Fermentation technology

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FERMENTATION TECHNOLOGY
AIM: To quantitatively estimate the amount of ethanol production
in the given fermentation reaction.
INTRODUCTION: Alcoholic fermentation, also referred to as
ethanol fermentation, is a biological process in which elements
such as glucose, fructose and sucrose are converted into cellular
energy and thereby productive ethanol and carbon-dioxide as
metabolic waste products.
Because yeasts perform this conversion in the absence of oxygen,
alcoholic fermentation is considered anaerobic.
C6H12O6 + H2) Zymase 2C2H5OH + 2CO2
The chemical equation submarrizes the fermentation of sucrose
into ethanol. Alcoholic fermentation converts one more of sucrose
into four moles of ethanol and four of carbon dioxide producing two
moles of ATP in the process.
Sucrose is a diamer of glucose and fructose molecules. In this first
step of alcoholic fermentation, the enzyme invertase cleaves the
glycosidic linkage between glucose and fructose molecules.
C12H22 O11 + H2O + Invertase  2C6H12O6
Next, each glucose molecule is broken down into two pyrvate
molecules in a process known as glycolysis. Glycolysis is
summarized by the equation:
C6H12O6 + 2ADP + 2Pi + 2NAD+  2CH3COOOO- + 2ATP + 2H+
+ 2NADH + 2H2O.

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The chemical formula of pyrivate is CH3OOOOO-, Pi standards for
the inorganic phosphate.
APPARATUS REQUIRED:
Conical flasks, braker, measuring cylinder, spectrophotometer,
standard flasks, test tubes, etc.
REAGENTS REQUIRED:
Absolute ethanol, Potassium dichromate solution, distilled water,
conc. Sulphuric acid.
PROCEDURE:
I. EXPERIMENTAL SETUP:
1. A specific quantity of grapes was washed and taken into a
mortar and pestle.
2. The grapes sere finely mashed and mascerated into a fine
paste.
3. To this, 100ml of distilled water was added in a flask.
4. One spatula of yeast extract was then added to the conical
flask.
5. The conical flask was dosed using a cotton plug in order to
maintain anaerobic condition.
6. This preparation is left for a period of 24 hours. the
appearance of bubbles indicate the generation of CO2 and
ethanol.
7. This simple becomes the unknown, whose amount is
estimated spectrophotometrically.

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II. QUANTITATIVE ESTIMATION OF ETHANOL
A. PREPARATION OF REAGENTS
1. Standard was prepared by dissolving absolute ethanol in
water to get 10mg/ml concentrations.
2. K2Cr2O7 solution was prepared by dissolving 10g of K2Cr2 O4
in distilled water and made up to 100ml in a standard flask.
B. STANDARD GRAPH FOR ETHANOL.
1. Standard solutions of 10mg/ml was prepared in different
concentrations 10mg/tube (0.1 – 1ml) and made upto 5ml in
distilled water.
2. Then 1 ml of K2Cr2O7 reagent was added.
3. All the test tubes were kept in ice cold water and 5ml of conc.
Sulphuric acid is added to each tube gently through the walls.
4. Then, the optical density was measured at 660nm and
standard graph was plotted with difference valves similarly
OD valves for unknown sample was measured.
5. The concentration of unknown samples corresponding to the
obtained OD values were taken.
RESULT:
The amount of ethanol produced by the fermentation of 30 grams of
grapes is 4.4.

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TABULATION:
S.
No
Concentration Amount
of
ethanol
Amount
of
distilled
water
Amount of
Potasium
Dischronate
Amount
of
sulphuric
acid
Total
volume
Absorbance
(at 660nm)
1. 0.5 0.5 4.5 1.0 5.0 11.0 0.119
2. 1.0 1.0 4.0 1.0 5.0 11.0 0.320
3. 1.5 1.5 3.5 1.0 5.0 11.0 0.350
4. 2.0 2.0 3.0 1.0 5.0 11.0 0.498
5. 2.5 2.5 2.5 1.0 5.0 11.0 0.350
6. 3.0 3.0 2.0 1.0 5.0 11.0 0.540
7. 3.5 35. 1.5 1.0 5.0 11.0 0.650
8. 4.0 4.0 1.0 1.0 5.0 11.0 0.600
9. 4.5 4.5 0.5 1.0 5.0 11.0 0.835
10. 5.0 5.0 0.0 1.0 5.0 11.0 0.935
11. Sample 1.0 4.0 1.0 5.0 11.0 0.769
ISOLATION OF PURE CULTURE IN FERMENTATION PROCESSES
AIM: To isolate pure culture of micro-organisms (yeast) in
fermentation processes.
INTRODUCTION: One of the most important of the microbial
eukaryotes to humans has been unicellular fungus saccharomyces
cerevisiae, the brewers and bakers’ yeast. By fermentation, the
yeast species converts carbohydrates into carbondioxide and
alcohols for thousands of years the carbondioxide has been used in
baking and the alcohol in alcoholic beverages.
Yeasts are chemoorganotrophs as they use organic compounds as a
source of energy. Yeats grow best in a neutral or slightly acidic PH
environment. In general, yaets are grown on solid growth media
or in liquid broths. Common media used for activation of yeasts
include potato dexbioze, agar or potato dexstrose both.

