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Industrial
Microbiology

Book list
1
...
Waites
2
...
Biotechnology
By U
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Microorganisms and Industry
Industrial microbiology: Industrial microbiology may be defined as the study of the large-scale and profit-motivated
production of microorganisms or their products for direct use or as inputs in the manufacture of other goods
...
Industrial microbiology is clearly a branch of biotechnology and includes traditional and nucleic
acid aspects
...
In a modern
industrial microbiology organization, these may include chemical or production engineers, biochemists, economists,
lawyers, marketing experts and other high-level functionaries
...

Despite the necessity for team work emphasized above, the microbiologist has a central and key role in his organization
...

2
...

4
...

6
...
e
...

During the actual production the microbiologist must monitor the process for the absence of contaminants,
and participate in quality control to ensure uniformity of quality in the products
The proper custody of the organisms usually in a culture collection, so that their desirable properties are
retained
The improvement of the performance of the microorganisms by genetic manipulation or by medium
reconstitution
...
Such
an organism is generally called as a producer strain
...

2
...

4
...

6
...


It should be able to grow on relatively cheaper substrates
...
this reduces the cooling costs
...

It should possess minimum reaction time with the equipment used in a fermentation process
...

It should yield only the desired substance without producing undesirable substances
...


Screening: Detection and isolation of a microorganism from a natural environment like soil containing large number
of microbial population is called screening
...
For example, Eli Lilly &
Co
...

Screening is of two types:
1
...


Primary screening and
Secondary screening

They are discussed below:
Primary screening: Primary screening may be defined as detection and isolation of the desired microorganism based
on its qualitative ability to produce the desired product like antibiotic or amino acid or an enzyme etc
...
Sometimes the desired microorganism has to be isolated from a large population of different species of
microorganisms
...

2
...

Primary screening determines which microorganisms are able to produce a compound without providing much
idea of the production or yield potential of the organisms
...

b
...

d
...


The crowded plate technique
Indicator dye technique
Enrichment culture technique
Auxanographic technique
Technique of supplementing volatile organic substrates

They are discussed below:
a
...

Process:
This technique starts with the selection of a natural substratum like soil or other source consisting of
microorganisms
...

↓
Suitable aliquot of the dilution is chosen which is able to produce 300 to 400 individual colonies when
plated on an agar plate, after incubation
...

↓
The antibiotic producing activity of a colony is indicated by no growth of any other bacterial colony
in its vicinity
...
This region is called as
growth inhibitory zone
...
The purified culture
is then tested for its antibiotic spectrum
...
It will not give indication of antibiotic producing organism against a desired organism
...

Procedure:
Suitable serially diluted soil suspension is spread on the sterilized agar plate to allow the growth of isolated
and individual microbial colonies (approximately 30 to 300 per plate) after incubation
...

↓
The formation of inhibitory zone around certain colonies indicates the antibiotic activity against the test
organism
...

↓

3

Antibiotic producing colonies are later on isolated from the plate and are purified before putting to further
testing to confirm the antibiotic activity of a microorganism
...


Indicator dye technique: Microorganisms capable of producing acids or amines from natural sources can be
detected by using this method by incorporating certain pH indicator dyes such as neutral red or bromothymol
blue into natural agar medium
...

Production of an organic acid can also be detected by an alternative method
...
The production of organic acid is indicated by the formation
of a clear zone around those colonies which release organic acid into the medium
...


c
...

They can be isolated if the nutrients required by them is incorporated into the medium or by adjusting the
incubation conditions
...
Auxanotrophic technique: This technique is employed for the detection and isolation of microorganisms
capable of producing certain extracellular substances such as growth stimulating factors like amino acids,
vitamins etc
...

Process:
A suitable aliquot is spread on the surface of a sterilized agar plate and allowed the growth of isolated
colonies, after incubation
...

↓
The production of particular metabolite required by the test organism is indicated by its increased
growth adjacent to colonies that have produced the required metabolite
...


e
...

Process:
Suitable dilution of a microbial like soil suspension are spread on the surface of sterile agar medium
containing all the nutrients except any one of volatile substrates like hydrocarbons, low molecular
weight alcohols and similar carbon sources
...

↓
Enough vapors from the volatile substrate spread to the surface of agar within the closed atmosphere
to provide the required specific nutrient to the microorganism, which grows and form colonies by
absorbing the supplemented nutrient
...


4

Secondary screening: The screening that allows further sorting out of microorganisms obtained from primary screening
having real value for industrial processes and discarding of those lacking this potential is called secondary screening
...

The quality of the yield product
...

Elimination of the organisms, which are not industrially important
...
The sensitivity of the test organism towards a newly discovered antibiotic is generally analyzed during
quantitative analysis
...

2
...

4
...

6
...

8
...

10
...


12
...

14
...

Secondary screening can be qualitative or quantitative in its approach
...

Secondary screening should determine whether microorganisms are actually producing new chemical
compounds not previously described
...

Secondary screening should also detect gross genetic instability in microbial cultures
...

Secondary screening should determine whether the product has a simple, complex or even a macromolecular
structure, if this information is not already available
...

Secondary screening should show whether the product possesses physical properties such as UV light
absorption or fluorescence or chemical properties that can be employed to detect the compound during the use
of paper chromatography or other analytical methods and which also might be of value in predicting the
structure of the compound
...

Secondary screening should reveal whether a product resulting from a microbial fermentation occurs in the
culture broth in more than one chemical form and whether it is an optically or biologically active material
...

Secondary screening helps in predicting the approaches to be utilized in conducting further research on the
microorganisms and its fermentation process
...


Giant colony technique: This technique is used for isolation and detection of those antibiotics, which diffuse
through solid medium
...

↓
The isolated Streptomyces culture is inoculated into the central area of a sterilized petri plates
containing nutrient agar medium and are selected
...

