Department of Biotechnology

Project Synopsis Presentation

Session I August-December 2020

Session II January – May 2021
Production and comparative analysis of Biofuel
produced from different fruit waste
using Saccharomyces cerevisiae.
Phaniraj Bijapur Nikhil L
PES1201701322 PES1201700586
7th Sem, B.Tech (Biotechnology) 7th Sem, B.Tech (Biotechnology)
Manikantha Hebballi
PES1201701711
7th Sem, B.Tech (Biotechnology)

Under the guidance of

Dr. Seema Tharannum
Department of Biotechnology, PESU

SESSION AUG 2020 – DEC 2020

HYPOTHESIS
Bio-Ethanol produced from fruit waste using Saccharomyces
cerevisiae by fermentation, can be used as an alternative biofuel.
OBJECTIVES:
1. Sample collection and processing
2. Production of ethanol.
3. Purification of ethanol.
4. Evaluation of ethanol produced.
5. Ethanol efficiency testing on motors.
PROPOSED METHODOLOGY:
• Phase I
• Collecting a variety of fruit wastes
• Preparation of media
• Incubation
• Test for the presence of ethanol
• Phase II
• Bio ethanol purification
• Comparison analysis of ethanol for different parameters with different
media contents.
• Using Produced ethanol for running motors and checking the efficiency.
PLAN OF WORK:
Phase Period Work to be executed

I 4 weeks Literature survey and selection of fruit waste

2 weeks Collecting a variety of fruit wastes

2 weeks Preparation of media

2 weeks Incubation

II 2 weeks Test for the presence of ethanol

5 weeks Bio ethanol purification

3 weeks Comparison analysis of ethanol for different parameters of media

contents.
2 weeks Using Produced ethanol for running motors and checking the
efficiency.
Paper – 1 [1]
Methods & materials:

• A total of 400 gm of apple pomace and rotten ripen banana was collected.
• Collected sample was washed in 5% Potassium permanganate (KMNO4) & rinsed in distilled water.

Preparation of the substrate for the fermentation process:

• Collected sample was first crushed using a mixer.

• In a separate beaker, 300ml of slightly warm distilled water, 50gm of sucrose and 10gm of Saccharomyces
cerevisiae was mixed well.
• The contents from the beaker and the collected sample was transferred into a 1.5L conical flask, mixed
together and the final volume was made up to 1000 ml.
• The flask was covered with dark paper and the mouth was sealed and kept in a shaker incubator and was
allowed to incubate for 36hrs at 36 ̊C with a speed of 100rpm.
Recovery of the product:

• After 36hrs of fermentation process a small amount the sample was taken out and centrifuged.
• The supernatant was collected and the volume of the alcohol was determined by the specific gravity
method.
• Then the rest of the sample was distilled using normal lab distiller to collect the concentrated alcohol .

Results & discussion:

• After 36hrs of incubation a total volume of 200ml of 48% ethanol was obtained from a total volume of
1000ml of substrate after distillation.
• The higher concentrated ethanol can be used as a biofuel and it releases no toxic gases to the environment.
The substrates used are very cheap raw material and the process was found to be very easy and less cost
effective.
• Using higher grade distillation assembly a more concentrated product can be recovered by re distillation.
Paper –2 [7]
Methods & materials:

• Watermelon waste was used for the extraction of bioethanol.

• High-performance liquid chromatography Waters 600 Controller was used to measure the convertible
sugars that exist in watermelon waste.
• An electric fruit juicer was used to turn watermelon waste into thick pulp.
Preparation of the substrate for the fermentation process:

• The pulp was then refined to get syrup with the concentration of 70%.
• For preculturing, the microorganisms were inoculated in Yeast extract-Peptone- Glycerol medium containing
yeast extract (3 g/L), peptone (10 g/L), and glucose (20 g/L).
• After 24 hr incubation time 5% (w/w) Saccharomyces cerevisiae were inoculated in the fermentation
medium, containing watermelon substrate as the carbon source.
• The process of fermentation took place under anaerobic conditions for 35 hr, which included the time
required to make the suspension at the constant fermentation temperature of 30 ̊C (28 ̊C–30 ̊C is an
appropriate and optimal temperature for the growth of S. cerevisiae yeast at different rates for the variables
in question).
Recovery of the product:

• Seven samples were taken in the span of 35 hours. After each sampling, the rate of the ferment growth and
production of alcohol in the substrate was measured using a Digital Alcohol Tester.
• The differences in the data obtained after measuring the alcohol in the substrate indicated that almost all of
the convertible sugar that existed in the substrate could be turned into alcohol through the Saccharomyces
cerevisiae yeast.

Results & discussion:

The amounts of fructose, glucose, and sucrose in each 1,000 g of watermelon waste were 46.55, 30.20, and
20.71, respectively. Therefore, due to the high amounts of transformable sugars (fructose, glucose, and sucrose)
in watermelon waste, this study suggests that watermelon waste could be used as one of the main sources of
bio-ethanol production . In various studies, the researchers have reported that bioethanol can be obtained from
cellulose, sugar and starch sources in agricultural products
Paper – 3[2]
Methods & materials:
• The banana waste was brought from the local market and washed and blended.
• The enzymes used were pectinase and cellulase. The enzymes used 0.3 units per mg.
• Yeast for this fermentation process was Saccharomyces cerevisiae type II. It was activated by hot water
bath at 37 ̊C and then dried fast to yield 90% active, viable yeast in sigma solid form.

Preparation of the substrate for the fermentation process:

• 100g banana mash was taken into 3 sterile bottles with 25 ml water in each bottle.
• 3 gm/lit of yeast Saccharomyces cerevisiae was added in each bottle and closely tightened so that an
anaerobic condition was provided and it was placed in incubator at 23 ̊C, 30 ̊C and 35 ̊C , each at
particular temperatures for 3 days of fermentation.
Recovery of the product:
• Bio-ethanol concentration was detected by potassium dichromate reagent solution ,s-diphenyl carbazide
solution and 40% potassium sodium tartarte solution.
• The absorbance value of the reducing sugar was measured using spectrophotometer at 575 nm.

Results & discussion:

TEMPERATURE EFFECT: The Bio-ethanol Concentration was increased with temperature. The highest
concentration 6.21%(v/v) of bio-ethanol at 35 ̊C during fermentation process.
EFFECT OF SHAKING HOURS: The concentration of bio-ethanol at different shaking hrs is tabulated .The
fermented banana mash without shaking only produced 5.86% followed by 3 hrs shaking period 6.35% and 6 hrs
produce 6.55%(v/v)ethanol.
Project Approach:
Methods & materials:

Collecting 4 different fruit wastes from local market, preparing samles by different combinationn of there pulp
and peel parts. Checking the glucose level, water content . Using grinding and powdering method form
preparation of broth

Preparation of the substrate for the fermentation process:

Preparation of broth for yeast growth. Adding yeast to the prepared media. Incubation. Testing for the
presence of ethanol.
Recovery of the product:

Doing distillation and concentrating the output. Using specific gravity method for knowing the concentration of
ethanol present in the final result.

Results & discussion:

Checking the quality of produced ethanol by using it in different machine and calculating the eqating the output
to different test charecterstics
1.Sample collection and processing.
The wastes of orange, mosambi, banana and watermelon were collected.

Collected Samples were washed with distilled water.

Sample was then kept under sunlight for drying.

The dried samples were then grinded into a powder.

The grinded powder was then weighed.

