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

To construct a growth curve using aspergillus niger.

Aim: To construct a growth curve using aspergillus niger.

Requirements: Master culture of Aspergillus niger
Prepared Potato dextrose agar(PDA) slants
Flasks
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:
Form master culture of Aspergillus niger, potato dextrose agar slants were incubated at room temp. for 4 days. The medium containing. Above composition was prepared and sterilized. The spores were suspended in sterile water and inoculated in to above medium.this kept on rotary shaker. After two days 10 ml of this broth was used as inoculum for another flask containing 100ml of medium and placed on rotary shaker.
Samples of 10ml were withdrawn using sterile pipette at 24hr interval and transplanted in to clean test-tubes. Samples were collected at 24,48,72,96hr. cell mass of each sample was determined by weighing the cell sediment. After centrifugation and decanting the supernant and weighing the empty tubes later. The differences in the two weight give the cell mass.
Prior to weighing the cell sediment, cells were dried by vacuum drier at 550c overnight. Dry weight of mycelium was found per each sample and noted against sampling hour. A graph plotted taking time on X-axis and weight of dried mycilium on Y-axis. A culture was drawn smoothly through the points, which is the fungal curve.

Observation:

Fungal growth curve:

Result:

As shown in curve we get the growth in the fungus till 72 hrs. Then after there was a decline in the growth of fungus.

Conclusion:

As shown in the graph we can see the four phases in the growth of the fungus.
Lag phase: Here the lag phase is for 24 hrs, which can be observed from the graph. There is a lag in the growth during these hours in fungus.

Log phase: Here the log phase is for 48 hrs in which the growth of fungus slowly increases by utilizing the nutrient from the media.

Stationary phase: After this there was a segment growth of fungus. This is due to lack of sufficient nutrient. Here the stationary phase last for small period of time.
Decline phase: After the stationary phase there is a decline in fungus growth due to total lack of nutrient or formation of some toxic material.