↓
Cultures of test organism, whose antibiotic sensitivity is to be measured are streaked from the edges
of plates but not touching the growth of Streptomyces and are further inoculated to allow the growth
of the test organisms
...
The relative inhibition of growth of different
organisms by the antibiotic is called inhibition spectrum
...

↓
Such species of Streptomyces, which have potentiality of inhibiting microorganisms is preserved for
further testing
...


Filtration method: This method is employed for testing those antibiotics which are poorly soluble in water or
do not diffuse through the soil medium
...

↓
Various dilutions of antibiotic filters are prepared and added to molten agar plating medium and
allowed to solidity
...

↓
The inhibitory effect of antibiotic against the test organisms is measured by their degree of growth in
different antibiotic dilutions
...


Liquid medium method: This method is generally employed for further screening to determine the exact
amount of antibiotic produced by a microorganism like Streptomyces
...

↓
They are also aerated by shaking continuously and vigorously during incubation period to allow
Streptomyces to produce the antibiotic in an optimum quantity
...

2
...

4
...

6
...


To check the suitability of different media for maximum antibiotic production
...

To check for contamination
...

To check the stability of the antibiotic at various pH levels and temperatures
...

To check about the toxicity of the antibiotic against the experimental animals
...

2
...

4
...


Effect of incubation temperature and antifoaming agents on fermentation
...

Checking the antibiotic for its bacteriostatic or bactericidal properties
...

Checking for possibility of inclusion of precursor of chemical of the antibiotic production in the medium
...


Strategies for isolation of industrially important microbes:

6

1
...

3
...


5
...

The first stage in the screening of microorganisms of potential industrial is their isolation
...

In some cases it is possible to design the isolation procedure in such a way that, the growth of producers is
encouraged or that they may be recognized at the isolation stage, whereas in other cases, organisms must be
isolated and producers recognized at a subsequent stage
...


Difference between primary and secondary screening:
Primary screening
1
...


Secondary screening
1
...


7

2
...
The word SCP is considered to be
appropriate, since most of the microorganisms grow as single or filamentous individuals
...
If the SCP is suitable for human consumption, it is considered as food grade
...

Single cell protein basically refers to the microbial biomass or protein extract used as food or feed additive
...
Thus, SCP is of high nutritional value for
human or animal consumption
...
Most of these
people live in developing countries
...
In addition to its utility as a nutritional supplement, SCP can also be used for the isolation of several
compounds e
...
carbohydrates, fats, vitamins and minerals
...
The cow can
produce about 200g protein per day
...
There are many advantages of using microorganisms for SCP
production
...

2
...

4
...


Microorganisms grow at a very rapid rate under optimal culture conditions
...

The quality and quantity of protein content in microorganisms is better compared to higher plants and animals
...

The culture conditions and the fermentation processes are very simple
...


Limitations for the widespread use of SCP:
1
...


3
...

5
...
This is highly hazardous, since humans have a limited capacity to degrade nucleic acids
...
These
include the hydrocarbons, heavy metals, mycotoxins and some contaminants
...

There is a possibility of contamination of pathogenic microorganisms in the SCP
...
This is frequently associated with indigestion and allergic
reactions in individuals
...
g
...
Of course,
this mainly depends on the cost of raw materials
...


The desirable properties of microbes for SCP production:
Microorganisms that are to be grown as protein source for animal and human food must have certain basic properties
...


2
...
The organisms should also not contain or
produce toxic or carcinogenic materials
...


8

3
...

5
...

Growth rate: It must grow rapidly in a cheap, easily available medium
...
Thus, strains which grow at low pH conditions or at high temperature are often chosen
...
g
...
These waste products, formed in varius industries and other
biological processes, largely contribute to environmental pollution
...
These include⸺ The conversion of low-cost organic wastes to useful products
⸺ Reduction in environmental pollution
...

Process: The Symba process is carried out in three phases:
Phase I: The waste material containing starch is sterilized by passing through a heat exchanger
...
The first reactor contains E
...
When this hydrolysate is passed to the second bioreactor, the organism C
...

Phase III: The microbial biomass can be separated, by centrifugation
...


Pekilo protein: A filamentous fungus, Paecilomyces variotii, with good fibrous structure was used for the production of
Pekilo
...
It can be produced by a continuous fermentation process
...
It was used as an animal feed in supplementing the diets of
calves, pigs, chickens and hens without any adverse effects
...

Mycoprotein: Mycoprotein is also a fungal protein derived from the growth of the fungus Fusarium on simple
carbohydrates
...

The main reason for choosing the mould is the fibrous texture of the product, which assists its recovery and make it
easier to manufacture final products with a meat like structure
...
It is produced by a continuous process with the organism Fusarium graminearum, using
glucose as a substrate, with other nutrients and ammonia and an ammonium salt as the nitrogen source
...

↓
Components are blended together and sterilized prior to addition to the fermenter
...

↓
pH is controlled at 6 by gaseous ammonia addition
...


9

↓
Following cyclone separation and an RNA reduction step, cells are recovered by rotary vacuum filtration and
formulated into a range of products
...

In comparison to animal proteins, the production of Mycoprotein shows several advantageous features
...
This is shown in the following table:
Cow
Pig
Chicken
Fusarium

Starting material
1 kg feed
1 kg feed
1 kg feed
1 kg carbohydrate

Protein
14 gm
41 gm
49 gm
136 gm

Product (Total)
68 gm beef
200 gm pork
240 gm meat
1080 gm wet cell mass

The main reason for choosing the mould is the fibrous texture of the product which assist its recovery and makes it
easier to manufacture final products with a meat-like structure which may be flavored and colored accordingly
...
The main
nutritional characteristics of Mycoprotein are shown in the following table which also gives a comparison with beef:
Component
Protein
Fat
Dietary fiber
Carbohydrates
Ash
RNA

Mycoprotein
47
14
25
10
3
1

Raw lean beefsteak
68
30
Trace
0
2
Trace

Microbial biomass protein (MBP): The term MBP is used to indicate a product, which contains residual substrates
along with microbial product
...


