Practical- 6: Fermentative Production of Ethanol

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1 Practical- 6: Fermentative Production of Ethanol Introduction The overall reaction in fermentation of hexose by Yeast particularly S. cereviceae, can be expressed as under C 6 H 12 O 6 2C 2 H 5 OH + 2CO 2 This reaction is considered as basic formula for calculation of alcohol fermentation. However, other than alcohol certain byproducts like glycerol, succinic acid, and acetic acid are produced along with ethyl alcohol. Haiden and Young discovered that free inorganic phosphate disappear during fermentation usually through EMP pathway. Ethyl alcohol is widely applicable as organic solvent, which is efficiently produced by growing Yeast. S. cereviceae obtained from cheap sources like corn syrup can be used in preparation of inoculum media. Principle of Alcohol Estimation Alcohol reacts with oxidizing agent i.e. potassium dichromate and gets oxidized to acetic acid. Remaining of the potassium dichromate will react with iodine. This is a light sensitive process, so incubation is carried out in dark. Liberated iodine is titrated with 0.1N sodium thiosulphate. In this process starch is used as an indicator. Reaction K2CrO7 + H2SO4 + 2C2H 5 OH 3CH 3COOH + 2K2SO4 + 4H2O + 2Cr2 (SO4)3 Requirements 500ml Production medium & 50ml inoculum medium ph meter & Water bath Methylene blue(1% w/v) NaHCO3 (Powder) Concentrated HCl Molar K2CrO7 25%(W/V) KI 1% (w/v) starch as indicator 0.1N Na2S2O3 25% ZnSO4 Staining reagents & Glassware

2 Composition of Media Inoculum medium Ingredients g/100ml Jaggery 5.00 (NH4)2SO KH2PO Yeast Extract 0.50 ph 4.5 Production medium Ingredients g/1000ml Jaggery KH2PO Yeast Extract 1.50 ph 4.50 Protocol Transfer aseptically 10% (50ml) of Inoculum in 500ml Production medium and Mix it well From Production medium aseptically withdraw approx ml (Measure ph and Calculate G % sugar from sample withdrawn) Acid Hydrolysis for 30 minutes (20ml of Broth +1ml of Conc. HCl in boiling water bath for 30 minutes+ neutralize with NaHCO3 Estimate the Gram percent of sugar by Cole s method After 5 days take broth Recover the alcohol as per procedure Procedure for Cole s method Take 20 ml of K3 (FeCN) 6 in 250 ml flask to that add 5 ml of NaOH Keep it for boiling under direct flame over the wire gauze add 2 drops of Methylene Blue Titrate with hydrolyzed sugar (Note that Reading as PR) From Pilot Reading Dilute the sample for TR (If PR is 2.0 then, we have to take 2 ml of neutralized sample and make the volume up to 10 with D/W) (For 3 TR reading total volume should be more than 30 ml) Again repeat the same procedure from step 1 and note down the titration reading as TR (TR reading should be between 9 11) Calculate Gram% sugar with the help of Sucrose Factor Purity of the culture is checked by Gram s staining

3 ph is measured ph meter Gram (w/v)% sugar is estimated Reference Microbiology- A laboratory Manual-International student Edition: 4th edition. By James G. Chappuccino & Natalie Sherman Bacteriological Techniques By F.J. Baker Introduction to Microbial Techniques By Gunasekaran Mannual of Industrial Microbiology and Biotechnology, 2nd Edition by Arnold L. Demain and Julian E. Davies, Ronald M. Atlas, Gerald Cohen, Charles L. Hershberger, Wei-Shou Hu, David H. Sherman, Richard C. Willson and J. H. David Wu

4 Cole s method for quantitative estimation of sugar Day Gram Staining ph Procedure for hydrolysis Measurement of Sugar by Cole s method Procedure for estimation of Sugar Pilot Reading (PR) Titration Reading (TR) Sugar (gm%) G% sugar consumed 1? 2? 20 ml of sample +1ml HCl hydrolysis for 30 min, neutralization with NaHCO 3 20 ml of K 3 (FeCN) 6 +5 ml of NaOH Boiling + 2 drops of M.B. titration with hydrolyzed sugar

5 Observation table for estimation of Ethanol: Sr. Ethanol Ethanol (mg/ml) Distilled water 0.025M K2CrO7 Incubation Distilled water 25% (w/v) KI Incubation 1% (w/v) Starch 0.1 N Na2S2O3 Blank - Test B S S S min in dark at 10 min at Room Temperature S Room 0.5 and transfer it to Temperature 250ml flask S D 3? D 3? D 3?

6 Calculation for G% sugar by Sucrose factor Sucrose G% = (0.036xTR) mg /TR ml where T R is the mean Titration Reading Procedure for Recovery of Alcohol Take 10ml of fermented broth, Neutralize with 1N NaOH Add 25% ZnSO4 allow it to react for 5-10 monutes Filter the Precipitated Proteins by Filter Paper Collect the Filtrated in distillation unit and set the temperature to 70 o C Collect the distillate and Dilute with Distilled water Procedure for Estimation of alcohol by potassium dichromate from fermented broth Take different aliquots of standard alcohol solution (0.1mg/ml) ranging from ml Take same aliquots of 1ml from Diluted sample of Distillate from Broth Make up the final volume to 5 ml by Distilled water Add 10 ml of 0.025M K2CrO7 solution in each tube. Allow the tubes to stand for 30 min in dark Transfer them into 250ml flask and Add 100 ml of Distilled water to each flask and cork it Add 4 ml of 25%(W/V) KI Allow them to stand at room temp for 10 min After incubation add 1% starch solution as an indicator and titrate against 0.1 N Na2S2O3 until it becomes colorless Observation table for Estimation of Alcohol Calculations for efficiency Theoretical Efficiency If X gm Total (which is present on Day 0) of sugar is consumed than Efficiency is 100% Then if Y gm is utilized (Total minus remaining amount of sugar on day 5) than Efficiency is X x 100 Y

7 Actual Efficiency Glucose 2mole of Ethanol + CO2 180gm 2(46) = 92gm If 180gm of Glucose is utilized then 92 gm of ethanol is produced that means efficiency is 100% But if Y gm is utilized then Efficiency is : Y x From Graph Calculate the amount of ethanol produced from Graph Result: After 5 days all the sugar will be consumed and alcohol will be produced which is estimated and efficiency of the Yeast cells can be calculated

8 Reference Microbiology- A laboratory Manual-International student Edition: 4th edition. By James G. Cappuccino& Natalie Sherman Bacteriological Techniques By F.J. Baker Introduction to Microbial Techniques By Gunasekaran Manual of Industrial Microbiology and Biotechnology, 2 nd Edition by Arnold ;. Demain and Julian, E. Davie Ronald M. Atlas, Gerald Cohen, Charles L. Hershberger, Wei-Shou Hu, David H. Sherman, Richard C. Wilson and J. H. David Wu

Some Basic Concepts of Chemistry

Q 1. Some Basic Concepts of Chemistry What weight of AgCI will be precipitated when a solution containing 4.77 g of NaCI is added to a solution of 5.77 g of AgNO 3? (IIT JEE 1978 3 Marks) Q 2. One gram