 Fruit 1: Citrus limetta (Mosambi)

Raw Sample After drying After grinding into fine powder

Raw weight of the sample: 1080grams

Final weight of the obtained powdered
sample :388grams
 Fruit 2: Musa (Banana)

After grinding into powder

Raw sample After drying

Raw weight of the sample: 386grams

Final weight of the obtained
powdered sample : 126grams
Fruit 3: Citrus X sinensis (Orange)

Before drying After drying After grinding into fine powder

Raw weight of the sample: 630 grams

Final weight of the obtained
powdered sample :221grams
Fruit 4: Citrullus lanatus (Watermelon)

Raw sample After drying of the sample After Grinding into the powder

Raw weight of the sample: 1180 grams

Final weight of the obtained powdered
sample :128grams
Preparation of Media [2]
Materials required:
1. Dry Yeast- 10g
2. Sucrose -50g
3. Luke warm water- 250ml

Procedure:
1. Dry yeast was added to luke warm water.
2. Sucrose was then added for the
activation of yeast.
Preparation of fermentation broth.
• Five different samples were taken.
• Each sample contained 100g of fruit wastes taken in a brown bottle.
• To each sample, 50 ml of the prepared yeast media was added.[2]
• 1500ml of distilled water was added to each sample.[3]
• Cotton plugs were inserted into the opening of the brown bottle to
ensure anaerobic fermentation.[3]
• All the samples were then kept on the rotatory shaker for 21 days.[3]
 Sample 1: Mixture of
powdered fruit wastes Sample 2: 100g of Sample 3: 100g of
of 25g each. powdered Mosambi powdered Orange
(Mosambi+banana+ waste. waste.
orange +watermelon)

Sample 4: 100g of Sample 5: 100g of

powdered Banana powdered
waste. Watermelon waste.
5 samples for fermentation Samples kept on rotary shaker for
incubation
Test for the presence of ethanol.
• The 5 samples were taken in different
centrifuge tubes.
• The samples were then centrifuged at
6000rpm for 20 mins.[4]
• Then a total of 10 ml of the
supernatant was retrieved for the
ethanol test.

5 samples in centrifuge tubes.

Presence of alcohol test by oxidation-
Dichromate test[6]
• Preparation of 0.015M K2Cr2O7 solution in 3M H2SO4
• 10 ml of the centrifuged supernatant was taken in a test tube.
• Then 10ml of 0.015M K2Cr2O7 was added to the test tube.
• Color change was observed from yellow and then to green and then
finally to blue which shows the presence of alcohol.
Supernatant After adding 0.015M 5 minutes later
K2Cr2O7 -BLUE GREEN
REFERENCES:
1] Borah, Debajit & Mishra, Vimalendr,”Production of Bio Fuel from Fruit
Waste”. International Journal of Advanced Biotechnology Research. 1. 71-74.(2011)
2] Shrinivas T. Kakade, Prashant L. Chaudhari,” Bio-ethanol Fuel Production from
Rotten Banana Waste”. International Journal of Science and Research.8.1332-1336.(2019)
3] Nosakhare, F.,Osemwengie, S.O. and Oiwoh, O.,” Comparative
analysis and optimization of bioethanol produced from orange and grape peels”.
Nigerian Research Journal of Engineering and Environmental Sciences 2. 563-570. (2017)
4] A.B.M.S.Hossain, A Hadeel, K. Mseddi, Nasir, A Ibrahim
& Vajid N V. “Comparative Studies of Bio-ethanol Production from Different
Fruits Biomasses”.Global journal of life sciences and biological
studies.1.1-6.(2015)1. Borah, Debajit & Mishra, Vimalendr,”Production of Bio Fuel from
Fruit Waste”. International Journal of Advanced Biotechnology Research. 1. 71-74.(2011)
5] K.Kavitha, P.Rajendran & S. Ambigai Balan., “A Comparative study of
ethanol production from various fruit wastes using Saccharomyces cerevisiae
and Candida albicans”. Indian journal of applied
microniology.17.65-72.(2014)

6] Burke, Labianaca ; “Oxidation of alcohols by K2Cr2O7-Breathalyzer Demo”.

Organic Chemistry, Dept. of chemistry, NCSU. 1-2(1990)

7] Ahmad Jahanbakhshi, Rouhollah Salehi.,”Processing watermelon waste

using Saccharomyces cerevisiae yeast and the fermentation method
for bioethanol production”., Food processing Engineering.1-10.(2019)