2
...
They can be divided into the
following two types:
a
...
It may be liquid hydrocarbon (e
...
paraffin,
petroleum), gaseous hydrocarbons (e
...
methane, propane and butane) or alcohols (e
...
methanol,
ethanol)
...
Renewable sources: The materials that make up waste should normally be recycled back to the
ecosystem
...

• Industrial wastes: Fruit pulp, waste form confectionary and canning industry
...
g
...


The advantages of using available organic wastes for SCP production:
1
...

3
...

5
...


Reduces environmental pollution
Most organic wastes are available at low cost in most countries, thus ensuring independence in supply
The wastes are upgraded in energy and protein level
Guards against a protein shortage in a community that may be largely dependent on imports
Allows for technological innovation which can often be transferred to developing countries
Many of the wastes such as cellulose and whey already form accepted parts of animal diet and will avoid the
acceptability problems of others unusual wastes e
...
human wastes and fossil fuels
...
Some of them are discussed below:
1
...
5-2 hours)
...
They can be easily genetically modified for varying the amino acid composition
...
A very high protein content (43-85%) in dry mass
...
They can utilize a broad spectrum of raw materials as carbon sources, which include even waste products
...

5
...

6
...

7
...

8
...

9
...
It can also be enhanced by easy regulation of physical
and nutritional factors
...
Algal culture can be done in space that is normally used and there is no need to compete for land
...

Disadvantages of MBP production of inedible substrates:

1
...

3
...

5
...

7
...


9
...

High capital cost for MBP factory
...

Requirements for oxygenation in large fermenters
...
They require further processing and addition of fats and other flavorings
...

Functional properties of MBP proteins e
...
flavor, color, solubility, viscosity etc
...

Flavour and texture acceptability of MBP proteins to the consumers
...
Advantages
of indigenous fermented food protein over MBP grown on inedible substrates include the following:

1
...

2
...
They are adaptable to modern large-scale processing (examples are Japanese shoyu, miso natto and sake; South
4
...

6
...

8
...


African sorghum, maize beer) all of which began as indigenous food fermentations that are large industries
today
...

Oxygen is easily supplied
...

There is no requirement to rupture the cells to recover the proteins
...

The indigenous fermented foods generally have decreased cooking fuel requirements, quicker cooking times,
better digestibility and improved nutritive value
...
There are few toxic problems and when such problems do exist, they generally are well known and can be
avoided
...
The production process of Quorn is discussed below:
The organism Fusarium venenatum is grown at 300 C in rigorously sterile conditions in air lift (pressure cycle)
fermenters
...

↓
The carbon source is glucose produced by the hydrolysis of corn starch and ammonium salts are included as
the source of nitrogen
...
5 – 7
...

↓
After fermentation, the cell suspension is heat-shocked to reduce the extent of development of RNA
degradation products, the presence of which will otherwise elevate the risk of gout in those partaking of the
foodstuff
...

↓
The cell suspension is harvested by centrifugation and the hyphae mixed with binding agents and flavourings
and heated to cause a gelling of the binder and a linking of the hyphae
...

[Food, Fermentation and Microorganisms, 2nd edition
Wiley Blackwell publication]
Baker’s yeast
In certain fermentation approaches, the microbial cells themselves are the desired fermentation product
...

Baker’s yeast is the common name for the strain of yeast commonly used as a leavening agent in baking bread and
bakery products, where it converts the fermentable sugars present in the dough into CO 2 and ethanol
...
Baker’s yeast is also a single celled microorganism found on and around the human body
...
Yeast growth is inhibited by both salt and sugar, but
more so with salt and sugar
...

Saccharomyces exiguous (also known as S
...
It is not however, generally used in pure form, but comes from being propagated in a sourdough starter
...

Characteristics of S
...

2
...

4
...

6
...


12

Role of baker’s yeast in bread making:

1
...

2
...


3
...


improved
...


Conditioning: Proteolytic enzymes from the yeast make the flour proteins in the dough mature; as a result the dough
become elastic and springy and therefore capable of retaining a maximal amount of CO2, gas produced by the yeasts
...
The principle carbon and energy source for the production of baker’s yeast is cane or beet molasses which
2
...

4
...


contain available forms of sugars, presumably glucose and dextrose
...
Ammonium sulfate
is also used as a source of sulfur
...

The fermentation medium is also supplemented with minerals (magnesium and trace elements) and vitamins
(biotin and pantothenic acid)
...


Advantages of molasses as raw material:

1
...

2
...

4
...


energy
...

It supplies the yeast with bios factor for growth
...

Beet molasses provides less filtration problems during media make-up
...

↓
The medium is adjusted to an acid pH (pH 4 to 5) which helps retard bacterial growth
...

↓
The cells are finally recovered on a filter press, small amounts of vegetable oil are added as a plasticizer and
then this mass of cells is molded into blocks
...

Fermented sausage
A sausage is a food usually made from ground meat with a skin around it
...
Some sausage
are cooked during processing and the casing may be removed after
...
Sausage may be preserved by curing, drying or smoking
...


13

Action of sausage organisms:
The potential starter culture organisms for fermented sausage include Lactobacillus pantarum, Leuconostoc
mesenteroides, Micrococcus aurantiacus and Pediococcus cerevisiae
...
Minor amounts of acetic acid and small quantities of butyric and propionic acids are also produced
...

Streptococcus lactic subspecies diacetylactis produces diacetyl and acetoin which impart a nutty flavour and aroma to
some sausages
...
Another function of lactic acid bacteria is the inhibition of Staphylococcus aureus also suppresses
enterotoxin A production
...
Air dried
b
...
10%

Smoked
Usually smoked

No
No

Summer sausage
German Teewurst

Ingredients and additives:

1
...

2
...


14

3
...
It is produced by fermentation of
barley or other cereals, by employing the yeasts (most frequently) S
...
carlsbergensis
...

Types of beer: There are two types of beer which are distinguished mainly on the basis of fermentation differences
...

2
...
Bottom fermented beers are generally called lagers
...
During this type of fermentation, the bottom yeast (Saccharomyces uvarum) tends to
flocculate and settle
...

Top fermented beer: Most top fermented beers are called ales
...
During
fermentation, top yeast (Saccharomyces cerevisiae) rises to the surface where it is recovered by skimming
...

Example: Weiss bier
...

2
...

4
...


Malt
Adjuncts
Water
Yeast and
A few miscellaneous additives

They are discussed below:
1
...
It is prepared from 2 species of barley
...

3
...
It provides carbohydrates and proteins
...
It also provides proteases which breakdown carbohydrate and proteins
...
The simple sugars and amino acid produced serve as nutrient for yeast growth and metabolism
...

Water: Water containing dissolved salt have profound influence on such thing as enzyme activity and stability
during mashing, hop extraction, the precipitation of proteins and tannins
...


Hops: The hop plant Humulus lupulus is a climbing herb
...

Culture yeast: For spontaneous fermentation, culture yeast is an essential ingredient
...

2
...


Used in fermentation
Flavor and aroma production

Miscellaneous additives: Miscellaneous additives are generally used during conditioning, aging, finishing or
some are used either in the process
...


1
...


Malting:
Dried barley is cleaned and soaked in water
for a period of two days
...

Germination
process
is
associated with the formation of enzymes
amylases (starch hydrolyzing) and proteases
(protein degrading)
...

↓
Malt is prepared by powdering the seeds
...

↓
During the course of mashing, the soluble materials from malt and malt adjuncts are extracted
...

↓
The temperature and pH influence the activities of enzymes
...

This is rich in sugars, amino acids, minerals and vitamins
...


4
...
Addition of hops to wort is often done to
provide characteristic flavour, aroma and stabilizing effect to the beer, besides providing a mild
antibacterial activity
...
cerevisiae
(usually top fermenting strains)
...
In some countries, the
bottom fermenting yeast, S
...
For this organism, the ideal temperature for optimal
fermentation is 10 – 150 C
...

↓
During the storage period (that may last for several weeks), cold storage maturation occurs
...

↓
This partially mature beer (usually with turbid appearance) is then subjected to chillproofing
...

↓
It is advisable to add antioxidants during cold storage maturation to prevent oxidative damage
...
Carbonation can also be accomplished by adding fermenting yeast, which is less
commonly done
...

↓
There is variation in the raw materials used for beer production
...
This is in contrast to barley beer produced in most Western
countries
...

2
...

4
...


The yeas rapidly uses the O2 and produce CO2
...

Hops have a strong antimicrobial effect on most Gram-positive organisms
...

Beer has an acidic pH (3
...
5)
The alcohol has a slight antibacterial effect
...


Names of yeast that can spoil beer:
1
...

3
...

5
...

7
...


Saccharomyces bayanus (S
...

Rhodotorula spp
...
uvarum, S
...
cerevisiae
Form a thick yeast hard on the surface
of the fermenter

4
...
Color

Light color

Dark color

6
...
Taste

Sweet

Bitter

8
...
Cost effectiveness

More expensive

Less expensive

Bock beer

Weis beer

1
...
Yeast
3
...
Example

Differences between bottom and top fermented yeast
Features

Bottom fermented yeast

Top fermented yeast

No

Yes

100%

33
...
Agglomeration forming ability
2
...
Optimal growth temperature

280 C

250 C

6
...
2 – 6
...
5 – 6
...


3
...
Cytochromes spectrum

0

7
...
Primary fermentation
9
...
Nevertheless, any
fruit with a good proportion of sugar may be used for wine production
...
may all be used to produce wine
...

Wine making is be made by a much simpler process
...
e
...

Types of wine: There are hundreds of different types of wines produced in different parts of the world
...

2
...

4
...

6
...
Red wines are commonly drunk by many people in the west, along with lunch and dinner
...

Rose wine: This can be produced with a limited contact to the skins of grapes during fermentation
...

Sweet wine: This is sweet to taste since it contains some residual sugars after fermentation
...
The wines can be fortified
with addition of alcohol (usually done by adding brandy or other distilled spirits) to the desired concentration
...


18

Method of wine production:
Quality of the grapes is very important for the production of wines
...

↓
This grape juice ready for fermentation process is technically referred to as must
...
SO2
which can kill other organisms can be tolerated by wine fermenting yeasts
...

↓
The must in suitable bioreactors is inoculated with desired strains of the yeast Saccharomyces cerevisiae
...

↓
The wine production normally takes a few days (2 – 5 days)
...
) are actually dependent on the type of wine produced
...

↓
Aging of wine is very important for the development of characteristic flavour and aroma
...

Wine spoilage: Wine spoilage is most commonly caused by microorganisms especially by yeasts and bacteria
...
Wine defects caused by yeasts: Yeasts represent a major cause of wine defects and spoilage
...

• Brattanomyces intermedius: They can spoil wine in various ways
...
Secondly, under aerobic conditions, it forms high concentrations
of acetic acid and ethyl acetate by oxidation of ethanol, which gives the wine a vinegar-like taste
...
They are mainly found in vats which have not yet been completely filled
...

B
...

• Acetic acid bacteria: The acetic acid bacteria are Gram negative, strict aerobes
...
Each genus contains only 1 species namely Acenatobacter aceti
and Gluconobacter oxydans
...

• Lactic acid bacteria: Lactic acid bacteria have either a homofermentive or heterofermentive metabolism,
or in some strains, the metabolic capacity for both
...
The acid causes the wine glycerol oxidation which reacts with polyphenols to form bitter compounds
...

Difference between white wine and red wine:

1
...

3
...

5
...
g
...
cerevisiae var
...
g
...
Fermentation

12 – 150 C

24 – 290 C

7
...

