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Alcohol fermentation
AIM: To produce Ethanol by fermentation using Bakers yeast and isolated strain of Saccharomyces cervisae. INTRODUCTION: Chemically, Ethanol (CH3CH2OH) is an organic compound, part of the alkanol family. It is a colourless liquid with a pungent taste and a characteristic odor. Its specific gravity at 20oC is 0.785g/ml and its boiling point is 78.4oC. Unlike water alcohol has a non-polar ethyl portion capable of Van-der Waals interactions and a polar OH group. Fermentation: SUBSTRATE AND ORGANISMS: Carbon Sources generally include saccharide material such as sugarcane juice, beet juice, blackstrap molasses, sweet sorghum, whey, ripe fruits. It also includes starchy material as cereal grains, potatoes, tubers. Cacti and cellulosic raw material as sulfite waste liquor, grasses and wood. Organisms include yeast such as S.cerevisae, S.uvarum, Candida utilis. Bacterial producers are Zymomonas mobilis. Clostridium thermocellum. USES OF ALCOHOL: Industrial alcohol has been valuable as a solvent, germicide, disinfectant, antifreeze, fuel, in cosmetic and chemical raw materials. REQUIREMENTS: 1) GYE MEDIUM (Inoculum development): Glucose 1% Peptone 0.5% Yeast extract 0.3% Distilled water 100ml PH 4.5-5.0 2) FERMENTATION MEDIUM: Jaggery 20% K2HPO4 1.5g/l (NH4)2SO4 1.5g/l Yeast extract 0.5g/l Distilled water 1 liter PH 4.5 Composition of jaggery: 65-85% sucrose, 10-15% invert sugars (hydrolyzed form: glucose, fructose) 3) Fresh culture slant of S. cerevisae 4) 250 ml flask, 10ml sterile pipettes. PROCEDURE:

Practical# Inoculum development: Grow S. cerevisae on GYE agar slant Prepare thick suspension of S.cerevisae from slant. Add 2ml of suspension in 100ml GYE broth. Incubate the flask at 3000C for 24 hrs for activation of culture. OR Commercially available dry Bakers yeast. Dissolve 3 beads in 5ml sterile D/W and make a homogenous suspension. Add 2ml of suspension in 100ml GYE broth. Incubate the flask at 300C for 24 hrs for culture activation.

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Fermentation: 1. Take a fresh culture of S.cerevisae and Bakers yeast and perform Grams staining and note its colony characteristics. 2. Prepare inoculum as mentioned above. Take 100ml active culture from the flasks inoculate into 900ml sterile fermentation broth and estimate sugar at 0hrs from broth flasks. Then at 300C in static condition. 3. At every 24hrs interval the sugar remaining is measured by Coles method (Both before hydrolysis and after hydrolysis). Alcohol estimation is carried out till sugar concentration becomes zero. % yield of alcohol is then calculated using the formula: % yield=alcohol produced (g)/ sugar utilized (g)*100. When sugar concentration becomes zero, the broth is centrifuged to remove the cell debris. After this the distillation of ethanol is done from the broth. Ethanol gets distilled at 78.40C. ALCOHOL FERMENTATION BY IMMOBILISATION. INTRODUCTION: Immobilisation refers to localization or confinement of cells during a process. Immobilisation can be applied to cells as well as enzymes. There are five markedly different techniques if immobilization: 1) Adsorption on a carrier surface. 2) Covalent coupling to a carrier surface. 3) Cross-linking between molecules. 4) Entrapment in a matrix; and 5) Encapsulation or confinement in a membrane structure. Of all the techniques enlisted above the physical entrapment in porous polymeric carriers is by far the most widely used technique for whole cell imobilisation. The size of the immobilized species makes it rather simple to prepare porous networks which combine complete cell retention with high enough porosity for substrate and product transport. Microencapsules are defined as usually spherical particles, where a liquid or solid suspension is enclosed by a dense, but semipermeable polymeric film. In this approach the cells are first entrapped in alginate beads and then treated with polycation solution like polylysine. An insoluble polyelectrolyte complex is formed. Finally the

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non-complexed alginate is redissolved from the microbead leaving the cell suspension in the microcapsule. REQUIREMENTS: Conical flasks, dropper, measuring cylinder, weigh balance. REAGENTS: Sodium alginate- 3% Calcium chloride- 0.5M Cell suspension of yeast cells (S.cerevisae) 10% sucrose solution. PROCEDURE: 1. Appropriately weighed sodium alginate solution is dropped into 0.5M CaCl 2 by means of a dropper. This results in formation of a bead matrix. This matrix is formed by cross-linking between the alginate ions of sodium alginate with calcium ions. These beads are allowed to stand for 30 minutes . 2. Add 4g of the cells in sucrose solution. Wash the beads with water and suspend in 1000ml of 10%sucrose solution. 3. Incubate the flask at 370C for 24hours. 4. After 24hrs separate the cells from the solvent by filtration and 5. Calculate the sugar concentration by Coles method as described above and alcohol estimation is done till sugar concentration becomes zero. 6. Following this, yield of alcohol is calculated and concentrated alcohol solution is distilled. OBSERVATION:

CONCLUSION: