PRACTICAL & Assignment of IB

Industrial Biotechnology
By Krunal Shah Semester-2 M.E. CAPD

L. D. College of Engineering Page 1

PRACTICAL-1
Aim:

To prepare agar plates and slants.

Procedure:
Nutrient Agar Plates:
1. Weigh out 23.0 grams of nutrient agar powder.
2. Add to 1.0 liter of distilled or deionized water in a 2.0 liter flask.
3. Sterilize at 121 C for 20-25 minutes.
4. Cool to 50 C.
5. Swirl thoroughly to mix agar and nutrients.
6. Pour 25-35 ml per petri plate.
7. Yields about 35 plates.

Nutrient Agar Slants:
1. Place screw cap test tubes in a test tube rack (without the caps).
2. Prepare a nutrient agar medium and boil it with stirring until all the agar is melted.
You must stir this very well so that the melted agar is distributed throughout the medium.
3. Use a pipette to transfer about 5 ml of molten agar to each test tube.
4. When all the tubes contain hot agar, place the caps loosely on the tubes.
5. Sterilize the tubes at 121 C for 15 minutes with caps loosely on.


6. While the medium is till hot, tilt the rack onto a thick book or other solid surface so that the
medium in the tubes is slanted. Allow the medium to harden in this position.
7. When the medium is cool, tighten the caps.
8. These tubes can be stored at room temperature or in the refrigerator.

Result:
After 10-15 min, we can observe solidify surface in the test tube & petry plate.
Conclusion:

Agar powder has ability to solidify when it cools from 100
0
c to 40
0
c. So because of agar
powder the media become solidify in the plates & tubes.



PRACTICAL-2
Aim:
Screening of Amylase & organic acid producing organism.
Introduction:
Amylases are enzymes that break down starch or glycogen. Amylases are produced by a variety
of living organism, ranging from bacteria to plants and humans. Bacteria and fungi secrete
amylases to the outside of their cells to carry out extra-cellular digestion. When they have broken
down the insoluble starch, the soluble end products such as (glucose or maltose) are absorbed
into their cells.
Amylase are classified based on how they break down starch molecules.
1. -amylase Reduced the viscosity of starch by breaking down the bonds at random, therefore
producing varied sized chains of glucose.
2. -amylase Breaks the glucose-glucose bonds down by removing two glucose units at a time,
thereby producing maltose.
3. Amyloglucosidase (AMG) Breaks successive bonds from the non-reducing end of the
straight chain, producing glucose.
Many microbial amylases usually contain a mixture of these amylases.
Organisms involved in Amylase production:
Although many microorganisms produce this enzyme, the ones most commonly used for their
industrial production are Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquifaciens and
Aspergillus niger.
Requirements:
Starch Agar plate
Sterile pipettes, Spreader, Flasks, Suspension tubes


Rotary shaker
Inoculum/fermentation medium
1% Starch plate:
Nutrient agar medium 5.6 g
Distilled water 175 ml
Agar-Agar 3 g
Starch 2 g
Total volume 200 ml
Organic acid:
Nutrient agar 5.6 g
0.04% Bromo Thymol Blue 10 ml
Agar-Agar 3 g
Total volume - 200 ml
Procedure:
1. Prepare above solution. Take a 6 test tube. Add 9 ml distilled water to each. Take petry
dish. First sterilize these in autoclave at 15 lb pressure n 121
0
C.
2. In first test tube add 1gm of soil sample and make serial dilution.
3. Make starch solution and organic acid solution petry plate.
4. Take a 0.1 ml sample from test tube & spread on this petry dish.
5. This spreading is to be done between burners i.e in sterile condition.
6. Observe for the zone of starch hydrolysis around the colonies.
7. In starch plat if we get clear solution around the colonies or yellow colonies it can be
produce amylase.


Result:
Starch plate
Dilution Actual no. of colony
10
-4
928
848
10
-5
163
180
10
-6
12
9
Organic acid plate
Dilution Actual no. of colony
10
-4
2216
1080
10
-5
174
181
10
-6
52
17



Conclusion:
We get more amylase production in 10
-4
dilution petry dish compare to 10
-5
and
10
-6
.



PRACTICAL-3
Aim:
To study Crowded plate method.
Procedure:
Plates
First, a sample of organisms from soil or some other source is diluted in water, then spread onto
Petri dishes containing agar gel rich in the nutrients the bacteria will need to grow. Scientists
select plates that have a large number of colonies, then look for microorganisms that have
inhibited the growth of other microorganisms in their vicinity. These microbes are possibly
secreting some kind of compound that is killing or inhibiting their neighbors.
Purification
Colonies that may be producing antibiotics are transferred to another plate so they can be
purified and grown in isolation. It's entirely possible, of course, that the colony was really just
altering the pH of its environment or making some other change that killed other bacteria, rather
than secreting an antibiotic, so further tests are needed to confirm that it is indeed an antibiotic-
producing strain. Nonetheless, the crowded plate technique was sometimes helpful in identifying
microorganisms that could serve as sources of new antibiotics.
Crowded Plate Technique:
For screening of antibiotic producing organisms, the simplest technique is crowded plate
procedure. This technique is used where one is interested only in finding microorganisms that
produces an antibiotic irrespective of its action against any specific organism. Hence the sample
is diluted only to such an extent that agar plates prepared from these dilutions will be crowded
with individual colonies on agar surface, i.e. 300 to 400 colonies or more. Colonies producing
antimicrobial activity are indicated by clear zone of growth inhibition surrounding the colony.
Such colony is later on sub cultured; purified and afterwards microbial inhibition spectrum is
tested against selective microorganisms.



Wilkinss method:
Another technique mainly use Wilkinss method. A Wilking medium which contains a pH
indicating dyes i.e. Bromo-thymol Blue which is green colored at neutral pH but colorless at
acidic pH. This method differentiate antibiotic producer from the acid producer. Those colonies
that produce antibiotics give zone of inhibition against sensitive organisms without changing
color surrounding it while in case of acid produce colony the we may find zone of inhibition due
to fail in pH along with colorless are due high acid production which result in lower pH,
ultimately change the color of dye from green to colorless.

Wilkinss agar composition:
Peptone 2 g
Nacl 0.5 g
0.04% Bromo Thymol Blue 5 ml
Agar-Agar 3 g
pH 7.4
\


Conclusion:



PRACTICAL-4
Aim:
Fermentation of Ethanol from glucose.
Introduction:
Chemically, ethanol is an organic compound part of the alcohol family. It is a colorless liquid
with a burning taste and a characteristic odor. Its specific gravity at 20
0
C is 0.785 g/ml, and its
boiling point is 78.4
0
C. In fact, distilled alcohol always contains traces of water from the
distillation mixture. Such compounds are referred to as azeotropes. Unlike water, ethanol has a
non-polar ethyl portion capable of Van-der-waals interactions. The chemical properties of
alcohol are thus a balance between its polar OH group and its non-polar hydrocarbon group.
Ethanol is currently the largest volume (liquid) industrial fermentation product worldwide.
Approximately 80% of the world supply of alcohol is produced by fermentation. Industrial
alcohol has been valuable as a solvent, germicide, fuel, in cosmetics and chemical raw material.
Many raw materials are used for alcohol fermentation as follows:
Saccharine materials: sugar cane juice, sugar beat juice, blackstrap molasses, whey, ripe
fruits.
Starchy material: cereal grains, potatoes, roots, tubers, cacti.
Cellulose raw material: sulfide waste liquor, grasses, wood.
Microorganisms involved in alcohol fermentation:
Yeast: Saccharomyces cerevisiae, S. uvarum, Candida utilis.
Bacteria: Zymomonas mobilis, Clostridium thermocellum
C
4
H
12
O
6
2 C
2
H
5
OH + 2 CO
2
+ energy (stored as ATP)

Alcohol fermentation is an anaerobic, exothermic process in which sugars break down into
ethanol and CO
2
gas, catalyzed by microbial or fungal enzymes.



Requirements:
GYE medium (inoculums development medium)
Glucose 1%
Peptone 0.5%
Yeast extract 0.3%
pH 4.5 5.0
Fermentation medium
Sugar/jeggary 20%
KH
2
PO
4
1.5 g/lit
(NH
4
)
2
SO
4
0.5 g/lit
Yeast extract 0.5 g/lit
Distilled water 1 lit
Ph 4.5
Composition of jeggary (g%)
65-85% sucrose, 10-15% invert sugars (hydrolyzed form of glucose, fructose), 2-5%
moisture, 3-6% protein, 5% unclassified.
Fresh culture slant o S. cerevisiae.
250 ml flasks, 10 ml sterile pipette
Procedure:
Inoculum developmet
s.cerevisiae on GYE agar slant

Prepare thick suspension of S. Cerevisiae from slant

Add 2 ml of suspension in 100 ml GYE broth


Incubate the flask at 30
0
C for 24 h for culture activation

Commercially available dry beads of bakers yeast

Dissolve 3 beads in 5 ml sterile D/W and make homogeneous suspension

Add 2 ml of the suspension in 100 ml GYE broth

Incubate the flask at 30
0
C for 24 h for culture activation

Fermentation
1. Take a fresh culture of S. cerevisiae and bakers yeast and perform gram staining
and note its colony characteristics.
2. After inoculums development process, take 10 ml active culture from the flasks
with the help of sterile 10 ml pipettes and inoculate them in to 90 ml sterile
fermentation broth in 250 ml flask and estimate 0 h, sugar from both flasks. Then
keep them on shaker for 24 h at 30
0
C.
3. After incubation of 24 h, add more sterile medium approximately 150 ml, so
finally each flask contain 250 ml broth and partially anaerobic condition is created
in the flask, which is best for the production of alcohol.
4. This flask is kept at 30
0
C in static condition and every 24 h interval the sugar
remaining and ethanol produced is measured by Coles method ( both before
hydrolysis & after hydrolysis) & alcohol estimation respectively till sugar
concentration becomes zero. % yield is calculated by the equation as follows:
% yield = [alcohol produced (g) / sugar utilized (g)] 100


5. When sugar concentration becomes zero, the broth is centrifuged to remove cells
and other particles. After this the distillation of ethanol is done from the broth.
Ethanol gets distilled at 78.4
0
C temp.
Result :
Sugar estimation from fermented medium
No. Time(h) Sugar present(gm) Sugar used
1 0 25 -
2 24 12.53 12.47
3 48 1.18 23.82
Alcohol estimation
sample D/W (ml) Na
2
S
2
O
3
(0.1 ml) Alcohol concentration
(mg/ml)
Blank 5 20.5 -
Original 0.5 ml 4.5 - -
1.2 dil. 0.5 ml 4.5 22 -
1.5 dil. 0.5 ml 4.5 12.2 9.5
Now the alcohol concentration = 95 mg/ml
= 9.5 g%
Alcohol concentration obtained after 48 hr is 9.5 g%.
Sugar concentration obtained is 23.82 g%.



Sugar to alcohol efficiency
Alcohol produced/ sugar consumed = y/x = 92/180 = 100%
9.5/23.82 = ?
= (9.5 100 180) / (23.82 92)
= 78% efficiency
Conclusion:
Alcohol concentration obtained after 48 hr is 9.5 g%.
Sugar concentration obtained is 23.82 g%.
Yield % = 78% efficiency





PRACTICAL-5
Aim:
To determine the oxygen transfer rate.
Introduction:
Majority of fermentation processes are aerobic and therefore require the provision of oxygen.
Due to its low solubility it is not possible to provide a microbial culture with all the oxygen it
will need for the complete oxidation of any carbon source in one addition. Therefore a microbial
culture must be supplied with oxygen during growth at a rate sufficient to satisfy the organisms
demand. The oxygen demand of an industrial fermentation process is normally satisfied by
aerating and agitating the fermentation broth. However, the metabolism of the culture is affected
by the concentration of dissolved oxygen in broth. The effect of dissolved oxygen concentration
on the specific oxygen uptake rate has been studied by Michaelis & Menten.
Specific oxygen uptake rate can be defined as mMoles of oxygen consumed per gram dry
weight of cells per hour. According to their study specific oxygen uptake rate increases with
dissolved oxygen concentration up to a certain point above which no further increase in oxygen
uptake rate occurs. Thus maximum biomass production may be achieved by satisfying the
organisms maximum specific oxygen demand by maintaining the dissolved oxygen
concentration greater than the critical level. If the dissolved oxygen concentration were to fall
below the critical level then the cells may be metabolically disturbed.
The oxygen is normally supplied to microbial cultures in the form of air, this being the cheapest
available source of the gas. Bartholomew et al. represented the transfer of oxygen from air to the
cell, during fermentation, as occurring in a number of steos:
The transfer of oxygen from an air bubble into solution.
The transfer of the dissolved oxygen through the fermentation medium to the
microbial cell.
The uptake of the dissolved oxygen by the cell.



Oxygen Transfer Rate By Sulphite Oxidation Method:
Cooper et al. were the first to describe the determination of oxygen transfer rate in aerated
vessels by the oxidation of sodium sulphite solution.
Principle:
Dissolved oxygen instantly converts sodium sulphite into sodium sulphate in the presence of a
copper or cobalt catalyst.
Na
2
SO
3
+ O
2
Na
2
SO
4

The residual SO
3
(unoxidized) are oxidized by addition of I
2
, depending upon amount of
unoxidized SO
3
. I
2
will be consumed & remaining I
2
can be treated with Na2S2O3 using starch
as an indicator.
Na
2
SO
3
(residual) + I
2
+ H
2
0 Na
2
SO
4
+ 2HI
I
2
(residual) + 2Na
2
S
2
O
3
Na
2
S
4
0
6
+ 2NaI
Now the oxygen transfer rate (OTR) can be calculated from the following formula:
OTR = TN 0.251000 / T Volume of abquot taken mMole o
2
/L/hour
Where,
T = B-E reading at time t, where B is blank reading and E is experimental reading
N = normality of solution
T = time interval in minute
Reagents
0.1 N Na
2
SO
3

Dissolve 12.9 gm Na
2
SO
3
in 1000 ml
0.1 N iodine solution
O.1% starch solution
0.1 gm starch in 100 ml.



Procedure:
Take 100 ml Na
2
SO
3
in 500 ml flask.

Immediately take out 5 ml of sample from this for 0 hour reading on put the flask on shaker.
Keep another same flask in static condition also.

With that 5 ml of sample add 1 ml of starch as an indicator.

Now titrate it with 0.1 N I
2
solutions. Color changes will be from colorless to black.

Take out samples at interval of 30 minutes.
Result:
Adding 2-3 drops
starch
Adding 0.5 ml starch
Time (min) Titration pt. Time (min) Titration pt.
0 10 0 11.6
30 9.5 30 9.8
60 9.3 60 9.3
90 8.5 90 8.5
120 7.8 120 7.6
Conclusion:
For aeration & agitation the OTR will be higher


PRACTICAL- 6
Aim:
To measure the optical density.
Biuret Assay
Material :
1. Protein standard (5gm albumin/ml). Prepare fresh.
2. Biuret reagent (dissolve 3 gm of Copper Sulphate (CuSO
4
.5H
2
O) and 9 gm of sodium
potassium tartrate in 500 ml of 0.2 mol/litre sodium hydroxide; add 5gm of potassium
iodide and make up to 1 litre with 0.2 mol/ litre of sodium hydroxide.)
3. Water bath at 37
o
C.
Method:
Add 3 ml of biuret reagent to 2 ml of protein solution mix and warm at 37
o
C for 10 min;
cool and read the extinction at 540 nm. Prepare of graph of extinction against albumin
concentration, this standard will prove useful in other experiments.

The Folin-Lowry method of protein assay
Principle:
Protein reacts with the Folin-Ciocalteau reagent to give a coloured complex. The colour
so formed is due to the reaction of alkaline copper with the protein as in the biuret test
and the reduction of phosphomolybdate by tyrosine and tryptophan present in the protein.
The intensity of colour depends on the amount of these aromatic amino acid present and
will thus vary for different proteins.

Materials:


1. Alkaline sodium carbonatesolution (20g/litre Na
2
CO
3
in 0.1 mol/litre NaOH).
2. Copper sulphate- sodium potassium tartratesolution (5g/litre CuSO
4
.5H
2
O in 10g/litre
Na, Ktartrate).Prepare fresh by making stock solutions.
3. Alkaline solution. Prepare on day of use by mixing 50 ml of (1) and 1ml of (2).
4. Folin-Ciocalteau reagent.(Dilute the commercial reagent with an equal volume of
water on the day of use. This solution of sodium tungstate and sodium molybdate in
phosphoric and hydrochloric acids.)
5. Standard protein (albumin solution 0.2 mg/ml).
Method:
Add 5 ml of Alkaline solution to 1 ml of the test solution. Mix throughly and allow to
stand at room temperature for 10 min or longer. Add 0.5 ml of diluted Folin-Ciocalteau
reagent rapidly with immediate mixing. After 30min read the extinction against the
appropriate blank at 750 nm.
Estimate the protein concentration of an unknown solution after preparing a standard
curve.
Result:
KMNO
4
solution
0.5 gm KMNO
4
IN 100 ml distilled water

KMNO
4
solution

Distilled water Total volume
Blank 2 2
0.4 1.6 2
0.8 1.2 2
1.2 0.8 2
1.6 0.4 2
2 - 2
Here, max = 550 nm


KMNO
4
solution

Wave length absorbance filter
0.4 550 0.760 4
0.8 550 1.381 4
1.2 550 1.923 4
1.6 550 2.388 4
2 550 2.468 4

Dye solution
Dye solution

Distilled water Total volume (add 3ml to
each)
Blank 2 5
0.4 1.6 5
0.8 1.2 5
1.2 0.8 5
1.6 0.4 5
2 - 5
Here, max = 500 nm
Dye solution

0.4 500 0.535 4
0.8 500 0.535 4
1.2 500 1.029 4
1.6 500 1.934 4
2 500 2.637 4



Protein solution
starch solution

B 660 0 6
0.2 660 0.178 6
0.4 660 0.037 6
0.6 660 0.072 6
0.8 660 0.035 6
2 660 0.111 6



ASSIGNMENTS



Assignment -1
LIPASES
A lipase is a water-soluble enzyme that catalyzes the hydrolysis of ester chemical bonds
in water-insoluble lipid substrates.

Lipases are a subclass of the esterases.
Lipases perform essential roles in the digestion, transport and processing of dietary lipids
(e.g. triglycerides, fats, oils) in most, if not all, living organisms. Genes encoding lipases are
even present in certain viruses.

Function
Most lipases act at a specific position on the glycerol backbone of lipid substrate (A
1
, A
2

or A
3
)(small intestine). For example, human pancreatic lipase (HPL), which is the main enzyme
that breaks down dietary fats in the human digestive system, converts triglyceride substrates
found in ingested oils to monoglycerides and free fatty acids.
Several other types of lipase activities exist in nature, such as phospholipases and
sphingomyelinases, however these are usually treated separately from "conventional" lipases.
Some lipases are expressed and secreted by pathogenic organisms during the infection. In
particular, Candida albicans has a large number of different lipases, possibly reflecting broad


lipolytic activity, which may contribute to the persistence and virulence of C. albicans in human
tissue.
Structure and catalytic mechanism
Although a diverse array of genetically distinct lipase enzymes are found in nature, and
represent several types of protein folds and catalytic mechanisms, most are built on an alpha/beta
hydrolase fold (see image) and employ a chymotrypsin-like hydrolysis mechanism involving a
serine nucleophile, an acid residue (usually aspartic acid), and a histidine.
Physiological distribution
Lipases are involved in diverse biological processes ranging from routine metabolism of
dietary triglycerides to cell signaling and inflammation. Thus, some lipase activities are confined
to specific compartments within cells while others work in extracellular spaces.
In the example of lysosomal lipase, the enzyme is confined within an organelle called the
lysosome.
Other lipase enzymes, such as pancreatic lipases, are secreted into extracellular spaces
where they serve to process dietary lipids into more simple forms that can be more easily
absorbed and transported throughout the body.
Fungi and bacteria may secrete lipases to facilitate nutrient absorption from the external
medium (or in examples of pathogenic microbes, to promote invasion of a new host).
Certain wasp and bee venoms contain phospholipases that enhance the "biological
payload" of injury and inflammation delivered by a sting.
As biological membranes are integral to living cells and are largely composed of
phospholipids, lipases play important roles in cell biology.
Malassezia globosa, a fungus that is thought to be the cause of human dandruff, uses
lipase to break down sebum into oleic acid and increase skin cell production, causing
dandruff.
Human lipases
The main lipases of the human digestive system are human pancreatic lipase (HPL) and
pancreatic lipase related protein 2 (PLRP2), which are secreted by the pancreas. Humans also
have several other related enzymes, including hepatic lipase (HL), endothelial lipase, and
lipoprotein lipase. Not all of these lipases function in the gut (see table).