Examples:
1
...

3
...

5
...

↓
The yeast fermentation normally produces an intermediate product containing a maximum of 14% ethanol
...

↓
In some instances, the intermediate are not related to a product of direct beverage use and it is only the final
distilled product that is of commercial interest
...
They are colorless neutral spirits with little innate flavor, but may be later
colored and flavoured with plant materials such as herbs and spices
...

Whiskey
Whisky is distilled from fermented grain-mash containing upto 50% ethyl alcohol
...
Malt whisky: This whisky is manufactured by microbial fermentation and subsequent distillation of malted
barley (barley grains are soaked in water and then germinated at 17°C)
...
Grain whisky: This whisky is manufactured by the microbial fermentation and subsequent distillation of a
mixture of malted and un-malted barley with un-malted maize
...
Scotch whisky: Malt whisky is matured (aged) in oak casks at least 3 years and then blended with grain
whisky
...

4
...

5
...

6
...
Since the rice is starchy, its starch is hydrolyzed by enzyme amylases derived from Aspergillus oryzae
before fermentation
...

↓

20

The malt and other cereals where used, are extracted and saccharified to form a wort, essentially as for beer
brewing, except that no hops are added
...
cerevisiae, which are sometimes
supplemented with surplus beer brewing yeast
...
cerevisiae and S
...
diastaticus which possess amyloglucosidase
(glucoamylase) activity
...

↓
Fermentations last for about 30⸺40 h and the emphasis is on the ethanol yield
...
Distillation residues may be used as an
animal feed supplement
...
Maturation is usually performed in oak casks for several years
...


21

4
...

2
...

4
...

6
...


2
...

4
...


Citric acid, due to its pleasant taste and palatability, is used as a flavoring agent in foods and beverages e
...

jams, jellies, candies, desserts, frozen fruits, soft drinks, wine etc
...

It is used in the chemical industry as an antifoam agent and for the treatment of textiles
...

In pharmaceutical industry, as trisodium citrate, it is used as a blood preservative
...
As iron citrate, it serves as a good source of iron
...
It forms a complex with
metal ions (iron, copper) and prevents metal catalyzed reactions
...

In detergent or cleaning industry, citric acid has slowly replaced polyphosphates
...
They are:
•

•

The surface process: This is characterized by growing the microorganisms as a layer on the film on a surface
in contact with the nutrient medium, which may be solid or liquid in nature
...

The submerged process: In this case, the organisms are immersed in or dispersed throughout the nutrient
medium
...


They are discussed below:
A
...


Solid surface fermentation: It takes about 80 – 100 hours for maximal production of citric acid
...

↓
The pH of the medium is adjusted to 4 – 5 and then sterilized
...
niger is sprayed as layers (3 – 6 cm thickness) and
inoculated at 280 C
...

↓
At the end of the process, citric acid is extracted into hot water and isolated
...


Liquid surface fermentation: It is the oldest method of citric acid production
...
It is done by the following steps:
The inoculum in the form of spores is spread over the aluminum trays filled with nutrient medium
...
The temperature is maintained around 300
C
...

↓
The pH of the medium falls to less than 2, as the mycelium grows in size and forms a thick layer on
the surface of the nutrient solution
...

↓
The mycelium and nutrient solution are separated
...

↓
The nutrient solution is subjected to processing for recovery of citric acid
...
The final yield of citric acid is in the range of 0
...
9 of per gram of
sugars
...
Submerged process: Around 80% of the world’s supply of citric acid is produced by submerged process
...
Steps are as follows:
Two types of bioreactors are in use: stirred tanks and aerated towers
...
The sparging of air occurs from the base of the fermenter
...
The
mycelium with forked and bulbous hyphae and branches with aggregate into pellets is ideal for citric
acid production
...

↓
An adequate supply of O2 is required for good production of citric acid
...
2 – 1 vvm (volume/volume/minute)
...
Antifoam agents (e
...
lard oil) and mechanical antifoam devices are used to prevent
foaming
...

2
...

Variations in O2 tension
...


Advanced control technology that requires highly trained personnel
...
For instance,
pure forms of citric acid are needed for use in food preparations, while for industrial use it can be crude form
...

↓
Oxalic acid is an unwanted byproduct and it can be removed by precipitation by adding lime at pH < 3
...
2 and temperature 70 – 900 C for precipitating citric acid
...

↓
The final steps for citric acid recovery are treatment with activated charcoal, cation and anion-exchangers and
crystallization
...

Gluconic acid: Gluconic acid can be produced by several bacteria and fungi
...

Applications:
1
...

3
...

5
...


Gluconic acid is used in the manufacture of metals, stainless steel and leather, as it can remove the
calcareous and rust deposits
...

Gluconic acid has pharmaceutical applications – calcium and iron therapy
...

Gluconate is used for desizing polyester or polyamide fabrics
...


Desizing is the process of removal of size material applied on warp threads of a fabric to facilitate the process
of weaving
...

Production process:
A
...
niger or Gliocladium suboxidans
↓
The culture medium contains glucose at a concentration of 12 – 15% (usually obtained from corn)
...
5 – 6
...

↓
Increasing the supply of O2 enhances gluconic acid yield
...
Chemical synthesis: By employing the immobilized enzyme glucose oxidase, gluconic acid can also be
produced
...

2
...

4
...


Chemical formula: C3H6O3
Physical appearance: Aqueous solution
Taste: Mild acid taste
Melting point: 530 C
Boiling point: 2000 C

24

6
...


