Effect of Aeration on Fermentation of Neomycin

 

Someswara Rao. B*, Ellaiah. P and Ramesh. B

Sree siddagnaga College of Pharmacy, B. H. Road, Tumkur (Karnataka)

*Corresponding Author E-mail: bsrao.borigey720@gmail.com

ABSTRACT:

In aerobic fermentations, the supply of oxygen to the microorganism is one of the variables which will affect the yield. An aerobic fermentation can be carried out only when the oxygen, which the organism is continuously absorbing from the mash, is continuously replaced and hence the problem of aeration is of considerable importance for successful fermentation.

 

In this study, the supply of air was initially maintained at a high level but was subsequently reduced to varying levels at different stages of the stationary phase and the results were observed and compared with control. The aeration was maintained at uniform level throughout the fermentation cycle.

 

A reduction in the aeration level from 1.0 vvm to 0.25 vvm at all stages of the stationary phase resulted in a decrease in the antibiotic production compared to control maintained at 1.0 vvm level. On the 7^th day  the difference in production of Neomycin narrowed to 400 units out of 7400 units between the sparged flask aerated at 1.0 vvm and the one where aeration was reduced to 0.25 vvm after 120 hours.

 

 

INTRODUCTION:

The production  of substances by aerobic microorganisms when growth on suitable substances has reached the stage, where mass cultivation is a common part of industrial process. On such a scale, the growth of aerobic organisms poses the problem of oxygen supply, that is, the problem of adequate aeration of the culture. Oxygen transfer and agitation are important in maintaining a desirable oxygen environment for mycelial  growth and antibiotic formation in  submerged cultures¹.

 

Since the respiratory enzymes are embedded within aqueous protoplasm, microorganisms can utilize only dissolved oxygen, even if grown at an air water interface. Furthermore; oxygen is insoluble that exist only a small reservoir of it in solution. Upon this reservoir, the microorganisms are continuously drawing and into the reservoir there flows a fresh supply of oxygen to balance the demand. Because the reservoir has such low capacity and oxygen demand of microbial tissue is so high, the rate of supply must equal the rate of demand in every portion  of the culture fluid. Otherwise there will be local or temporary depletion of oxygen which damages the respiratory cells.

 

Such damage was shown by Hromatka, Ebrner and Croklich¹ who found that interruption of air flow to Acetobacter for 15 seconds caused death and disrupted metabolism. Sulphite method was used for measuring aeration efficiency.

 

MATERIALS AND METHODS:

i)       Organism: The organism is Streptomyces marinensis, a streptomycete producing Neomycin and maintained on jowar starch and stored in refrigerator.

ii)      Jowar starch medium contained- jowar starch, cornsteep liquor, (NH₄)₂SO₄, Nacl, agar and P^H adjusted at 6.5.

iii)     Inoculum medium contained soluble starch, cornsteep liquor, (NH₄)₂SO₄, and CaCo₃.

iv)     Production medium contained dextrin, soyabean meal, cornsteep liquor, sesame meal, (NH₄)₂SO₄, and CaCo₃.

v)      Sterile air filters: Calcium chloride tubes were packed with non-absorbent cotton wool to a density of 17 lbs/cu.ft., and sterilized by heating in an oven at 160⁰ C for 1 hour.

 

Method:

Fermentaion of Neomycin:

Inoculum was added to the fermentation flask containing sterile production medium. Then the outlet of sterile air filter was connected to the inlet tube of the sterile water flask.

Table No. I- Effect of aeration at different time intervals of Neomycin fermentation.

 

Sl. No.	Description	PH	S.V. for 10 ml	Z.D.(I) in mm	C.F.	Z.D© in mm	Dilution	Units/ml
1.	Test-A	6.9	4.5	17.0	+0.2	17.2	200	4400
2.	Control	7.2	5.2	16.4 (R=16.2)	+0.2	16.6	300	5760
3.	Test-B	7.6	3.2	15.8	+0.4	16.2	200	3360
4.	Test-C	7.8	2.4	14.4 (R=16.0)	+0.4	14.8	200	2400

 

Test-A: Volume of air adjusted to 1.0 vvm and continued up to 6 days. Control: Control (250ml/500ml) closed with two layers of lint cloth.