Name

Location Description Disorder
bile salt
dependent
lipase

pancreas,
breast milk
aids in the digestion of fats

pancreatic
lipase
digestive juice
In order to exhibit optimal enzyme activity
in the gut lumen, HPL requires another
protein, colipase, which is also secreted by
the pancreas.

lysosomal
lipase
interior space
of organelle:
lysosome
Also referred to as lysosomal acid lipase
(LAL or LIPA) or acid cholesteryl ester
hydrolase
Cholesteryl ester
storage disease
(CESD) and Wolman
disease are both
caused by mutations
in the gene encoding
lysosomal lipase.
hepatic
lipase
endothelium
Hepatic lipase acts on the remaining lipids
carried on lipoproteins in the blood to
regenerate LDL (low density lipoprotein).
-
lipoprotein
lipase
endothelium
Lipoprotein lipase functions in the blood to
act on triacylglycerides carried on VLDL
(very low density lipoprotein) so that cells
can take up the freed fatty acids.
Lipoprotein lipase
deficiency is caused
by mutations in the
gene encoding
lipoprotein lipase.
hormone-
sensitive
lipase

intracellular - -
gastric
lipase
digestive juice
Functions in the infant at a near-neutral pH
to aid in the digestion of lipids
-
endothelial
lipase
endothelium - -
pancreatic
lipase
related
protein 2

digestive juice - -
pancreatic
lipase
related
protein 1

digestive juice
Pancreatic lipase related protein 1 is very
similar to PLRP2 and HPL by amino acid
sequence (all three genes probably arose
via gene duplication of a single ancestral
pancreatic lipase gene). However, PLRP1
is devoid of detectable lipase activity and
its function remains unknown, even though
it is conserved in other mammals.
-


lingual
lipase
digestive juice - -
There also are a diverse array of phospholipases, but these are not always classified with
the other lipases.
Industrial uses
Lipases from fungi and bacteria serve important roles in human practices as ancient as
yogurt and cheese fermentation. However, lipases are also being exploited as cheap and versatile
catalysts to degrade lipids in more modern applications. For instance, a biotechnology company
has brought recombinant lipase enzymes to market for use in applications such as baking,
laundry detergents and even as biocatalysts in alternative energy strategies to convert vegetable
oil into fuel.
Applications of Lipases
Dairy Industry
Lipases are extensively used in the dairy industry for the hydrolysis of milk fat. Current
applications include the flavour enhancement of cheeses, the acceleration of cheese ripening , the
manufacturing of cheese like products, and the lipolysis of butterfat and cream. The free fatty
acids generated by the action of lipases on milk fat endow many diary products, particularly soft
cheeses, with their specific flavour characteristics. Thus the addition of lipases that primarily
release short chain (mainly C4 and C6) fatty acids lead to the development of a sharp,tangy
flavour, while the release of medium chain (C12,C14) fatty acids tend to impart a soapy taste to
the product. In addition, the free fatty acids take part in simple chemical reactions, as well as
being converted by the microbial population of the cheese. This initiates the synthesis of flavour
ingredients such as acetoacetate, beta-keto acids, methyl ketones, flavour esters and lactones.
The intensive use of lipases in cheese making started in the U.S.A after the Second
World War. It was engendered by the Food and Drug Administration's ban on the import of
rennet paste from Europe because of the impurity and unsatisfactory microbiology of the
product. This paste was used by the manufacturers of traditional Italian cheeses ( Provolone,
Romano, Mozzarela, Parmesan) and the first lipase cocktails were introduced as a substitute to
create the typical lipolytic flavour of these varieties.
The traditional sources of lipases for cheese flavour enhancement are animal tissues,
especially pancreatic glands(bovine and porcine) and the pre-gastric tissues of young
ruminants(kid,lamb,calf). The latter are more commonly used in cheese making. The commercial
pre-gastric lipases are available in the form of liquid extracts, pastes and vaccum or freeze dried
powders. Each type of pre-gastric lipase gives rise to its own charecteristic flavour profile : a


buttery and slightly peppery flavour(calf) ; a sharp 'piccante' flavour (kid); a strong 'pecerino'
also described as 'dirty sock' flavour(lamb).
A whole range of microbial lipase preparations has been developed for the cheese
manufacturing industry: Mucor meihei(Piccnate, Gist-Brocades; Palatase M, Novo Nordisk),
Aspergillus niger and A.oryzae (Palatase A, Novo Nordisk; Lipase AP, Amano; Flavour AGE,
Chr. Hansen) and several others. These microbial lipases are used not only for flavour
enhancement and the acceleration of the ripening of specific cheeses such as blue, but in some
cases they have also successfully replaced pre-gastric lipases . A range of cheeses of good
quality was produced by using individual microbial lipases or mixtures of several preparations.
Apart from substitution of rennet paste and flavour enhancement, lipases are widely
used for imitation of cheeses made from ewe's or goat's milk. Addition of lipases to cow's milk
generates a flavour rather similar to that of ewe/goat milk. This is used for producing cheeses
like Feta, Manchego and Romano from cow's milk. When added to certain blue cheeses, lipase
imitate the taste of Roquefort, which is normally produced from sheep's milk. Similarly, the
addition of lipases to pasteurised milk leads to the development of the normal flavour of Ras or
Konpanisti, which traditionally are produced from raw milk.
Lipases also play a crucial role in the preparation of so-called enzyme modified cheeses
(EMC). EMC is a cheese that is incubated in the presence of enzymes at elevated temparature in
order to produce a concentrated flavour for use as an ingredient in other products
(dips,sauses,dressings,soups,snacks etc.). Typically the concentration of free fatty acids is ten
times higher in EMC than in that of the corresponding young cheese. EMC technology is widely
used in the U.S.A.
Detergents
Enzymes can be used in the laundry detergents and automatic dish washing machines
detergents. Enzymes can reduce the environmental load of detergent products, since they save
energy by enabling a lower wash temperature to be used; allow the content of other, often less
desirable, chemicals in detergents to be reduced; are biodegradable, leaving no harmful residues;
have no negative impact on sewage treatment processes; and, do not present a risk to aquatic life.
Other enzymes are currently widely used in household cleaning products. A great deal of
research is currently going into developing lipases which will work under alkaline conditions as
fat stain removers.
Oleochemical Industry
The scope for the application of lipases in the oleochemical industry is enormous. Fats
and oils are produced world wide at a level of approximately 60 million tonnes per annum and a
substantial part of this(more than 2 million tonnes per annum) is utilised in high energy


consuming processes such as hydrolysis, glycerolysis and alcoholysis. The conditions for steam
fat splitting and conventional glycerolysis of oils involve high temparatures of 240-260 degree C
and high pressures (methanolysis is currently performed under slightly milder conditions). The
resulting products are often unstable as obtained and require re-distillation to remove impurities
and products of degradation. In addition to this, highly unsaturated heat sensitive oils cannot be
used in this process without prior hydrogenation.
The saving of energy and minimisation of thermal degradation are probably the major
attractions in replacing the current chemical technologies with biological ones. However, in spite
of their apparent superiority, enzymic methods have not as yet attained a level of commercial
exploitation commensurate with their potential. There have been several communications about
relatively small scale enzymic fat splitting processes for the production of some high value
polyunsaturated fatty acids and the manufacture of soap. For instance Miyoshi Oil & Fat Co.,
Japan, reported the commercial use of Candida cylindracea lipase in the production of soaps. The
company claimed that the enzymic method yielded a superior product and was cheaper overall
than the conventional Colgate-Emery process.
There are probably several reasons for the generally disappointing level of commercial
applications of lipase in this sector at present. First of all, the oleochemical industry is very
conservative, owing to huge capital investments involved. Therefore one cannot expect rapid
changes. Secondly until recently the high cost of lipases remained prohibitive for the
manufacturing of bulk products. The introduction of the new generation of cheap and very
thermostable enzymes should change the economic balance in favour of lipase use. Thirdly,
some concern has been expressed by chemical engineers with regard to running and controlling
enzymic processes on the required scale. However the recent commercialisation of several lipase
based technologies has proved their feasibility unambiguosly. The future of lipases look rather
promising in the context of oleochemistry.
Some fats are much more valuable than others beacuse of their structure. Less valuable
fats can be converted into more useful species using blending of chemical methods but these tend
to give quite random products. Lipase catalysed transesterification of cheaper oils can be used,
for example to produce cocoa butter from palm mid-fraction.
Pharmaceutical Industry
The vast variety of synthetic pharmaceuticals and agrochemicals containing one or
more chiral centres, are stll being sold as racemates. This is despite the fact that the desired
biological activity resides in one particular enantiomer. A single isomer is preferable to a
racemate, but there are severe technical and/or economic problems with the production of single
isomers.


The usefulness of lipases in the preparation of chiral synthons is well recognised. The
resolution of 2-halopropionic acids, starting materials for the synthesis of phenoxypropionate
herbicides is being carried out on a 100-kg scale by Chemie Linz Co.(Austria) under a license
from the Massachusetts Institute of Technology. The process is based on the selective
esterification of (S)-isomers with butanol catalysed by porcine pancreatic lipase in anhydrous
hexane. Typically , > 99% enantiomeric excess(e.e) is obtained at 75% of the theoritical yield
and the resolution is complete in several hours.
Generally, the lipase mediated resolution of 2-substituted propionic acids, and
especially 2-aryl derivatives, have been the subject of intensive investigations. A substantial
body of literature exists on the production of both (R) and (S) isomers of alpha(*sub)-
phenoxypropionic acids, which are useful synthons for the preparation of enantiomerically pure
herbicides and non-steroidal anti-inflammatory drugs (naproxen,ibuprofen) respectively. The
required optically pure derivative can be obtained directly via the (trans)esterification or
hydrolysis of the corresponding ester. These resolutions have been performed on a multi-
kilogramme scale by several companies world-wide.
Another instance of commercial application of lipases to the resolution of racemic
mixtures is the hydrolysis of epoxy alcohol esters. The highly enantioselective hydrolysis of
(R,S)-glycidyl butyrate has been developed by DSM-Andeno (the Netherlands). The reaction
products, (R)-glycidyl esters and (R)-glycidol, are readily converted to (R)-and (S)-
glycidyltosylates, which are very attractive intermediates for the preparation of optically active
beta-blockers and a wide range of other products.
A similar technology has been commercialized by Sepracor Inc.(USA). This company
has successfully operated a multi-kilogramme scale membrane bioreactor to produce the
2(R),2(S) methyl methoxyphenyl glycidate, the key intermediate in the manufacture of the
optically pure cardiovascular drug diltiazem. 2(S),3(R)-methoxyphenyl glycidic acid, the product
of enzymic hydrolysis, was found to be unstable under the conditions of the reaction, and the
resultant aldehyde inhibited the lipase activity and reduced the lifetime of the enzyme. Both
problems were overcome by the introduction of a multi-phase membrane reactor where the
aldehyde by-product reacted in situ with bisulphite to form a non-inhibitory, water soluble
adduct, extracted into the aqueous phase.
Lipases have been found useful as industrial catalysts for the resolution of racemic
alcohols. Enantiomerically pure endo-2-norborn-2-ol is an important chiral intermediate in the
preparation of some prostaglandins, steroids and carbocyclic nucleoside analogues. Bend
Research Inc(USA) have developed a two-step resolution process . The process involved
acylation of the (R)-alcohol with butyric anhydride, mediated by Candida cylindracea lipase,
followed by the hydrolysis of the (R)-ester catalysed by the same enzyme. The first resolution
resulted in the enantiomerically pure (S)-alcohol (e.e > 98%) and (R)-ester(e.e-78%) which was


further enriched by the back conversion to (R)-alcohol(e.e > 98%). the resolution was performed
on a multi-kilogramme scale in a permselective membrane bioreactor specially designed to
facilitate product recovery and to minimise product inhibition.
Lipases are currently being used by many pharmaceutical companies world-wide for
the preparation of optically active intermediates on a kilo-gramme scale. A number of relatively
small biotechnological companies, such as Enzymatix in the U.K, specialise in
biotransformations and offer a whole variety of intermediates prepared via lipase mediated
resolution.
Regioselective modifications of polyfunctional organic compounds is yet another area
of expanding lipase application. In may cases, lipases have been shown to acylate or deacylate
selectively one or several hydroxyl groups of similar reactivity in carbohydrates,
polyhydroxylated alkaloids and steroids.Apart from the synthesis of sugar based surfactants,
lipases wer successfully applied in the regioselective modification of castanospermine. a
promising drug for the treatment of AIDS.
Thus lipases have become a conventional research tool in many organic chemistry
laboratories. As a result they are readily incorporated into synthetic routes especially when
optical purity of the final product is essential.
Cosmetic Industry
Although the cost of lipase catalysed esterification remains too high for the
manufacturing of bulk products, the synthesis of several speciality esters has found its way in the
market place. Unichem International has launched the production of isopropyl myristate,
isopropyl palmitate and 2-ethylhexylpalmitate for use as an emollient in personal care products
such as skin and sun-tan creams, bath oils etc. Immobilised Rhizomucor meihei lipase was used
as a biocatalyst in the solvent free esterification, which was driven to completion by vaccum
distillation of the water produced during the reaction. The company claims that the use of the
enzyme in place of the conventional acid catalyst gives products of much higher quality,
requiring minimum downstream refining. Batches of several tonnes have been successfully
produced at Unichem's factory in Spain.
Wax esters (esters of fatty acids and fatty alcohols) have similar applications in
personal care products and are also being manufactured enzymically (Croda Universal Ltd). The
company uses Candida cylindracea lipase in a batch bioreactor. According to the manufaturer,
the overall production cost is slightly higher tha that of the conventional method, but the cost is
justified by the improved quality of the final product.



Medical applications
Possible medical applications of lipases are under consideration, for example inhibition
of the human enzyme as a method of reducing fatty acid absorption is being investigated as a
possible treatment for obesity.



Assignment - 2
Direct Dual Fermentation
What is Direct Dual Fermentation ?
A method of continuous product formation using at least two continuous fermentation units and a
microorganism capable of being induced, in response to environmental conditions, to undergo a
genetic alteration from a state favoring microorganism growth to a state favoring product
production by the microorganism.

THEORETICAL CONSIDERATIONS
Description
The Dual Fermentation process is comprised of three parts: two nonidentical fermentation units
(designatedFermentation 1 and Fermentation 2) and a recovery unit (designated Recovery).
Fermentation 1 is when the salt of astrong acid is converted by fermentation to the salt of a
weakacid. Fermentation 2 is when a carbohydrate is fermented toacidify the fermentation broth
with formation of a strongacid. The strong acid formed acidifies the salt of the weakacid.
Recovery is when the weak acid is recovered bysolvent extraction; distillation, carbon
adsorption, etc. Thesalt of the strong acid is recycled to Fermentation 1 for conversion.for the
production of acids

Direct dual fermentation processfor the production of acids

The acid produced in Fermentation 2 should have a pK at recovery options suggested here are
is sufficient to drive the acidification of the weaker acid and give adequate selectivity.The acid
produced in Fermentation 1 should not be consumed by the organism used for Fermentation 2.
This is not a concern when an anaerobic fermentation is used for Fermentation 2, since the acids
from Fermentation 1 will not be further metabolized. However, many aerobic organisms are able
to degrade acetate, propionate and butyrate; care would be needed in selecting a suitable aerobic
organism for Fermentation selective for the unionized form of acid molecules. To maximize
extraction of only the weak acid, the strong acid should be completelydissociated. A one-unit pK
difference 2.The overall fermentation yield should be identical to that of the single fermentation.
This, in effect, eliminates the combining of an anaerobic fermentation in Fermentation 1with
oxidative fermentations in Fermentation 2. For example, an aerobic, citric acid-producing
fermentation in Fermentation 2 would maximally yield one citrate molecule per dextrose
fermented. Fermentation of citrate to acetate in Fermentation 1 would result in a maximum
formation of 2.25 acetate molecules per citrate. The net result of this combination would be 2.25
acetate per dextrose,substantially below the 3.0 acetate per dextrose obtained by direct
fermentation.Equivalent amounts of protons and acid anions should be produced in the system.


Since only the undissociated form of the acid is removed from the system, any excess anion
which is formed will accumulate in the system. When an acetate-lactate couple is used, 1
molprotons and 3 molacetate are generated in Fermentation 1; 2 rnol protons are generated in
Fermentation 2. The net process yields 3 molacetate and 3 rnol protons. Essentially all of the
formed acetate can then be extracted as the undissociated acid. A case where there would not be
this equivalence would be one where base was added to maintain constant pH in thefermentation
of lactate to acetate. In this case, only twothirdsof the acetate would be recoverable and
increasing amounts of acetate would be recycled through the fermentations until growth ceased.
The organism used in Fermentation 2 should be able to tolerate the high levels of organic acids
produced in Fermentation 1.

EXAMPLES OFDIRECT DUALFERMENTATIONS

Acetic Acid

Acetic acid involves the following dual-fermentation

Fermentation 1: 2 lactate + 2 acetate + 1 acetic acid

Fermentation 2: 1 dextrose + 2 lactic acid

2 lactic acid + 2 acetate + 2 lactate + 2 acetic acid

Overall reaction: I dextrose + 3 acetic acid

Propionic Acid

Propionic acid involves the following fermentation process:

Fermentation 1: 3 lactate + 2 propionate + 1 acetate

Fermentation 2: 1.5 dextrose + 3 lactic acid

3 lactic acid + 2 propionate + 1 acetate +

2 propionic acid + 1 acetic acid + 3 lactate

Overall reaction: 1.5 dextrose + 2 propionic acid +1 acetic acid

APPLICATION

1.Complex fruit wine produced from dual culture fermentation.



2.Production of aliphatic acids

ADVANTAGES
controlled conditions in the provision of substrates during the fermentation, particularly
regarding the concentration of specific substrates as for ex. the carbon source
alternative mode of operation for fermentations leading with toxic substrates (cells can only
metabolize a certain quantity at a time) or low solubility compounds
DISADVANTAGE
it requires a substantial amount of operator skill for the set-up, definition and development of the
process

References

I ..R. D. Schwartz and F. A. Keller, Jr., Appl. Environ. Microbiol., 43,117 (1982)
.
2.G. Wang and D. I. C. Wang, Appl. Environ. Microbiol.,47, 294
(1984).



ASSIGNMENT-3
Classification of micro organism

The chart above shows how microorganisms are related. The three most general groups into
which the organisms are placed are prokaryotes, eukaryotes, and non-living organisms.
prokaryotes are more primitive organisms than eukaryotes. Only bacteria are prokaryotes; the
rest of the organisms considered in this course are either eukaryotes or viruses.
Prokaryotes
Introduction



Prokaryotes are organisms which do not contain nuclei or membrane-bound organelles. All
prokaryotes are unicellular, which means that each organism is made up of only one cell.
Another trait common to all prokaryotes is their small size - a typical cell is only about 2 um
long. A micrometer, abbreviated as um and sometimes known as a micron, is equal to one
millionth of a meter. It would take about 13,000 prokaryotes lying end to end to stretch the
length of one inch. Under a light microscope, bacteria are so small that they are usually visible
only as tiny dots.
Although there are two kingdoms which contain prokaryotes (Eubacteria and Archaebacteria), all
prokaryotes are commonly known as bacteria. In the past, some prokaryotes have been called
blue-green algae, but these organisms are now known as cyanobacteria.
Importance
Bacteria are present in large numbers in raw wastewater, in biological treatment plants, in plant
effluent, in natural waters, and throughout our environment. In the wastewater treatment plant,
they form part of the slime on trickling filters and on the discs of rotating biological contactors.
They are also present in activated sludge.
Bacteria are heterotrophs, meaning that they get their food from eating other organisms or from
eating organic matter. (In contrast, organisms like plants which make their own food are known
as autotrophs.) As a result, bacteria are important to the wastewater operator since the bacteria
are able to digest a large amount of the waste in wastewater. On the other hand, some bacteria
get their food from living inside organisms such as humans, in which case they can cause
disease.
Cell Structure
A cell is the fundamental unit of all life. In the case of unicellular organisms, a cell is the body
of the organism. In the case of multicellular organisms (organisms which consist of more than
one cell), the cell is the building block from which the organism's body is made.


