Research on the Procedure of Yeast Ethanol Fermentation Procedure

The process which consumes sugar in absence of oxygen i.e., anaerobic condition is called fermentation. The products list out to be organic acids, gases, or alcohol. It happens in yeast (Saccharomyces Cervisiae)because it has capability of producing ethanol and bacteria(microorganisms), and additionally in oxygen-starved muscle cells. In straightforward manner fermentation is the chemical changes of sugar into ethanol. Ethanol is a transparent, uncolored fluid which is quickly retained from the gastrointestinal tract.

This method takes place once there are useful bacteria present that smash down the starch and sugars inside the food. Because the microorganisms divide, lactic acid is made, that stops the further growth of unhealthy bacterium. The lactic acid is also what provides fermented foods that very specific ‘tangy’ or ‘acidic’ flavor. These fermented foods will last for several months (some products even enduring numerous years) as long as they're hold on, under a cool, unlit place and stored in the suspension of salt and water referred to as brine.

Here I have taken JACKFRUIT’S (Artocarpus heterophyllus) stone and pulp because it is easily available in market and cheap also. It has very good sugar content and have antioxidant activity. It acts as the cancer prevention agent. It is one of the most profitable and well paid fruit in India and its production is shooting up day by day because of its demand in the market and benefits for health. Outset of jackfruit is inherent to India but at present it is widely spread to tropical parts. It is produced in Malaysia, Burma and in some areas of Brazil. At the ready stage, the fruit weight is accounted to vary in a range from 3-7kg in which the mash contains 29%,seed 12%, and it’s peel is 54%.The big bland seeds which are encapsulated in the fleshy mash of consumable part of the fruit are often disposed of. But these seeds were accounted to have 25-52% starch, 9.2% protein and 24.4% lipid in it.

The value of ethanol is incalculably boosting as a beneficial propellant earlier than the present era .The production of ethanol can be used more with the existence of affordable substrate. There are number of sources which can be reused such as rice husk, wheat straw, coffee pulp, sugarcane juice, grains for the manufacture of alcohol. Molasses have been used more in the past years for the production of ethanol but due to increase in price so more preference is given to other substrates. This is on grounds that these substrates are prepared for the bioconversion to ethanol with restricted pretreatments when contrasted with the starchy or cellulosic substance.

Ripened jackfruit is taken and after peeling it off, pulp and seeds are separated. The seeds extracted are blended with the help of blender and the paste is further dissolved in water. Then this mixture is used for the preparation of ethanol.

Instrumentation

• Spectrophotometer
• Weighing balance
• Autoclave
• Incubator
• Shaker incubator
• Hot plate
• Laminar Air Flow
• Water bath
• pH meter
• Vortex Mixture
• Blender
• Micropipette

Glasswares (borosil)

• Conical flask
• Beakers
• Test tubes

Chemicals

• Nutrient broth
• Agar
• Chromic Acid
• Di-nitro salicylic acid reagent solution
• 40% potassium tartrate
• Yeast strains

Methods

-Composition of media for the Growth of Yeast Culture

Components Grams/litre

Nutrient Broth 1.68

Nutrient Agar 0.52

Procedure for the Growth Media of Yeast culture

I. Weigh required amount of nutrient broth and nutrient agar and dissolve them in 500 ml of distilled water

II. Make up the volume to 1000ml.

III. Autoclave the mixture for 45 minutes at 121°C.

IV. Measure 100 ml media and pour in 2 different conical flasks, one for pulp and other for seed.

V. To prepare a inoculum, take 2 test tubes and pour 10ml of media in each one of it.

Steps for Serial dilution

I. Take 4 test tubes and mark it as 101,102,103,104.

II. Add 9ml distilled water to all of the test tubes marked as 101,102,103,104.add 10 ml

III. Then with the help sterilized micropipette, add 1ml of yeast from the stock.

IV. From test tube marked as101, with the help of micropipette, take 1ml sample and transfer it to the test tube named as 102, and repeat the same process for 103,104 test tubes. Note: This process should be done under clean aseptic condition inside laminar air flow.

Streak plating method for growth of yeast

I. After the culture is autoclaved pour it into the petri dish and allow it to cool down.

II. Then sterilize the glass rod with spirit lamp and with the help of the glass rod make streaks using the media in 104 test tube.

III. Cover the petri dish tightly with the help of Para film wax so that outside microorganism do not enter into it and keep it in the incubator for the growth of colonies for 2 days.

