrial Biotechnology Lab in the Department of Chemistry & Biochemistry.


3.1 Production of citric acid from Aspergillus niger

3.1.1Fermentative Organism



Pure wild type culture of Aspergillus niger was obtained from Industrial Biotechnology Lab (IBL). The stock culture was raised on Potato dextrose agar (PDA). The pH of the medium was adjusted to 4 with 1 M HCl/ 1M NaOH and autoclaved for 15 min at 121 oC under the pressure of 15 lb/in2. Fungal culture was transferred aseptically to PDA medium in Laminar airflow. The slants will be incubated at 28oC for 7 day and preserved at 4oC for two months.

Composition of PDA medium for sporulation of Aspergillus niger

S. no Components Quantity g/100ml


1 Potato Starch 2.0

2 Glucose 2.0

3 Urea 0.3

4 ZnSO4.7H2O 0.001

5 KH2PO4 0.008

6 KCl 0.015

7 Agar 2.0

8 MgSO4.7H2O 0.05

9 Distilled water 100ml

PH, 5.5; temperature, 37oC.


pH of the medium was adjusted to 5.5 with 1 M HCl/ 1M NaOH and autoclaved for 15 min at 121oC under the pressure of 15 lb/in2. The media was poured into the autoclave test tubes and the test tubes were left undisturbed in slanting position at 250C for solidification. Fungal culture was transferred aseptically to PDA medium slants in laminar airflow. The slants were incubated at 28oC for 3 days for sporulation and preserved at 4oC for subsequent use for citric acid production.