The diagram above illustrates a typical bacterial cell. As with every other kind of cell,
a membrane serves as a sac holding the parts of the cell together. The membrane also regulates
what passes into and out of the cell.
Inside the membrane, the cell is filled with a fluid known as cytoplasm. Floating in the
cytoplasm are various organelles (subcellular structures with specific functions.) Notice that the
the DNA, which contains the genetic material of the cell, is floating freely in a mass within the
cell. In addition to the main mass of DNA, the bacterial cell contains plasmids, which are small
loops of DNA which can be transferred to other bacteria, or in some cases into other
organisms. Ribosomes are the sites of protein synthesis.
Outside the membrane, most bacteria are surrounded by two other layers. The first of these,
the cell wall, is a rigid layer made up of proteins, polysaccharides, and lipids. The cell wall gives
the bacterium a set shape. Outside the cell wall is the capsule, a gelatinous slime layer which
allows the bacterium to attach to surfaces and also protects the bacterium. In the treatment plant,
bacterial capsules are responsible for clumping the organisms into flocs, or aggregations, which


can settle out of water. In order for disinfecting agents such as chlorine to be effective, they
must penetrate this protective slime layer.
The bacterium can also have various appendages. Pili are hollow, hair-like structures which
allow the bacterium to attach to other cells. Flagella are longer projections which can move and
push the bacterium from place to place.
Endospores

Some bacteria are able to survive in harsh environments by forming endospores. Endospores are
small spores which develop asexually inside the bacterial cell. An endospore consists of the
bacterium's DNA surrounded by a protective cell wall. Once the endospore has formed, the
parent cell bursts open and releases the endospore.

An endospore is able to survive in very harsh environments because it is in a dormant state and
does not attempt to eat, grow, and reproduce. Bacteria typically form endospores when they
encounter an undesirable pH, electrolyte content, amount of food, or amount of oxygen in the
environment.



Classification
There are thousands of species of bacteria on earth, many of which have not yet been identified.
When attempting to classify a bacterium, a variety of characteristics are used, including visual
characteristics and laboratory tests.
Some bacteria can be identified through a simple visual perusal. First, the operator considers the
appearance of the bacterial colony (a group of the same kind of bacteria growing together, often
on a petri dish.) The operator also views individual bacteria under a microscope, considering
their shape, groupings, and features such as the number and location of flagella.
A variety of laboratory techniques can be used to narrow down the identity of a bacterial species
if a visual survey is not sufficient. The operator can stain the bacteria using a gram stain or an
acid-fast stain. The bacteria can be cultured on a specific medium which promotes the growth of
certain species, as in the membrane filter method of testing for coliform bacteria. Other tests can
detect bacterial by-products, while yet more advanced tests actually analyze the DNA of the
bacteria.

Bacterial Shapes
The most basic method used for identifying bacteria is based on the bacterium's shape and cell
arrangement. This section will explain the three morphological categories which all bacteria fall
into - cocci, bacilli, and spirilla. You should keep in mind that these categories are merely a way
of describing the bacteria and do not necessarily refer to a taxonomic relationship.

Cocci (or coccus for a single cell) are round cells, sometimes slightly flattened when they are
adjacent to one another. Cocci bacteria can exist singly, in pairs (as diplococci), in groups of


four (as tetrads), in chains (as streptococci), in clusters (as stapylococci), or in cubes consisting
of eight cells (as sarcinae.)

Bacilli (or bacillus for a single cell) are rod-shaped bacteria. Since the length of a cell varies
under the influence of age or environmental conditions, you should not use cell length as a
method of classification for bacillus bacteria. Like coccus bacteria, bacilli can occur singly, in
pairs, or in chains. Examples of bacillus bacteria include coliform bacteria, which are used as an
indicator of wastewater pollution in water, as well as the bacteria responsible for typhoid fever.

Spirilla (or spirillum for a single cell) are curved bacteria which can range from a gently curved
shape to a corkscrew-like spiral. Many spirilla are rigid and capable of movement. A special
group of spirilla known as spirochetes are long, slender, and flexible.
Eukaryotes
Introduction
Except for bacteria and viruses, all other organisms considered in this course are
eukaryotes. Eukaryotes are unicellular or multicellular organisms which contain a nucleus and
membrane-bound organelles. A nucleus is a membrane sac within the cell which holds all of the
cell's DNA. Membrane-bound organelles within the cell can include chloroplasts, mitochondria,
and several other organelle types which we will not discuss here.


The diagram above shows some of the parts found in a typical eukaryotic cell. Since there are so
many different kinds of eukaryotes, several of the parts shown in the cell above will not be
present in certain species. Also, some species may contain additional parts not shown above.
Eukaryotic cells are always bounded by a membrane, just as prokaryotic cells are. Some
eukaryotic cells are also surrounded by a cell wall, but eukaryotic cells do not have capsules.
Although they are not shown in the diagram above, eukaryotic cells can have protuberances such
as flagella or cilia (tiny hairs which typically form a fringe all the way around a cell.)
Like the prokaryotic cell, the eukaryotic cell is filled with cytoplasm. Ribosomes and various
other organelles can be found floating in the cytoplasm. The two additional organelles shown in
the diagram above are membrane-bound and are found only in eukaryotic
cells. Mitochondria (mitochondrion when referring to a single organelle) are present in nearly all
eukaryotic cells and produce the cell's energy by breaking down food. Chloroplasts, in contrast,
are present only in plants and algae and are used in photosynthesis, the process through which
the organism uses energy from the sun to build sugars.



Fungi
Fungi are organisms which typically cannot move, which cannot make their own food
(heterotrophic), and which contain a chemical known as chitin in their cell walls. They can be
multicellular or unicellular, with the unicellular organisms having relatively large cells.
Although some fungi live in salt or fresh water, most fungi are terrestrial. Many species
are saprophytic, feeding on dead organic matter. Others are parasites which live inside or on
host animals, primarily feeding on plants though a few also live on animals. The aquatic fungi
are important in treating wastewater.

Classification of fungi is based primarily on reproductive structures, with all of the aquatic fungi
being found in the Mastigomycota group. We use several common names to refer to groups of
fungi, but these groupings refer only to morphology and not to any relationship or scientific


classification. Yeast are single-celled fungi, molds are filamentous fungi consisting of multiple
cells in threads known as hyphae, and mushrooms are the fruiting bodies of filamentous fungi.
Algae
Introduction
Algae are distinguished from animals, fungi, and protozoans by their ability to make their own
food through photosynthesis and are distinguished from plants by their relative simplicity of
structure. All algae contain the green pigment chlorophyll and the organelles chloroplasts, both
of which are essential for photosynthesis.
Algae may be either unicellular (in which case they are known as phytoplankton) or
multicellular. The algae which are important to water treatment are generally unicellular. All
algae contain a rigid cell wall and some also have sheaths (or thin gelatinous coatings) outside
the cell wall. Algae may be non-motile, but many are able to move using a flagella, in which
case they are known as flagellates (a term based on morphology rather than taxonomy.)
Importance

Most algae are aquatic, living in salt or fresh water, though a few live in soil or on the bark of
tres. In natural waters, algae are an important source of food for other organisms. They also
produce oxygen during photosynthesis, adding to the dissolved oxygen content of the water.

Since algae require light for growth, they are restricted mostly to the top surfaces of trickling
filters and ponds. They are seldom found in large numbers except in tertiary treatment units,
such as clear wells, and usually are not important for water treatment. However, in oxidation
ponds, the algae may represent a substantial portion of the microorganism population and may
play a significant role in treatment.



protozoa
The Protozoa are considered to be a subkingdom of the kingdom Protista, although in the
classical system they were placed in the kingdom Animalia. More than 50,000 species have been
described, most of which are free-living organisms; protozoa are found in almost every possible
habitat. The fossil record in the form of shells in sedimentary rocks shows that protozoa were
present in the Pre-cambrian era. Virtually all humans have protozoa living in or on their body at
some time, and many persons are infected with one or more species throughout their life. Some
species are considered commensals, i.e., normally not harmful, whereas others are pathogens and
usually produce disease. Protozoan diseases range from very mild to life-threatening. Individuals
whose defenses are able to control but not eliminate a parasitic infection become carriers and
constitute a source of infection for others. In geographic areas of high prevalence, well-tolerated
infections are often not treated to eradicate the parasite because eradication would lower the
individual's immunity to the parasite and result in a high likelihood of reinfection.
Many protozoan infections that are inapparent or mild in normal individuals can be life-
threatening in immunosuppressed patients, particularly patients with acquired immune deficiency
syndrome (AIDS). Evidence suggests that many healthy persons harbor low numbers
of Pneumocystis carinii in their lungs. However, this parasite produces a frequently fatal
pneumonia in immunosuppressed patients such as those with AIDS. Toxoplasma gondii, a very
common protozoan parasite, usually causes a rather mild initial illness followed by a long-lasting
latent infection. AIDS patients, however, can develop fatal toxoplasmic
encephalitis. Cryptosporidium was described in the 19th century, but widespread human
infection has only recently been recognized. Cryptosporidium is another protozoan that can
produce serious complications in patients with AIDS. Microsporidiosis in humans was reported
in only a few instances prior to the appearance of AIDS. It has now become a more common
infection in AIDS patients. As more thorough studies of patients with AIDS are made, it is likely
that other rare or unusual protozoan infections will be diagnosed.
Acanthamoeba species are free-living amebas that inhabit soil and water. Cyst stages can be
airborne. Serious eye-threatening corneal ulcers due to Acanthamoeba species are being reported
in individuals who use contact lenses. The parasites presumably are transmitted in contaminated


lens-cleaning solution. Amebas of the genus Naegleria, which inhabit bodies of fresh water, are
responsible for almost all cases of the usually fatal disease primary amebic meningoencephalitis.
The amebas are thought to enter the body from water that is splashed onto the upper nasal tract
during swimming or diving. Human infections of this type were predicted before they were
recognized and reported, based on laboratory studies of Acanthamoeba infections in cell cultures
and in animals.
The lack of effective vaccines, the paucity of reliable drugs, and other problems, including
difficulties of vector control, prompted the World Health Organization to target six diseases for
increased research and training. Three of these were protozoan infectionsmalaria,
trypanosomiasis, and leishmaniasis. Although new information on these diseases has been
gained, most of the problems with control persist.
References:
1) http://water.me.vccs.edu/courses/env108/lesson2_print.htm
2) http://www.ncbi.nlm.nih.gov/books/NBK8325/