IV. Growth of yeast colonies was observed. Note: This process should be done under clean aseptic condition inside laminar air flow.

Preparation of Inoculum

I. Nutrient broth was taken as the media for the preparation of inoculum media and was sterilized at 121oC for 15 minutes at 15 psi with the help of autoclave.

II. With the help of inoculum needle extract the streak colonies of yeast.

III. In the laminar air flow, take the test tubes containing the media; sterilize its mouth by heating its mouth by the burner.

IV. Take the inoculation loop and sterilize it also with the burner.

V. With the help of the loop take out a small colony of yeast and inoculate it in the test tubes.

VI. Then apply the cotton plugs on the mouth of the test tubes so that no microbes enter into it and leave it in the incubator for 2-3 days.

VII. Then after 3 days, transfer 5ml of inoculum to each of the media in the conical flask.

VIII. Now the media is ready for fermentation process which is done in anaerobic condition.

IX. Now the media is ready for the glucose and alcohol test.

X. After taking the sample for test keep it back in the shaker incubator so that the temperature is maintained.

Glucose estimation by Di nitro salicyclic acid (DNS) methods

Using the pure glucose solution as a standard, the purity of glucose was determined by 3,5- Dinitrosalicylic acid (DNS) method. It is an aromatic compound which assures the presence of carbonyl group. Principle: A few reagents have been utilized which measure sugar by utilizing their decreasing properties one such compound in 3,5 dinitrosalicyclic acid which in soluble arrangement diminishes to 3-amino 5-nitro salicylic acid. Reagents:

Glucose solution- In distilled water 1mg/ml aqueous solution.

DNS solution- Mix 10.6 gm. 3, 5 dinitrosalicyclic acids, 14.16ml distilled water and 19.8gm NaOH. Dissolve these then add about 30.6gm Rochelle’s salt(sodium Potassium tartrate),7.6 ml phenol(melt at 50°C) and 8.3gm sodium meta-bisulphite. With 0.1 N HCl titrate 3ml sample (with Phenolphthalein). The sample should take HCl of about 5.6ml .if required add NaOH. Procedure:

I. Take 1ml sample from each of the conical flask.

II. Add 1ml DNS reagent to the sample and keep it in the water bath at 90oC for 15 mins to develop a reddish brown colour.

III. Now after removing from water bath, add 1ml of 40%potassium tartrate solution.

IV. Cool at room temperature and then record the optical density ar 545nm wavelength.

Standard curve for Glucose

A solution of standard glucose (1mg/ml) was prepared and in different test tube different concentration was prepared by taking 0.1ml to1ml of glucose solution and afterwards made up to 1ml with distilled water except in 1ml solution. Instead of standard solution 1ml distilled water was used for the blank. Then 1ml DNS reagent was added and kept for boiling in the beaker containing water for 5minutes. In vortex mixture, mix the solution of each test tube. After standing for couple of minutes absorbance was estimated at 540nm and a curve was plotted against absorbance vs. glucose concentration (mg/ml).

Alcohol estimation by potassium dichromate test

Many of the chemical oxidation are usually based on the complete oxidation of ethanol by dichromate in the presence of H2SO4 with the acetic acid formation. As K2 Cr2O 7 is easily assessable in high clarity and is stable in air. I have used chromic acid for alcohol testing. It cannot be used for testing tertiary alcohol but can be used only to identify aldehydes, primary alcohol and secondary alcohol. Reagents- Chromic acid (conc.H2SO4, 20mg K2Cr2O7), distilled water

Procedure-

I. For preparation of chromic acid K2Cr2O7 is taken in 300ml conc.H2SO4.

II. Take 0.5ml sample and add 12.5ml of chromic acid to it

III. Then keep it in water bath for 15 minutes at 70°C

IV. After taking out from the water bath quickly add 12 ml distilled water to it to stop further reaction.

V. Then with the help of spectrophotometer draw the optical density of the sample at 600 nm and compare it with the standard graph.

Alcohol estimation using fermented sugarcane juice as a substrate

Purification of Fermented sugarcane broth

Removal of insolubilities

To remove the insolubilities from the medium, a three-step process is done.

Filtration

To filter out bigger and insoluble particles, a muslin cloth or filter paper is used. The biomass is collected on the muslin cloth/ filter paper.

Centrifugation

To remove other smaller impurities, the medium containing enzyme and impurities is centrifuged at 10000 rpm for 15 minutes. Supernatant is collected in a clean beaker after measuring and is neatly labelled for further experiments. The beakers are stored in refrigerators.