Test-B: Volume of air adjusted to 1.0 vvm. and continued up to 8 days. Test-C: Volume of air adjusted to 1.0 vvm and after 60 hours it was reduced to 0.25 vvm and continued  up to 8 days.

 

P^H: P^H at the end of fermentation; S.V.: Sediment volume; Z.D.(I): Inhibition zone diameter; C.F. : Correction factor; Z.D©: Corrected zone diameter

                                     

Table No.-II- The effect of aeration at 72, 96, and 120 hours of Neomycin fermentation

Sl. No.	Description	PH	S.V. for 10 ml	Z.D.(I) in mm	C.F.	Z.D.(C) in mm	Dilution	Units/ml
5th day samples
1.	Test-A	6.42	4.5	13.2	-	13.2	400	3280
2.	Test-B	6.4	4.7	14.4	-	14.4	400	4480
3.	Test-C	6.42	5.8	13.8	-	13.8	600	5760
4.	Control	6.62	6.6	15.0 (R=16.4)	-	15.0	600	7680
6th day samples
1.	Test-A	6.8	4.9	15.2	-0.4	14.8	400	4960
2.	Test-B	6.76	5.2	15.4	-0.4	15.0	400	5120
3.	Test-C	6.84	6.2	15.0	-0.4	14.6	600	6960
4.	Control	7.10	6.5	15.8 (R=16.8)	-0.4	15.4	600	8640
7th day samples
1.	Test-A	7.10	4.7	16.4	-0.4	16.0	400	6560
2.	Test-B	7.39	4.8	16.6	-0.4	16.2	400	6880
3.	Test-C	7.29	5.2	15.4	-0.4	15.0	600	7680
4.	Control	7.78	6.0	13.6 (R=16.8)	-0.4	13.2	600	4920
8th day samples
1.	Test-A	7.8	4.1	19.0	-1.6	17.4	200	4800
2.	Test-B	7.75	4.0	19.6	-1.6	18.0	200	5600
3.	Test-C	8.10	4.7	16.2 (R=18.0)	-1.6	14.6	500	5800

Test-A: Volume of air adjusted to 1.0 vvm and after 72 hours, it was reduced to 0.25 vvm and continued.

Test-B: Volume of air adjusted to 1.0 vvm and after 96 hours it was reduced to 0.25 vvm and continued.

Test-C: 1.0 vvm of air was adjusted initially and after 120 hours, it was reduced to 0.25 vvm and continued.

Control: Control (50ml/250ml) flask with two layers of lint cloth

                                  

Table No.-III- Comparison of yield of Neomycin by reducing the aeration on 6^th, 7^th and 8^th days of fermentation

Sl. No.	Description	PH	S.V. for 10 ml	Z.D.(I) in mm	C.F.	Z.D.(C) in mm	Dilution	Units/ml
6th day samples
1.	Test-A	6.74	4.4	13.8	-0.6	13.2	500	4100
2.	Test-B	6.78	5.3	15.2	-0.6	14.6	500	5900
3.	Test-C	6.92	5.7	15.6	-0.6	15.0	500	6400
4.	Control	7.26	6.1	16.4 (R=17.0)	-0.6	15.8	500	7800
7th day samples
1.	Test-A	7.18	4.3	15.0	-0.4	14.6	500	5800
2.	Test-B	7.24	4.8	15.8	-0.4	15.4	500	7000
3.	Test-C	7.42	5.5	16.0	-0.4	15.6	500	7400
4.	Control	7.90	5.2	15.6 (R=16.8)	-0.4	15.2	500	6800
8th day samples
1.	Test-A	7.90	4.1	15.8	-1.8	14.0	400	4000
2.	Test-B	7.82	4.4	17.0	-1.8	15.2	400	5440
3.	Test-C	8.10	4.8	17.2	-1.8	15.4	400	5600
4.	Control	8.35	4.7	16.8 (R=18.2)	-1.8	15.0	400	5120

Test-A     : Volume of air adjusted to 1.0 vvm and after 96 hours it was reduced to 0.25 vvm and continued

Test-B     : Volume of air adjusted to 1.0 vvm and after 120 hours it was reduced to 0.25 vvm and continued

Test-C     : Volume of air adjusted to 1.0 vvm and continued up to the end of the fermentation

Control    :  Control(250ml/500ml) flask with two layers of lint cloth.