ASSIGNMENT-4
INTRODUCTION TO FERMENTATION

Fermentation

Fermentation in progress

The term Fermentation is derived from the Latin verb "fervere" which means "to boil".
Fermentation is the process of deriving energy from the oxidation of organic compounds, such
as carbohydrates, and using an endogenous electron acceptor, which is usually an organic
compound, as opposed to Respiration where electrons are donated to an exogenous electron
acceptor, such as oxygen, via an electron transport chain. Fermentation does not necessarily have
to be carried out in an anaerobic environment. For example, even in the presence of abundant
oxygen, yeast cells greatly prefer fermentation to oxidative phosphorylation, as long as sugars
are readily available for consumption.
Sugars are the most common substrate of fermentation, and typical examples of fermentation
products are ethanol, lactic acid, and hydrogen. However, more exotic compounds can be
produced by fermentation, such as butyric acid and acetone. Yeast carries out fermentation in the
production of ethanol in beers, wines and other alcoholic drinks, along with the production of
large quantities of carbon dioxide. Fermentation occurs in mammalian muscle during periods of
intense exercise where oxygen supply becomes limited, resulting in the creation of lactic acid.



Energy source in anaerobic conditions
Fermentation products contain chemical energy (they are not fully oxidized) but are considered
waste products, since they cannot be metabolized further without the use of oxygen (or other
more highly-oxidized electron acceptors). A consequence is that the production of adenosine
triphosphate (ATP) by fermentation is less efficient than oxidative phosphorylation, whereby
pyruvate is fully oxidized to carbon dioxide. Water temperature must be warm for fermentation.
The yeast cells will die if it is too hot.

Ethanol fermentation (performed by yeast and some types of bacteria) breaks the
pyruvate down into ethanol and carbon dioxide. It is important in bread-making, brewing,
and wine-making. Usually only one of the products is desired; in bread-making, the
alcohol is baked out, and, in alcohol production, the carbon dioxide is released into the
atmosphere or used for carbonating the beverage. When the ferment has a high
concentration of pectin, minute quantities of methanol can be produced.

Homolactic Fermentation breaks down the pyruvate into Lactate. It occurs in the muscles
of animals when they need energy faster than the blood can supply oxygen. It also occurs
in some kinds of bacteria (such as lactobacilli) and some fungi. It is this type of bacteria
that converts lactose into lactic acid in yogurt, giving it its sour taste. These lactic acid
bacteria can be classed as homofermentative, where the end product is mostly lactate, or
heterofermentative, where some lactate is further metabolized and results in carbon
dioxide, acetate or other metabolic products.

Hydrogen gas is produced in many types of fermentation (mixed acid fermentation,
butyric acid fermentation, caproate fermentation, butanol fermentation, glyoxylate
fermentation), as a way to regenerate NAD
+
from NADH. Electrons are transferred to
ferredoxin, which in turn is oxidized by hydrogenase, producing H
2
. Hydrogen gas is a
substrate for methanogens and sulfate reducers, which keep the concentration of
hydrogen sufficiently low to allow the production of such an energy-rich compound.
Fermenting


Modern fermentation tanks

After the wort is cooled and aerated usually with sterile air yeast is added to it, and
it begins to ferment. It is during this stage that sugars won from the malt are metabolized into
alcohol and carbon dioxide, and the product can be called beer for the first time. Fermentation
happens in tanks which come in all sorts of forms, from enormous cylindro-conical vessels,
through open stone vessels, to wooden vats.
Open fermentation vessels are also used, often for show in brewpubs, and in Europe in wheat
beer fermentation. These vessels have no tops, which makes harvesting top fermenting yeasts
very easy. The open tops of the vessels make the risk of infection greater, but with proper
cleaning procedures and careful protocol about who enters fermentation chambers, the risk can
be well controlled.
Fermentation tanks are typically made of stainless steel. If they are simple cylindrical tanks with
beveled ends, they are arranged vertically, as opposed to conditioning tanks which are usually
laid out horizontally. It is often put on the tanks to allow the CO
2
produced by the yeast to
naturally carbonate the beer. This bung device can be set to a given pressure to match the type of
beer being produced. The more pressure the bung holds back, the more carbonated the beer
becomes.



Fermentation methods

There are three main fermentation methods, warm, cool and wild or spontaneous.
Fermentation may take place in open or closed vessels. There may be a secondary fermentation
which can take place in the brewery, in the cask or in the bottle.

Warm fermenting

Ale yeasts ferment at warmer temperatures between 1520 C (5968 F), and
occasionally as high as 24 C (75 F). Pure ale yeasts form a foam on the surface of the
fermenting beer, because of this they are often referred to as top-fermenting yeastthough there
are some British ale yeast strains that settle at the bottom. Ales are generally ready to drink
within three weeks after the beginning of fermentation, however, some styles benefit from
additional aging for several months or years. Ales range in colour from very pale to an opaque
black. England is best known for its variety of ales. Ale yeasts can be harvested from the primary
fermenter, and stored in the refrigerator or freezer.

Cool fermenting

While the nature of yeast was not fully understood until Emil Hansen of the Carlsberg
brewery in Denmark isolated a single yeast cell in the 1800s, brewers in Bavaria had for
centuries been selecting these cold-fermenting lager yeasts by storing ("lagern") their beers in
cold alpine caves. The process of natural selection meant that the wild yeasts that were most cold
tolerant would be the ones that would remain actively fermenting in the beer that was stored in
the caves. Some of these Bavarian yeasts were brought back to the Carlsberg brewery around the
time that Hansen did his famous work.
Traditionally, ales and lagers have been differentiated as being either a top fermentor or
bottom fermentor, respectively. The main difference between the two is lager yeast's ability to
process raffinose. Raffinose is a trisaccharide composed of galactose, fructose, and glucose.


Lager yeast tends to collect at the bottom of the fermenter and is often referred to as
bottom-fermenting yeast. Lager is fermented at much lower temperatures, around 10 C (50 F),
compared to typical ale fermentation temperatures of 18 C (64 F). It is then stored for 30 days
or longer close to freezing point. During storage, the beer mellows and flavours become
smoother. Sulfur components developed during fermentation dissipate.

Spontaneous fermentation

These beers are primarily brewed around Brussels, Belgium. They are fermented by
"open" fermentation, allowing wild yeast and bacteria to ferment the wort(unfermented beer).
The beers fermented from yeast and bacteria in the Brussels area are called Lambic beers. These
bacteria add a sour flavour to the beer. Of the many styles of beer very few use bacteria, most are
fermented with yeast alone and bacterial contamination is avoided. However, with the advent of
yeast banks and the National Collection of Yeast Cultures, brewing these beers, although not
through spontaneous fermentation, is possible anywhere. Specific bacteria cultures are also
available to reproduce certain styles.

Secondary fermentation

Secondary fermentation is an additional fermentation after the first or primary
fermentation. For the secondary fermentation, the beer is transferred to a second fermenter, so
that it is no longer exposed to the dead yeast and other debris (also known as "trub") that have
settled to the bottom of the primary fermenter. This prevents the formation of unwanted flavours
and harmful compounds such as acetylaldehydes, which are commonly blamed for hangovers.
During secondary fermentation, most of the remaining yeast will settle to the bottom of the
second fermenter, yielding a less hazy product. Some beers may have three fermentations, the
third being the bottle fermentation.



Bottle fermentation

Some beers undergo a fermentation in the bottle, giving natural carbonation. This may be
a second or third fermentation. They are bottled with a viable yeast population in suspension. If
there is no residual fermentable sugar left, sugar may be added. The resulting fermentation
generates CO
2
which is trapped in the bottle, remaining in solution and providing natural
carbonation.

Cask conditioning

Cask ale or cask-conditioned beer is the term for unfiltered and unpasteurised beer which
is conditioned (including secondary fermentation) and served from a cask without additional
nitrogen or carbon dioxide pressure.

Conditioning
When the sugars in the fermenting beer have been almost completely digested, the
fermentation slows down and the yeast starts to settle to the bottom of the tank. At this stage, the
beer is cooled to around freezing, which encourages settling of the yeast, and causes proteins to
coagulate and settle out with the yeast. If a separate conditioning tank is to be used, it is at this
stage that the beer will be transferred into one. Unpleasant flavours such as phenolic compounds
become insoluble in the cold beer, and the beer's flavour becomes smoother. During this time
pressure is maintained on the tanks to prevent the beer from going flat.
Conditioning can take from 2 to 4 weeks, sometimes longer, depending on the type of beer.
Additionally lagers, at this point, are aged at near freezing temperatures for 16 months
depending on style. This cold aging serves to reduce sulfur compounds produced by the bottom-
fermenting yeast and to produce a cleaner tasting final product with fewer esters.
If the fermentation tanks have cooling jackets on them, as opposed to the whole
fermentation cellar being cooled, conditioning can take place in the same tank as fermentation.
Otherwise separate tanks (in a separate cellar) must be employed. This is where aging occurs.