Solubility in water: Soluble
Volatility: Low

Uses of lactic acid:
A
...
Meat, poultry and fish: Lactic acid can be used in meat, poultry and fish in the form of sodium or
potassium lactate to extend shelf life, control pathogenic bacteria, enhance and protect meat flavor,
improve water-boiling capacity and reduce sodium
...
Beverages: Because of its mild taste, lactic acid is used an acidity regulator in beverages such as soft
drinks and fruit juices
...
Pickled, vegetables: Lactic acid is effective in preventing the spoilage of olives, gherkins, pearl onions
and other vegetables preserved in brine
...
Confectionary: Formulating hard-boiled candy, fruit gums and other confectionary products with
lactic acid results in a milder flavor while maintain microbial stability and safety
...
Dairy: The natural presence of lactic acid in dairy products, combined with the dairy flavor and good
antimicrobial action of lactic acid, makes it an excellent acidification agent for many dairy products
...
Baked goods: Lactic acid is a natural sourdough acid, which gives the bread its characteristic flavor
and therefore it can be used for direct acidification in the production of sourdough
...
Savory flavors: Lactic acid is used to enhance a broad range of savory flavors
...

8
...

B
...
Pharmaceutical: The primary functions for the pharmaceutical applications are: pH regulation, metal
sequestration, chiral intermediate and calcium lactate can be used for calcium deficiency and anti
carries agent
...
Biomaterial: Lactic acid is a valuable compound in biomaterials such as resorbable screws, sutures
and medical devices
...
Detergents: Lactic acid is well known for its descaling properties and widely applied in household
cleaning products
...

4
...
Examples are manufacture of leather and textile products
and computer disks, as well as coating
...
Animal feed: Lactic acid is a commonly used additive in animal nutrition
...
Lactic acid can be used as an additive in
food and/or drinking water
...
Biodegradable plastics: lactic acid is the principle building block for Poly Lactic Acid (PLA)
...

7
...

8
...

Types of lactic acid fermentation: Lactic acid fermentation are carried out by number of bacteria
...
They are –
1
...
This also occurs in animal muscle deprived of O2
...

Pyruvate → Lactate

2
...
For example: Leukonostoc mesenteroides
...


Mixed acid fermentation: The products include lactate, acetate, small quantities of ethanol and formate
...
For example, E
...

Pyruvate → 2 lactate + Ethanol + 2 CO2 + 2 H2 + Acetate

25

Microorganisms involved in lactic acid fermentation:
Homolactic fermentation:
1
...

3
...

5
...

7
...


Lactobacillus acidophilus
Lactobacillus casei
Lactobacillus plantarum
Lactobacillus bulagaris
Lactobacillus leichmannii
Lactobacillus delbrueckii
Pediococcus cerevisiae
Streptococcus spp
...

2
...


Leuconostoc mesenteriods
Bifidobacterium bifidum
Lactobacillus brevis

Along with these, thermophiles are preferred because they have high tolerance to temperature, allow minimum
concentration, produce low by-products
...

Raw materials:
1
...


3
...


Glucose: Best raw material but not feasible
...
It is a byproduct of cheese industry and contains a good amount of lactose
...

• It is not expensive
...

Corn and potato starch: These must first be hydrolyzed to glucose by acid or enzymatic treatment
...
Corn and potato starch comes as waste material from different industry
...


Production process:
The fermentation medium contains 12⸺15% of glucose, nitrogen and phosphate containing salts and
micronutrients
...
5⸺6
...

↓
Generally, the strains operating at higher temperature (45 – 600 C) are preferred, since it reduces the need for
medium sterilization
...
This can be
achieved either by a continuous culture technique or by removal of lactic acid by electrodialysis
...

Microorganisms used for production of acetic acid: The commercial production of acetic acid is carried out by a
special group of acetic acid bacteria, which are divided into two genera:
•
•

Gluconobacter: They oxidize ethanol exclusively to acetic acid
...
These over-oxidizers are
Gram-negative and acid tolerant e
...
A
...
peroxidans, A
...


Production process:
For every molecule of ethanol oxidized, one molecule of acetic acid is produced
...

↓
For optimal production, adequate supply of O2 is very essential
...

↓
Surface fermentation or submerged fermentation processes can be carried out to produce acetic acid
...

↓
Recovery: The acetic acid produced is clarified by filtration and then subjected to decolorization by Potassium
ferrocyanide (K4(FeCN)6)
...
It is very widely used as a flavoring agent for processed liquid foods such as sauces
and ketchups
...
Surface process:
The fermentation material is sprayed over the surface which trickles through the shavings that that
contain the acetic acid producing bacteria
...

↓
Vinegar is produced in about 3 days
...
Submerged process:
The fermentation bioreactors are made up of stainless steel
...
The production rate in the submerged process is
about 10 times higher than the surface process
...

2
...
The proteins can be subjected to
hydrolysis and the requisite amino acids can be isolated e
...
cysteine, tyrosine, leucine
...
Most of the amino acids
required for commercial applications are of L-category
...


27

3
...
There are three different approaches:
a
...
g
...
Certain industrial
byproducts like molasses and starch hydrolysate can also be used
...

b
...
g
...

c
...
Some
examples are given below:
• Amino acid dehydrogenases from certain bacteria (e
...
Bacillus megaterium) can be used for
the amination of α-keto acids to produce L-amino acids e
...
alanine from pyruvate, leucine
from phenylpyruvate
...

• Enzymes or immobilized cells are also employed for the production of several other amino
acids e
...
tryptophan, tyrosine, lysine, valine
...

2
...


4
...
glutamicum is used from the stock
culture for the production of inoculum
...

Preparation of medium: Medium contains reducing sugar and soyabean meal hydrolysate
...
The amount of growth
factors, homoserine or threonine and methionine should be appropriate for the production of L-lysine and
suboptimal quantity to support the optimal growth
...

Recovery: After significant quantities of L-lysine is formed, lysed bacterial cells are removed from the
fermentation broth by centrifugation
...
Preparation at the isoelectric point
b
...
Electrophoresis
d
...

2
...

4
...

It is used as supplementary for cereal proteins
...

It is used as a nutraceutical
...
Therefore, a natural overproduction of
amino acids is a trace occurrence
...
g
...