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MATERIALS REQUIRED:
1. Fruit or any rotten samples.
2. Loops, slides, coverslips, etc.
3. Metal spatula, tweezers, pasteur pipets.
PREPARATION OF MEDIA
1. To one liter of water, the following was added:
20g dextrose
10g peptone
5g yeast extract.
The pH was adjusted to 5 with dilute Hcl.
2. For tubes, 10ml was dispensed into each of about 16x150mm
tubes. It was capped and autoclated. It was allowed to
cool, and 40ul 25mg/ml ampicillin was added to each tube.
3. For plates, 15g of agar was added per liter, it was mixed and
autoclaved. It was cooled to 50oC and then 0.1g ampicillin
powder was added, mixed and poured into plates.
PROCEDURES:
1. A sterilized loop was used to innocutate 10ml tube of YPD-
Amp. A small piece of fruit was sufficient. It was incubated
for 1-3 days at 30oC.
2. If the tube has grown up, 1 drop of enrichment was
transferred into a fresh tube of YPD-Amp.
3. It was incubated again for 1-2 days at 80oC.

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4. A sample was streaked onto YPD-Amp Plate and Incubate 1-2
days at 30oC.
5. The isolated colonies were checked for microscopically to
identify yeast.
6. From this, a well isolated colony was picked and restreaked
onto a fresh plate.
OBSERVATION: The sample of enrichment was observed at low
power yeast and its bunds were identified. Other moulds could be
spotted but the distinguishing trait is the presence of buds. Yeasts
will bud, spores will germinate and these were readily distinguished.
RESULT: Pure cultures of yeast were isolated from the given
fermented sample.
PHYTOREMEDIATION
AIM: To study the process of phytoremediation by estimating the
amount of heavy metal uptake by the plants.
INTRODUCTION: Phytoremediatin is the direct use of living green
plants for in-site or in place removal, degradation or contamination
in Soils, sludges, sediments, surface water and ground water.
Phytoremediation is a low cost, solar energy driven clean up
technique. There are several different types of phytoremediation
mechanisms. These are:
1. Rhizodegradation: In this process, plants release enzymes
through roots, supplying nutrients to micro-organisms in the
soil. They enhance biological degradation.

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2. Phytostabilization: In this process, chemical compounds
produced by the plant immobilize contaminants, rather than
degrade them.
3. Phytovolatalvation: In this process, plants take up water
containing organic contaminants and release the
contaminants into the air through their leaves.
4. Phytodegradation: In this process, plants actually metabolize
and destroy contaminants within plant tissues.
MATERIALS REQUIRED:
1. Coriander seeds
2. Fresh garden soil
3. Pots (small size)
4. Chromium solution (10ppm, 15 ppm, 20 ppm)
5. Perchloric acid
6. Conc. Nitric acid
7. Conc. Hydrochloric acid
8. Distilled water
APPARATUS REQUIRED:
1. Burette, pipette, conical flask, beaker
2. Hot plate
3. Spectrophotometer
PROCEDURE:
I. EXPERIMENTAL SETUP:

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1. 50g of soil was taken 10ppm of chromium solution was
added. This was repeated with adding 15ppm and 20ppm of
chromium solution.
2. Control was also maintained with no addition of chromium
solution.
3. Coriander seeds were taken, soaked for 1 hour in water. Late
they were sown in the soil (10 seeds in each pot).
4. They were allowed to grow.
5. After 25 days, the plants were taken separately. They were
washed with distilled water and kept in hot air oven at 70oC
for 24 hours.
6. The material was powdered and made into fine powder
separately and 10mg dry weight of this material was taken
for each concentration of heavy metal.
II.ACID DIGESTION:
1. 5ml of perchloric acid, 1ml of conc. HNO3 was added. This
mixture was kept on the hot plate. The sample was boiled
until they dry.
2. 1:1 (Hcl: HNO3) was added to the sample till they dried.
3. 5ml of Hcl was added and boiled till they become dry.
4. After they were dried, m3 of water.
III.ESTIMATION OF CHROMIUM
1. 1ml of sample was added with 1ml H2SO4 and 1ml
diphenycarbazide solution.

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2. The solution turned pink.
3. A blank was also prepared in the similar manner.
4. The absorbance was read at 540nm in the spectrophotometer.
BIOINDICATORS – USE OF BIOLOGICAL ORGANISMS
1. MICROALGAE:
1. Microalgae are indicators of water quality.
2. Within different organisms that can be used as models for
bioarrays, microalgae have gained a lot of attention in the
last years due to several reasons.
 Abundantly occurring organisms.
 Play a vital role in the food web of aquatic systems.
 Higher sensitivity than invertebrates and fish.
 Easy to perform; not difficult to perform auctures, do not
demand a lot of area for algae growth, easy to handle
when comparing ecotoxicological assays with invertebrates
or fish.
 Ethical reasons, it avoid the use of experimental animals
like rats and rabbits.
2. EUGLENA
1. Euglena gracilis is a freshwater photosynthetic flagellate.
2. Although Euglena is rather tolerant to acidity, it responds
rapidly and sensitivity to environmental stressors like heavy
metals or inorganic and organic compounds.

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3. Typical responses are the inhibition of movement and the
change of orientation parameters.
4. One very useful particularity of this organism is the
gravitactic orientation, which is very sensitive to pollutants.
5. Euglena gracilis orients itself in the water column phototaxis
and gravitaxis.
6. The gravireceptors are impaired by pollutants like heavy
metals and organic or inorganic compounds.
7. Therefore the presence of such substances is associated with
random movement of the cells in the water column.
3. MACROPHYTES:
1. A macrophyte is an aquatic plant that grows in or near water
and is either emergent, submergent or floating.
2. In lakes and rivers macrophytes provide cover for fish and
substrate for aquatic invertebrates produce oxygen, and act
as food for some fish and wild life.
3. A decline in a macrophyte community may indicate water
quality problems and changes in the ecological status of the
water body.
4. Such problems may be the result of excessive turbidity,
herbicides or salinization.
5. Conversely, overly high nutrient levels may create an
overabundance of macrophytes which may in turn interfere
with lake processing.

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BIOFERTILIZERS
1. BLUE GREEN ALGAE:
1. Blue Green algae is a type of biofertilizer used as an alga.
2. Important species are cyanobacteria, anaebena, Nostoc, and
Tolypothrix. Blue green algae occurs naturally and comes up
well under moist conditions.
3. The role of BGA as biofertilivesr are:
 BGA elaborates vitamin Biz and growth factors that makes
the plant grow vigorously.
 It oxygenates the water compounded in the field.
 It excretes organic acids that renders phosphorus
solubilization.
 The algal mat in paddy fields also protects loss of moisture
from the soil.
 It is a very efficient and ecofriendly biofertilizer.
2. AZOLLA
1. Azolla is a free-floating water fern having an algal symbiant
Anabaena azolla.
2. Azoalla can be multiplied by constructing nurseries with
10cm deep standing water and adding superphosphate Azolla
can be used immediately after harvest.
3. It can be applied as green manure prior to rice planting or
can be grown as dual crop with rice.
4. The role of azolla as bioferilizer is:

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 It excretes organic nitrogen in water during its growth and
immediately upon trampling.
 Fern friends are soft and rapidly decomposed.
 It absorbs traces of potassium from irrigation water.
 Azolla provides nitrogen, potassium, organic carbon.
 It prevents weed growth in rice field water.
3. VERMICOMPOST
1. Vermicompost is the product or process of composting using
various worms and other earthworms to create a
heterogenous mixture of decomposing vegetable or food
waste, bedding materials and vermicast.
2. The role of vermicompost as a biofertilizer are:
 Improves soil aeration.
 Enriches soil with micro-organisms (adding enzymes such
as phosphates and cellubase)
 Microbial activity increases the water holding capacity of
the soil.
 Enhances germination and plant growth, in turn increases
crop yield.
 Improves root growth and structure.
 Enriches soil with micro-organisms (adding plant harmones
such as auxins and gibberellic acid.
3. It is a low capital investment and relatively simple
technologies making vermicomposing practical for less
developed agricultural regions.