Filtering

A mixture of diatomaceous earth and yeast after filtering.

Filtering the beer stabilizes the flavour, and gives beer its polished shine and brilliance.
Not all beer is filtered. When tax determination is required by local laws, it is typically done at
this stage in a calibrated tank.
Filters come in many types. Many use pre-made filtration media such as sheets or
candles, while others use a fine powder made of, for example, diatomaceous earth, also called
Kieselguhr, which is introduced into the beer and recirculated past screens to form a filtration
bed.
Filters range from rough filters that remove much of the yeast and any solids (e.g. hops,
grain particles) left in the beer, to filters tight enough to strain colour and body from the beer.
Normally used filtration ratings are divided into rough, fine and sterile. Rough filtration leaves
some cloudiness in the beer, but it is noticeably clearer than unfiltered beer. Fine filtration gives
a glass of beer that you could read a newspaper through, with no noticeable cloudiness. Finally,
as its name implies, sterile filtration is fine enough that almost all microorganisms in the beer are
removed during the filtration process.

Sheet (pad) filters
These filters use pre-made media and are relatively straightforward. The sheets are


manufactured to allow only particles smaller than a given size through, and the brewer is free to
choose how finely to filter the beer. The sheets are placed into the filtering frame, sterilized (with
hot water, for example) and then used to filter the beer. The sheets can be flushed if the filter
becomes blocked, and usually the sheets are disposable and are replaced between filtration
sessions. Often the sheets contain powdered filtration media to aid in filtration.
It should be kept in mind that pre-made filters have two sides. One with loose holes, and
the other with tight holes. Flow goes from the side with loose holes to the side with the tight
holes, with the intent that large particles get stuck in the large holes while leaving enough room
around the particles and filter medium for smaller particles to go through and get stuck in tighter
holes.
Sheets are sold in nominal ratings, and typically 90% of particles larger than the nominal
rating are caught by the sheet.

Kieselguhr filters

Filters that use a powder medium are considerably more complicated to operate, but can
filter much more beer before needing to be regenerated. Common media include diatomaceous
earth, or Kieselguhr, and perlite.

Packaging

Packaging is putting the beer into the containers in which it will leave the brewery.
Typically this means putting the beer into bottles, aluminum cans and kegs, but it may include
putting the beer into bulk tanks for high-volume customers.

Industrial fermentation
Fermentation has many important uses in industry. Though the word fermentation can
have stricter definitions, when speaking of it in industrial fermentation it more loosely refers to
the breakdown of organic substances and re-assembly into other substances. Somewhat


paradoxically, Fermenter culture in industrial capacity often refers to highly oxygenated and
aerobic growth conditions, whereas fermentation in the biochemical context is a strictly
anaerobic process. A very old method is ABE fermentation.
Food fermentation
Ancient fermented food processes, such as making bread, wine, cheese, curds, idli, dosa,
etc., can be dated to more than 6,000 years ago. They were developed long before man had any
knowledge of the existence of the microorganisms involved. Also, fermentation is a powerful
economic incentive for semi-industrialized countries, in their willingness to produce bio-ethanol.
Pharmaceuticals and the biotechnology industry
There are 5 major groups of commercially important fermentation:
1. Microbial cells or biomass as the product, e.g. single cell protein, bakers yeast,
lactobacillus, E. coli, etc.
2. Microbial enzymes: catalase, amylase, protease, pectinase, glucose isomerase, cellulase,
hemicellulase, lipase, lactase, streptokinase, etc.
Microbial metabolites :
1. Primary metabolites ethanol, citric acid, glutamic acid, lysine, vitamins,
polysaccharides etc.
2. Secondary metabolites: all antibiotic fermentation
3. Recombinant products: insulin, HBV, interferon, GCSF, streptokinase
4. Biotransformations: phenyl acetyl carbinol, steroid biotransformation, etc.
Nutrient sources for industrial fermentation
Growth media are required for industrial fermentation, since any microbe requires water,
(oxygen), an energy source, a carbon source, a nitrogen source and micronutrients for growth.
Carbon & energy source + nitrogen source + O
2
+ other requirements Biomass + Product +
byproducts + CO
2
+ H
2
O + heat


Nutrient Raw material
Carbon
Glucose corn sugar, starch, cellulose
Sucrose sugarcane, sugar beet molasses
glycerol
Starch
Maltodextrine
Lactose milk whey
fats vegetable oils
Hydrocarbons petroleum fractions
Nitrogen
Protein soybean meal, corn steep liquor, distillers' solubles
Ammonia
pure ammonia or ammonium salts
urea
Nitrate nitrate salts
Phosphorus source phosphate salts
Vitamins and growth factors
Yeast, Yeast extract
Wheat germ meal, cotton seed meal


Beef extract
Corn steep liquor

Trace elements: Fe, Zn, Cu, Mn, Mo, Co
Antifoaming agents : Esters, fatty acids, fats, silicones, sulphonates, polypropylene glycol
Buffers: Calcium carbonate, phosphates
Growth factors: Some microorganisms cannot synthesize the required cell components
themselves and need to be supplemented, e.g. with thiamine, biotin, calcium pentothenate
Precursors: Directly incorporated into the desired product: Phenyl ethylamine into Benzyl
penicillin, Phenyl acetic acid into Penicillin G
Inhibitors: To get the specific products: e.g. sodium barbital for rifamycin
Inducers: The majority of the enzymes used in industrial fermentation are inducible and are
synthesized in response of inducers: e.g. starch for amylases, maltose for pollulanase, pectin for
pectinase,olive oil and tween are also used at times.
Chelators: Chelators are the chemicals used to avoid the precipitation of metal ions. Chelators
like EDTA, citric acid, polyphosphates are used in low concentrations.
Sewage disposal
In the process of sewage disposal, sewage is digested by enzymes secreted by bacteria. Solid
organic matters are broken down into harmless, soluble substances and carbon dioxide. Liquids
that result are disinfected to remove pathogens before being discharged into rivers or the sea or
can be used as liquid fertilizers. Digested solids, known also as sludge, is dried and used as
fertilizers. Gaseous by-products such as methane can be utilized as biogas to fuel generators. One
advantage of bacterial digestion is that it reduces the bulk and odor of sewage, thus reducing


space needed for dumping, on the other hand, a major disadvantage of bacterial digestion in
sewage disposal is that it is a very slow process.
Phases of microbial growth
When a particular organism is introduced into a selected growth medium, the medium is
inoculated with the particular organism. Growth of the inoculum does not occur immediately, but
takes a little while. This is the period of adaptation, called the lag phase. Following the lag phase,
the rate of growth of the organism steadily increases, for a certain period--this period is the log or
exponential phase. After a certain time of exponential phase, the rate of growth slows down, due
to the continuously falling concentrations of nutrients and/or continuously increasing
(accumulating) concentrations of toxic substances. This phase, where the increase of the rate of
growth is checked, is the deceleration phase. After the deceleration phase, growth ceases and the
culture enters a stationary phase or a steady state. The biomass remains constant, except when
certain accumulated chemicals in the culture lyse the cells (chemolysis). Unless other micro-
organisms contaminate the culture, the chemical constitution remains unchanged. Mutation of
the organism in the culture can also be a source of contamination, called internal contamination.

References:

http://en.wikipedia.org/wiki/Fermentation_(biochemistry)

http://en.wikipedia.org/wiki/Industrial_fermentation



ASSIGNMENT-5
The application of recombinant DNA techniques
The transfer of DNA between different species of bacteria has been achieved experimentally
using both in vivo and in vitro techniques. Thus, genetic material derived from one species may
be incorporated into another where it may be expressed. In vivo techniques make use of phage
particles which will pick up genetic information from the chromosome of one bacterial species,
infect another bacterial species and in so doing introduce the genetic information from the first
host. The information from the first host may then be expressed in the second host. Whereas, the
in vivo techniques depend on vectors collecting information from one cell and incorporating it
into another, the in vitro technique involve the insertion of the information into the vector by in
vitro manipulation followed by the insertion of the carrier and its associated extra DNA into the
recipient cell. Because the DNA is incorporated into the vector by in vitro methods the source of
the DNA is not limited to that of the host organism of the vector. Thus, DNA from human or
animal cells may be introduced into the recipient cell. Atherton et al.(1979) listed the basic
requirements for the in vitro transfer and expression of foreign DNA in a host micro organism as
follows:
1) A vector DNA molecule (plasmid or phage) capable of entering the host cell and
replicating within it. Ideally the vector should be small, easily prepared and must contain
at least one site where integration of foreign DNA will not destroy an essential function.
2) A method of splicing foreign genetic information into the vector.
3) A method of introducing the vector/foreign DNA recombinants into the host cell and
selecting for their presence. Commonly used simple characteristics include drug
resistance, immunity, plaque formation, or an inserted gene recognizable by its ability to
complement a known auxotroph.
4) A method of assaying for the foreign gene product of choice from the population of
recombinants created.
The initial work focused on E.coli but subsequently techniques have been developed for the
insertion of foreign DNA into a range of bacteria, yeasts, filamentous fungi and animal cells. The


insertion of information into the vector molecule is achieved by the action of restriction
endonucleases and DNA ligase. Site specific endonucleases produce specific DNA fragments
which may be joined to another similarly treated DNA molecule using DNA ligase. The
modified vector is then normally introduced into the recipient cell by transformation. Because
the transformation process is an inefficient one, selectable genes must be incorporated into the
vector DNA so that the transformed cells may be cultured preferentially from the mixture of
transformed and parental cells. This is normally accomplished by the use of drug resistant
markers so that those cells containing the vector will be capable of growth in the presence of a
certain antimicrobial agent. The process is shown diagrammatically in figure.

