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 0c to 40

0c. 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 0C is 0.785 g/ml, and its

boiling point is 78.4 0C. 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

C4H12O6 2 C2H5OH + 2 CO2 + energy (stored as ATP)

Alcohol fermentation is an anaerobic, exothermic process in which sugars break down into

ethanol and CO2 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%

KH2PO4 1.5 g/lit

(NH4)2SO4 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 0C 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 0C 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 0C.

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 0C 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 0C 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) Na2S2O3(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.

Na2SO3 + O2 Na2SO4

The residual SO3 (unoxidized) are oxidized by addition of I2, depending upon amount of

unoxidized SO3. I2 will be consumed & remaining I2 can be treated with Na2S2O3 using starch

as an indicator.

Na2SO3 (residual) + I2 + H20 Na2SO4 + 2HI

I2 (residual) + 2Na2S2O3 Na2S406 + 2NaI

Now the oxygen transfer rate (OTR) can be calculated from the following formula:

OTR = TN 0.251000 / T Volume of abquot taken mMole o2/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 Na2SO3

Dissolve 12.9 gm Na2SO3 in 1000 ml

0.1 N iodine solution

O.1% starch solution

0.1 gm starch in 100 ml.



Procedure:

Take 100 ml Na2SO3 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 I2 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 (CuSO4.5H2O) 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 37oC.

Method:

Add 3 ml of biuret reagent to 2 ml of protein solution mix and warm at 37oC 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 Na2CO3in 0.1 mol/litre NaOH).

2. Copper sulphate- sodium potassium tartratesolution (5g/litre CuSO4.5H2O 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:

KMNO4 solution

0.5 gm KMNO4 IN 100 ml distilled water

KMNO4 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



KMNO4 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 (A1, A2

or A3)(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 H2. 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 CO2 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 CO2 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

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