S.V.: Sedimentation volume;  Z.D.(I) : Inhibition zone diameter; C.F. : Correction factor;  Z.D.(C): Corrected zone diameter

 

 

Then the flasks were arranged on the shaker, air was admitted into fermentation vessel and shaker switched on. The fermentation was run for 6 days at room temperature. After 6 days the broth was centrifuged and the Neomycin in

 

the broth was estimated by microbiological assay using Bacillus pumilus as test organism. The inhibition zones were measured using Thermonik antibiotic zone reader. The difference between the reference zone of the plate and reference zone of the standard graph was correction factor for that solution. From the standard curve, the potency of the test solution was calculated.

 

Experiment No.1:

Three sets were arranged out of which 1.0 vvm of air was put into one set and continued up to 6 days, another set aerated as above but continued up to 8 days and third set where in aeration was reduced to 0.25 vvm after 60 hours and continued up to 8 days. Along with these one control flasks with lint cloth was run for 6 days. The results are tabulated in Table-I.

 

Experiment No.2

In this experiment, three sets were arranged for which the supply of air was reduced to 0.25 vvm at different stages of stationary phase. i.e. commencement, middle and end of the stationary phase(72,96 and 120 hours). Samples were drawn from 120 hours to 192 hours and shown in Table-II.

 

Experiment No.3:

In this experiment, aeration level was reduced to 0.25 vvm after 96 and 120 hours as in experiment No.2. One flask was kept with 1.0 vvm of air throughout and another flask was run with lint cloth. Samples were drawn similarly as above and the results are expressed in Table-III.

 

RESULT:

The experimental results in table-I showed that antibiotic produced at the end of 6^th  day even in continuously aerated flask at 1.0 vvm level was less compared to the shake flask closed with lint cloth. Further, the antibiotic production at the end of 8^th day, the flask in which aeration was reduced to 0.25 vvm after 60 hours was less compared to control with aeration at 1.0 vvm level up to 8 days.

 

On observation of results in table-II, the productivity in a flask covered with lint cloth was more than in flask aerated with sparger at 1.0 vvm level, while maximum production was obtained on 6^th day for control, which decreased on the 7^th day, for the rest production reached a maximum on the 7^th day. Further the data shows that the differences between test A,B and C narrowed down by 7^th day when the production in all the three reached a maximum.

 

The maximum antibiotic titre was observed at the end of 7^th day in aerated flasks and at the end of 6^th day in shake flask with lint cloth. But observing the 7^th day data, it appears that the difference between the productivity  in the aerated flask with 1 vvm and one aerated with 0.25 vvm from 120 hours is much less(Table-III).

 

 

DISCUSSION:

The expenditure incurred on aeration in submerged industrial fermentations constitutes about 20% of the production cost of an antibiotic. It is, therefore, possible to bring down the production cost by restricting the aeration levels at any feasible stage in the course of fermentation.

 

CONCLUSION:

The results indicated that production in the shake flask covered with lint cloth is more and reaches earlier by a day compared to the aerated shake flask continuously with 1.0 vvm of air. On the 7^th day, the difference in production of Neomycin narrowed down to 400 units out of 7400 units between the sparged flask aerated at 1.0 vvm level and the one where in aeration was reduced to 0.25 vvm after 120 hours.

 

The data indicates that reduction of air supply towards the end of the stationary growth phase does not very much affect the production of Neomycin.

 

REFERENCES:

1.     Hromatka.O., Ebner.H and Croklinch.C., Enzymolgia, 15,134, 1951,

2.     Cooper.C.M., Fernstrom.G.A. and Miller.S.A., Ind.Engg.Chem. 36.504,1944,

3.     Bigelow.R.H., Physik.Z., Chem.26,493,1898,

4.     Pirt.S.J. et al., Chem. and Ind.,730,1957,

5.     Yoshida.L.I. et al., Ind. Engg.Chem, 52, 435,1960.

 

 

Received on 03.03.2010       Modified on 05.04.2010

Accepted on 07.04.2010      © RJPT All right reserved