Thursday, April 19, 2007

To determine the yield constant of Aspergillus niger.

Aim: To determine the yield constant of Aspergillus niger.

Requirements: slant culture of A. niger 250ml and 500ml indented flask Centrifuged bottles Sterile water

Medium composition:
Sucrose 3gm
Sodium nitrate 0.2gm
Potassium dihydrogen phosphate 0.1gm
Magnesium sulphate 0.05gm
Potassium chloride 0.05gm
Ferrous sulphate 0.0001gm
Distilled water 1000ml
pH 5.6

Procedure:
150ml of above medium was prepared and 100ml was taken into 500 ml indented flask and 50ml into 250ml flask, these two flask were sterilized.
A slant culture of A.niger was taken and spores were suspended in sterile water by scarapping the surface of slant. The spore suspension was inoculated into the flask, were kept on rotary shaker. After 4days whole broth was centrifuged and supernatane was transferred to measuring cylinder. The volume was noted the cells were washed with water and centrifuged again and later cell weighed was determined and noted. Similarly cell weight was determined.

Estimation of glucose content by DNS method:
To 1ml of the sample , 1ml of distilled water and 3ml of the DNS reagent were added tubes, were shaken and heated in boiling water bath for 5-10min. Tubes were cooled at room temp. To this 5ml of distilled water was added and tubes were shaken well and optical density of the resulting solution at 600nm against blank. Blank was prepared by omitting the sample glucose content of the sample was calculated from the standard graph.


Preparation of DNS (cinitrisalicylic acid) reagent:

Solution A: 250gm of Rochelle salt was added to a mixture of 300ml 45% sodium hydroxide and 880ml of 1% DNS solution.
Solution B: 10gm of crystalline phenol and 22ml of 10% sodium chloride solution were mixed and dissolved in distilled water and mixed thoroughly.
Solution C: 69ml of solution B was mixed with 6.9ml of sodium bisulphate thoroughly. Finally solution C was mixed thoroughly with solution A until all rochell salt was dissolved. Then reagent was kept in a closed brown colored glass bottle.



Observation:
Calculation:
Value of culture broth- 10ml
Initial glucose content- 300mg
(uninoculated broth)
Glucose content after 24hr incubation- 29mg
Glucose consumed in 24hr=300-29=271mg
Glucose content after 96hr=300-11=289mg
Glucose in cell between
24hr and 96hr intervals=3.07-0.82=2.25gm
Glucose consumed between
24hr and 96hr interval=289-271=18mg

Yield constant=2.25/18=0.125gm cell/mg of glucose

Result:
Yield constant of A. niger= 0.125g cell/g of glucose

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