Distillation unit

For the extraction of pure ethanol distillation is done in the distillation unit in low temp so that there would be no water vapour in the extraction of ethanol.

Distillation unit

And the pure ethanol is collected in the beakers or tube and stored in the refrigerators.

Result and discussion

Kinetics study of Substrate Utilization

The kinetic study of substrate utilization was done to observe the amount of glucose consumed for the growth of yeast during the production of alcohol from waste jackfruit seed and pulp samples.

Glucose Content in Samples S. No Incubation time (hr) Glucose content(mg/ml) Sample 1 Sample 2

1. 0 12.10843 12.63855
2. 24 9.13253 12
3. 48 6.879518 11.87952
4. 72 6.987952 11.75904
5. 96 6.927711 10.73494
6. 120 5.795181 7.481928

The kinetic study of substrate utilization shows that glucose is reducing as the incubation days were increases, which indicates that yeast has utilised the carbohydrate source for performing its activities like microbial growth, cellular maintenance and production of ethanol.

Kinetics study for alcohol Production

The alcohol production was carried out on waste sugar cane juice using yeast as microbial source which was isolated from ripen grapes.

Kinetic study of alcohol produced S No. Incubation time Pulp Seed

• 1 0 0 0
• 2 24 0 0
• 3 48 0.242779 0.234408
• 4 72 0.816241 0.355797
• 5 96 1.276685 0.774383
• 6 120 0.8581 0.598577

The kinetic study for alcohol production was carried out for 120 hr at the interval of 1 hr using spoiled jackfruit samples of seed and pulp. The best yield was observed after 96 hr and after that it started reducing till 120 hr. The glucose concentration was reducing almost. Comparatively pulp sample shows higher production of alcohol than that of the seed as in the pulp the production is 1.276685 whereas in seed sample the production is 0.774383% v/v.

Purification of Ethanol

The filtration and centrifugation was done for the removal of insoluble and cell debris, after the removal of cell debris the ethanol was purified by distillation process. After distillation the sample was freezed in refrigeration at 4 0C due to which the water was converted into ice and purified alcohol was recovered.

Ethanol’s chemical formula is C2H5OH.

Microorganisms

The significant part of production of ethanol is the association of microorganisms in fermentation of sugars. Very few microorganisms have the ability to use glucose, without oxygen for their energy, for producing carbon dioxide and ethanol. Due to this property they are potential bio agents in fermentation technology since the beginning. Sugar fermentation which uses single cell microorganism, that is, Yeast, which is widely used for drinking alcohol production and is the most settled practices in Biotechnology.

Microorganisms like dried yeast or Saccharomyces have been studied for ethanol Production from juices which contains sugar. It is the most appealing choice In fermentation because of its higher efficiency in conversion of sugar to alcohol.

Temperature is an crucial factor which has to be carefully controlled during fermentation as it has a fundamental effect on the procedure and production of ethanol. It was reported that Production of ethanol highly depends on fermentation temperature like it depends upon pH and to some degree with increase in temperature concentration also increases.

Microbial action and fermentation process are managed by various protein and enzymes which are sensitive to high temperature since it destroy their tertiary Structure resulting the inactivity. It is generally believed that the ideal fermentation temperature range is between 20 and 35oC. The optimum fermentation temperature for microorganisms of S. cerevisiae is near 30oC . Sugar Concentration. The most Important influencing parameter is the initial sugar concentration as it straightly effect on fermentation rate and microbial cells. The actual relationship between initial sugar content and the fermentation rate is complex. Generally, with the increase in initial sugar concentration fermentation rate will be increased up to a crtain level. But very high concentration of sugar will increase the upholding capacity of the microbial cells leading to a slow fermentation rate. According to these facts, in fermentation the optimum sugar concentration was determined as 190g/L. Likely, the ratio between sugar and Microorganism concentration was observed as 200.0 g/L and 30.0 g/L, respectively.

Inoculum Size

Inoculum concentration has a significant effect on the resulted ethanol concentration and also affects sugar consumption rate and productivity of ethanol. However, it was reported that ethanol production was increased with increase in the initial cell numbers from 1 × 104 to 1 × 107 cells/mL ethanol production is also increased. Fermentation time can be reduced with the increased cell concentration in a certain range also because of the increase in the production of cells in the fermentation media that instantly consumes provided sugars which produces ethanol.