In order to achieve an overproduction of any amino acid by a microorganism, methods have to be devised for the
elimination of the metabolic regulatory or control processes
...
The following are the major ways of strain development
...


Auxotrophic mutation: These mutants are characterized by a lack of the formation of regulatory end product
(i
...
repressor or regulatory effector)
...


28

2
...


Genetic recombination: Mutants can be developed by genetic recombination for overproduction of amino
acids
...
g
...

Recombinant DNA technology: The classical techniques of genetic engineering can be used for strain
development
...
In one of the
techniques, E
...
g
...


Production of solvents
The commercially important organic solvents are:

1
...

3
...


Ethanol
Acetone
Butanol and
Glycerol

Ethanol
Biosynthesis of ethanol

Production process of ethanol

29

Production process of acetone and butanol
Organism: Clostridium acetobutylicum
Fermentation: Fermentation is usually carried out in corn or molasses based medium
...
6 – 8
...
It occurs in three phases:
• Phase I: It is characterized by rapid growth of the organism and production of acetic acid and
butyric acid
...
2
...

↓
Product recovery: Acetone and butanol are recovered through continuous distillation and fractionation
...

Glycerol
Glycerol is widely used in industry and commerce
...
Glycerol is usually not produced by fermentation
...
It can also be chemically synthesized
from propylene or propane
...
However, in the normal
process, the quantity of glycerol formed is very low
...

↓
Sodium sulfite reacts with CO2 in the medium and gets converted to sodium bisulfate which blocks alcohol
synthesis
...

↓
The fermentation is run for 2 – 3 days
...

↓
This yield is about half of the theoretical value due to the fact that ethanol fermentation cannot be completely
inhibited
...

Enzymes
Commercial microbial enzymes are increasingly replacing conventional chemical catalysts in many industrial
processes
...
This results in the consumption of less energy and there is usually no
requirement for expensive corrosion-resistant equipment
...
Consequently, there is less need for extensive refining and purification of the
target product
...

Production procedure:

31

32

5
...
Most of the antibiotics are produced by microorganisms (i
...
product of one
organism that can kill other organism)
...

Penicillins:
Production process of penicillins:
Penicillin production is an aerobic process and therefore, a continuous supply of O 2 to the growing culture is very
essential
...
5 and the optimal temperature is in the range of 25 – 270 C
...

↓
The medium used for fermentation consists of corn steep liquor and carbon source (usually lactose)
...
Sometimes ammonium sulfate is
added for the supply of nitrogen
...

↓
Further, continuous feeding of sugar is advantageous for a good yield of penicillin
...

The mycelium is removed by filtration
...
The penicillin-containing solvent is treated
with activated carbon to remove impurities and pigments
...
The yield of penicillin is around 90%
...
To
overcome these allergic problems, cephalosporins were developed
...
Cephalosporins are broad-spectrum antibiotics with
low toxicity
...
The
other ingredients of the media include sucrose, glucose and ammonium salts
...

↓
The fermentation is carried out at temperature 25 – 280 C and pH 6 – 7
...

↓
Cephalosporin C from the culture broth can be recovered by ion-exchange resins, and by using column
chromatography
...

Steroids

Vaccines

33

Recombinant vaccines
Vaccination is the phenomenon of preventive immunization
...

Types: Vaccines are mainly of three types:
1
...
Live non-viral or weakened (attenuated) bacteria or viruses
3
...
This provides surveillance against future exposure to such an organism and thus protects
the body
...

Types of recombinant vaccines:
1
...

3
...
Subunit vaccines
include proteins, peptides and DNA
...

Vector recombinant vaccines: These are the genetically modified viral vectors that can be used as vaccines
against certain pathogens
...
g
...

The gene encoding for hepatitis B surface antigen (HBsAg) is identified
↓
The gene for HBsAg is inserted into yeast vector which is linked to the alcohol dehydrogenase promoter
↓
These plasmids are then transferred to yeast cells and cultured
↓
The cells grown in tryptophan-free medium are selected and cloned
↓
The yeast cells are cultured
↓
The HBsAg gene is expressed to produce 2 nm sized particles similar to those found in patients infected with
hepatitis B (these particles are immunoreactive with anti – HBsAg antibodies)
↓
The subunit HBsAg as 22 nm particles can be isolated and used to immunize individuals against hepatitis B
Antibodies
Monoclonal antibodies
Antibodies or immunoglobulins are protein molecules produced by a specialized group of cells called B lymphocytes
(plasma cells) in mammals
...
Each antigen has specific antigen determinants (epitopes) located on it
...

In response to an antigen (with several different epitopes), B lymphocytes gear up and produce many different
antibodies
...
The
polyclonal antibody production is variable and is dependent on factors such as epitopes, response to immunity etc
...


34

Monoclonal antibody is a single type of antibody that is directed against a specific antigenic determinant (epitope)
...
In the early years, animals were
immunized against a specific antigen, B lymphocytes were isolated and cultured in vitro for producing monoclonal
antibodies
...

It is interesting that immortal monoclonal antibody producing cells do exist in nature
...
It was in 1975
...
They could successfully hybridize antibodyproducing B lymphocytes with myeloma cells in vitro and create a hybridoma
...
The hybridoma
cells possess the growth and multiplying properties of myeloma cells but secrete antibody of B lymphocytes
...

Principle for creation of hybridoma cells
The myeloma cells used in hybridoma technology must be capable of synthesizing their own antibodies
...
The
mammalian cells can synthesize nucleotides by two pathways – de novo synthesis and salvage pathway
...
The formation of
tetrahydrofolate and therefore nucleotides can be blocked by the inhibitor aminopterin
...

Hypoxanthine guanine phosphoribosyl transferase (HGPRT) is a key enzyme in the salvage pathway of purines
...

Thymidine kinase (TK), involved in the salvage pathway of pyrimidines converts thymine to thymine monophosphate
(TMP)
...