Once the desired gene has been introduced into the recipient cell the problem of expression of
the gene arises. This is particularly difficult when a eukaryotic gene is introduced into a
bacterium. In the late 1970s a large number of mammalian genes were successfully introduced
into bacterial cells, but there was little evidence of any gene expression. The problem of
eukaryotic gene expression in prokaryotic cells is due to the different structure of eukaryote
genes which contain non-coding segments of DNA. Thus, the production of a eukaryotic product
by a prokaryotic cell necessitates the incorporation of the genes coding for the product in a form
that may be translated by the recipient cell. Two approaches have been adopted to construct
eukaryotic genes in a prokaryotic form: the first is to synthesize DNA corresponding to the
primary structure of the protein product of the gene, although this method is suitable only for the


construction of genes coding for small peptides of known structure. Itakura et al. synthesized the
gene coding for the human hormone, somatostatin, and succeeded in incorporating it in E. coli
where it was expressed. The alternative technique is to synthesize DNA from the messenger
RNA corresponding to the gene, using the enzyme, reverse transcriptase. Eukaryote messenger
RNA is similar to bacterial RNA in that it does not contain non coding segments so that DNA
synthesized from an eukaryotic messenger RNA template should be in a form which is
transcribable by a prokaryotic cell. Nagata et al. used the reverse transcriptase method to produce
the genes coding for human interferon. Complementary single stranded DNA was prepared from
a mixture of messenger RNA extracted from virus induced human lymphocytes. The DNA was
then introduced into E. coli at random using a plasmid vector and the recipient cells screened for
the presence of the interferon gene. Those colonies that contained the gene were then examined
for interferon production. Using this method, Nagatas group succeeded in demonstrating the
expression of the interferon gene in E. coli.
The most publicized application of recombinant DNA technology in the context of fermentation
is the construction of strains capable of synthesizing foreign proteins. Also the use of
recombinant DNA technology for the improvement of native microbial products.

References
Book PRINCIPLE OF FERMENTATION TECHNOLOGY
By PETER F. STANBURY
ALLAN WHITAKER
STEPHEN J. HALL



ASSIGNMENT-6
STREAK PLATE METHOD
Materials Required:
1. Mixed culture of bacteria.
2. Sterile petri dish with appropriate bacterial media(such as trypticase soy agar, nutrient agar).
3. Inoculating loop (usually nichrome, a nickel-chromium alloy, or platinum; it may also be a
single-use disposable plastic loop, which would be discarded between sectors rather than
resterilized).
4. Bunsen burner.
5. Marking pen.

Procedure:
All the process is done in a laminar air flow cabinet aseptically.

Label a Petri dish:
Petri dishes are labelled on the bottom rather than on the lid. Write close to the edge of the
bottom of the plate to preserve area to observe the plate after it has incubated. Labels usually
include the organism name, type of agar, date, and the plater's name or initials. Using sterile
cotton swabs, remove any visible water on the agar in the plate or around the inner rim of the
petri plate. Observe the plate and mentally divide it into three sectors. The plate will then be
turned clockwise (if you are right handed) with the agar side up. The second sector will then be
at the top for streaking and then the plate is turned again so that the third sector can be streaked.

Sterilize the Transfer Loop before Obtaining a Specimen:

To streak a specimen from a culture tube, metal transfer loops are first sterilized by flaming the
wire loop held in the light blue area of a Bunsen burner just above the tip of inner flame of the
flame until it is red-hot. If a hot incinerator is available, the loop may be sterilized by holding it
inside the incinerator for 5 to 7 seconds. Once sterile, the loop is allowed to cool by holding it


still. Do not wave it around to cool it or blow on it. When manipulating bacteria, transfer loops
are usually held like a pencil. If plastic disposable loops are being utilized, they are removed
from the packaging to avoid contamination and after being used, are discarded into an
appropriate container. A new loop is recommended for each sector of an isolation streak plate.

Open the culture tube and collect a sample of specimen using the sterile loop:
Isolation can be obtained from any of a variety of specimens. This protocol describes the use of
a mixed broth culture, where the culture contains several different bacterial species or strains.
The specimen streaked on a plate could come in a variety of forms, such as solid samples, liquid
samples, and cotton or foam swabs. Material containing possibly infectious agents should be
handled appropriately in the lab using bio safety procedure.
Remove the test tube cap. It is recommended that the cap be kept in your right hand (the hand
holding the sterile loop). Curl the little finger of your right hand around the cap to hold it or hold
it between the little finger and third finger from the back. Modern test tube caps extend over the
top of the test tube, keeping the rim of the test tube sterile while the rim of the cap has not been
exposed to the bacteria. The cap can also be placed on the disinfected table, if the test tube is
held at an angle so that air contamination does not fall down into the tube. Insert the loop into the
culture tube and remove a loopful of broth. Replace the cap of the test tube and put it back into
the test tube rack.

Streak the Plate:
The lid of the agar plate has to be opened just sufficiently enough to streak the plate with the
inoculation loop. Minimize the amount of agar and the length of time the agar is exposed to the
environment during the streak process.

Importance of Streaking:
The human body has billions of bacteria which constitutes the normal flora fighting against the
invading pathogens. It is tedious to isolate a particular type of bacteria from a clinical sample.
Streak plate technique is used to grow bacteria on a growth media surface so that individual
bacterial colonies are isolated and sampled. Isolated colonies indicate a clone of cells, being
derived from a single precursor cell. When the selected culture media is inoculated using a single


isolated colony, the resulting culture grows from that selected single clone. The modern streak
plate method has evolved from the efforts by Robert Koch and other microbiologists to obtain
pure culture of bacteria in order to study them. The dilution or isolation by streaking procedure
was originally developed by Loeffler and Gaffky in Koch's laboratory, which involves the
dilution of bacteria by systematically streaking them over the surface of the agar in a petri dish to
obtain isolated colonies which will subsequently grow into mass of cells, or isolated colonies. If
the agar surface grows microorganisms which are all the genetically same, the culture is then
considered as a pure culture.

The commonly used petri dishes are of hundred millimetre diameter. The agar surface of the
plate should be dry without any moisture such as condensation drops. The source of inoculums
can be clinical specimen, environmental swab, sedimented urine, broth or solid culture.

In the streaking procedure, a sterile loop or swab is used to obtain an uncontaminated microbial
culture. The process is called "picking colonies" when it is done from an agar plate with isolated
colonies and is transferred to a new agar or gelatin plate using a sterile loop or needle. The
inoculating loop or needle is then streaked over an agar surface. On the initial region of the
streak, many microorganisms are deposited resulting in confluent growth or the growth of culture
over the entire surface of the streaked area. The loop is sterilized by heating the loop in the blue
flame of the Bunsen burner, between streaking different sections, or zones and thus lesser
microorganisms are deposited as the streaking progresses. The streaking process will dilutes out
the sample that was placed in the initial region of the agar surface. There are two most
commonly used streak patterns, a three sector "T streak " and a four quadrant streak methods.

Three Sector Streak (t streak):
1. Sterilize the wire loop.
2. Cool the loop by touching it on the edge of the sterile agar plate.
3. Dip the loop into the broth culture containing the mixture of bacteria.
4. Lift the lid of the plate just enough to insert the loop. Drag the loop over the surface of the top
one-third of the plate back and forth in a "zig-zag" formation.
5. The loop has picked up thousands of bacteria which are spread out over the surface of the agar.


6. Sterilize the loop in the flame.
7. Turn the plate 90 degrees and drag the loop through the area you have just streaked two to three
times and continue to drag the loop in a "zig-zag" formation in the remaining half of the plate
without touching that area again.
8. Sterilize the loop in the flame.
9. Turn the plate 90 degrees. Repeat the procedure. Drag the loop two to three times through the
area you just streaked, and fill in the remaining area of the plate (zig-zag formation), being very
careful not to touch any of the areas you previously streaked.
10. Incubate the plate for 24 hours. If you streaked correctly, you will see isolated colonies in the
third sector. The heaviest growth will be in the first sector. There will be less growth and some
isolated colonies in the second sector. The third area should have the least growth with isolated
colonies.

A three sector T streak of Serratia marcescens grown on TSA

Four Quadrant Streak :
1. Loosen the cap of the bottle containing the inoculum.
2. Hold an inoculation loop in your right hand.
3. Flame the loop and allow it to cool.
4. Lift the test tube containing the inoculum with your left hand.
5. Remove the cap/ cotton wool plug of the test tube with the little finger of your right hand.


6. Flame the neck of the test tube.
7. Insert the loop into the culture broth and withdraw. At all times hold the loop as still as possible.
8. Flame the neck of the test tube again.
9. Replace the cap/ cotton wool plug of the test tube using the little finger of your right hand. Place
the test tube in a rack. For a liquid culture, dip the loop into the broth, or for solid media, lightly
touch a colony with the loop.
10. Partially lift the lid of the Petri dish containing the solid medium.
11. Place a loopful of the culture on the agar surface on the area 1. Flame the loop and cool it for 5
seconds by touching an unused part of the agar surface close to the periphery of the plate, and
then drag it rapidly several times across the surface of area1.
12. Remove the loop and close the Petri dish.
13. Reflame and cool the loop, and turn the petri dish 90C then touch the loop to a corner of the
culture in area1 and drag it several times across the agar in area 2, hitting the original streak a
few times. The loop should never enter area 1 again.
15. Reflame and cool the loop and again turn the dish 90C anticlockwise. streak area 3 in the same
manner as area 2, hitting last area several times.
17. Flame the loop, again turn the dish 90C and then drag the culture from a corner of a area3
across area 4, contacting area 3 several times and drag out the culture as illustrated. Using a
wider streak. Do not let the loop touch any of the previously streaked areas. The flaming of the
loop at the points indicated is to effect the dilution of the culture so that fewer organisms are
streaked in each area, resulting in the final desired separation.
19. Tape the plate closed and incubates the plate in an inverted position in an incubator for 24-48
hours.
20. Flame the loop before putting it aside.


Quadrant Streak Method

1. Area of initial inoculation and the first streak yields heavy growth.
2. Area of the second streak from the area 1 yields gives dense growth.
3. Area of the third streak from the area2 yields weak growth.
4. Area of the fourth streak from the area3 yields single colonies.

Staphylococcus aureus colony on nutrient agar

References:
1) http://amrita.vlab.co.in/?sub=3&brch=73&sim=213&cnt=2
2) http://amrita.vlab.co.in/?sub=3&brch=73&sim=213&cnt=1

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