Experiment no.1-Isolation of industrially important microorganism

Isolation of Amylase producing micro organism

Materials: Starch agar medium, soil sample, iodine solution.

Preparation of starch agar medium:-

1. Dissolve 0.5 gm soluble starch in some amount of water by kept it on a

magnetic hot plate stirrer. Then add in to this1.3gm nutrient broth then make

up the volume to100 ml.

2. Adjust the pH 7.2.

3. Perform the steps from 4 to 7 as for the preparation of skim milk agar.

Procedure:-

1. Label the plate on the backside using a marker.

2. Take a loopful of each soil suspension and streak it on the agar

plate.

3. Incubate the plate in inverted position at 37°C for 24 hours.

4. Flood the agar surface with iodine solution. Keep it for a minute

and then decant the iodine solution.

5. Wash the plate with water if necessary.

6. Observe the plate carefully.

Result:-

Iodine reacts with starch to form a blue compound. The colorless/light brown

zone surrounding colonies indicates production of extra cellular amylase enzyme.

Experiment No. 2 –Preparation of Culture media

Potato Dextrose agar

This is used to cultivate fungal cultures. The composition of the media per liter is:

Potato infusion : 200g

Dextrose : 20g

Agar : 15g

pH : 5.6 ± 0.2

Sterilize by autoclaving at 15 lbs pressure (1210C) for 15 minutes. After cooling the

media to about 450C the plates are poured. These plates are kept overnight in the

incubator to check contamination and are then streaked.

This media (PDA) can also be prepared by using the following protocol:

Two hundred grams of peeled potatoes are cut into small pieces and suspended in

1000ml of distilled water and steamed for 30 minutes. Decant the extract or filter the

material through sieve and make the final volume to 1000ml. Add 20g of dextrose and

15g of agar (Sometimes 0.1g of yeast extract is also added). Sterilize by autoclaving at 15

lbs pressure (1210C) for 15 minutes. After cooling the media to about 450C the plates are

poured. These plates are kept overnight in the incubator to check contamination and are

then streaked.

Experiment No. 3-Production run for Yeast in a bioreactor

Media Preparation:

Prepare glucose yeast extract peptone media by adding 0.5% glucose, 0.5% yeast extract and 0.5% peptone. Adjust the pH 5.

Sterilization:

In case of autoclavable fermentor, put the media in the fermentor

vessel and then plug all the openings with cotton. And close the all the

valves. Then put the vessel in the autoclave and sterilize by autoclaving at

15psi pressure (121oC) for 15 minutes. Allow it to cool for inoculation.

In case of in situ fermentor, put the media in the fermentor vessel and close

all the valves. Heat the water jacket to a temperature of 90oC by

flowing steam, from the steam generator/boiler, through it. Then open

the valve to allow steam to pass through the vessel containing the

medium. Hold it at 15psi for 15 minutes and then release it through

the exhaust valve. Allow the medium to cool. Once the temperature

reaches 60 oC, allow chilled water to flow through the water jacket. Program the

system to maintain the temperature according to the culture requirement.

Inoculation:

Inoculate the media in the fermentor vessel through the inoculation port of the fermentor

under flame with the inoculums of yeast culture generated by incubating overnight at

30oC at 200rpm.

Set the various parameters such as aeration, agitation, pH according to the culture. And

run the fermentor.

Sample Collection:

After each 2 hours of inoculation, collect the sample through the sample port by creating

a positive pressure inside the vessel and perform the following steps with each sample.-

1. Take the growth O.D of the sample against blank medium at 600nm and plot the

growth curve.

2. Perform the Grams staining of each sample to check the contamination.

3. Record the cell pellet weight:

Weigh an empty labeled micro centrifuge tube.

Put 1ml of the sample in it and centrifuge at 10,000rpm for 10 minutes at

4oC.

Then discard the supernatant and allow the pellet to dry.

Take the weight of the micro centrifuge tube containing the dry pellet.

4. Put rest of the sample in a centrifuge tube and centrifuge it at 10,000rpm for

10minutes at 4oC.

5. Separate the supernatant and perform the glucose estimation and alcohol

estimation with it.

Experiment No.4 –Estimation of Ethanol by Potassium Dichromate method

Chromic acid reagent:-

1. Potassium dichromate 34g

2. Chilled concentrated H2SO4 325 ml

3. Distilled Water 675 ml

Dissolve potassium dichromate in chilled concentrated H2SO4. Add this into distilled

water.

Standard Solution:-

Make 10% standard solution of absolute alcohol. And make 1 ml of each 1 to 9%

solutions from it.

Procedure:-

1. Add 1ml of sample / standards in a test tube/s

2. To it add 25 ml of chromic acid reagent .Mix thoroughly.

3. Place the tubes in water bath at 70oC for 15minutes.

4. Make its volume up to 50 ml with water.

5. Measure absorbance at 600 nm against reagent blank.

Experiment No.5 –Determination of total sugars by Anthrone method

Anthrone reagent

1. Anthrone : 0.2g

2. Concentrated sulphuric acid :100ml

3. Standard stock glucose solution :100g/ml

4. Standard solution :0-100g/ml

Procedure:

1. Take 1 ml of sample/glucose in clean dry test tube/s.

2. Add 1 ml of distilled water and cool in ice bucket for 15 minutes.

3. Add 4 ml of anthrone reagent slowly along the glass wall of the test tube.

4. Place the tubes in boiling water bath for 10 minutes.

5. Cool the tubes for 10 minutes. Let these stand at room temperature for 10 minutes.

6. Measure absorbance at 625 nm against a reagent blank.

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