When cells deficient (mutated cells) in HGPRT are grown in a medium containing hypoxanthine aminopterin and
thymidine (HAT medium), they cannot survive due to inhibition of de novo synthesis of purine nucleotides (salvage
pathway is not operative due to lack of HGPRT)
...

The hybridoma cells possess the ability of myeloma cells to grow in vitro with a functional HGPRT gene obtained from
lymphocytes (with which myeloma cells are fused)
...

Production of monoclonal antibodies:
Steps:
1
...
Cell fusion
3
...
Screening the products
5
...
Characterization of storage
They are discussed below:
1
...
The antigen, along with an adjuvant like Freud’s complete or incomplete adjuvant is
injected subcutaneously (adjuvants are non-specific potentiators of specific immune responses)
...
This enables increased stimulation of B lymphocytes which are
responding to the antigen
...
The immune stimulated cells for synthesis of antibodies have grown maximally by this approach
...

When the serum concentration of the antibodies is optimal, the animal is sacrificed
...
The lymphocytes of the spleen
are separated from the rest of the cells by density gradient centrifugation
...


3
...


5
...


Cell fusion: The thoroughly washed lymphocytes are mixed with HGPRT defective myeloma cells
...

PEG is removed by washing and the cells are kept in a fresh medium
...

Selection of hybridomas: When the cells are cultured in HAT medium, only the hybridoma cells grow, while
the rest will slowly disappear
...
Selection of a single antibody producing
hybrid cells is very important
...
The
suspension of the hybridoma cells is so diluted that the individual aliquots contain on an average one cell each
...

Screening the products: The hybridomas must be screened for the selection of the antibody of desired
specificity
...
The two techniques namely ELISA and RIA are commonly used for this purpose
...
Thus, the hybridoma cells producing the desired
antibody can be identified by screening
...

Cloning and propagation: The single hybrid cells producing the desired antibody are isolated and cloned
...

• Limiting dilution method: In this procedure, the suspension of hybridoma cells is serially diluted and
the aliquots of each dilution are put into microculture wells
...
This ensures that the antibody produced is
monoclonal
...
It is possible to
simultaneously grow many cells in the semisolid medium to form colonies
...

In actual practice, both the above techniques are combined and used for maximal production of
monoclonal antibodies
...
It is also important to elucidate the monoclonal antibody for the
immunoglobulin class or sub-class, the epitope for which it is specific and the number of binding sites it
possesses
...
Recombinant Products
Insulin: Insulin is a peptide hormone produced by beta cells of the pancreatic islets; it is considered to be the main
anabolic hormone of the body
...

↓
The plasmids are first inserted into a non-harmful type of E
...
Plasmids enter the bacteria in
a process called transfection
...

↓
The bacteria synthesizing the insulin then undergo a fermentation process
...
The millions of bacteria replicate roughly every 20
minutes through mitosis and each expresses the insulin gene
...

↓
The two chains are then mixed together and joined by disulfide bonds through the reduction-reoxidation
reaction
...
The batch is then placed in a centrifuge
...
Purification of the mixture can be
done by chromatography or other separation techniques
...
A marker protein lets
to detect E
...

Interferon
Interferon is the name given to a group of protein known primarily for their role in inhibiting viral infections and in
stimulating the entire immune system to fight disease
...
Interferons can promote or hinder the ability of some cells to differentiate,
that is, to become specialized in their function
...
Recent studies have also found that one interferon may play an important role in
the early biological processes of pregnancy
...

The first interferon was discovered in 1957 by Alick Isaacs and Jean Lindenmann
...
They named the protein interferon because it interferes
with infection
...

In addition to altering a cell’s ability to fight off viruses, interferons also control the activities of a number of specialized
cells within the immune system
...
Interferon γ can also stimulate the production of a class of T lymphocytes known as suppressor CD8
cells, which can inhibit B cells from making antibodies
...
These scavenger cells attack
infected cells while also stimulating other cells within the immune system
...


37

Mechanism of action: Interferons work to stop a disease when they are released into the blood stream and then bind
to cell receptors
...
Scientists have identified over 30 disease-fighting proteins produced by
interferons
...
Type I interferon: These include α, β, τ (tau) and ω
...
Type II interferon: This is γ
...
ILs can be divided into four major groups based on distinguishing structural features
...
The human genome encodes
more than 50 interleukins and related proteins
...
The majority of interleukins are
synthesized by helper CD4 T lymphocytes, as well as through monocytes, macrophages, and endothelial cells
...


38

7
...

Biomass: Biomass is the total cellular and organic mass, produced by the living organisms
...
e
...
Broadly speaking, biomass represents all forms of matter
derived from biological activities
...

Options for the conversion of biomass to energy:

The biomass is utilized for the production of biofuels and various other compounds
...

Combustion: Low moisture containing biomass (wood, straw, bran) can be directly burnt by a process, referred to as
combustion to generate electricity
...

Aqueous processes: The biomass with high water content is used to aqueous processes such as fermentation to produce
ethanol, oils and methane
...
It can also
be directly used for various domestic and industrial processes
...

It occurs in four phases
...


2
...


4
...

Acidifying phase: This phase is characterized by more
formation of organic acids, besides H2, CO2 and alcohol
...
These bacteria also generate acetate from
H2 and CO2
...
The methanogenic bacteria
(e
...
Methanobacterium omelianskii, M
...

bryantii, Methanosarcina barkeri) convert acetate, CO2
and H2 into methane
...
In the normal process of methanogenesis, approximately 50% of the complex polysaccharides contribute
to methanogenesis
...

2
...

4
...

Slurry composition: The ratio between solid and water composition in the slurry should be around 1:1
...
Good mixing and
solubilization of the organic constituents is required
...

Presence of inhibitors: Ammonium sulfate and antibiotics inhibit methane production
...


Limitations for large scale production of methane:
1
...

3
...


Methane is abundantly available in natural oil and gas fields (produced by the same mechanism of
methanogenesis, over a period of years)
...

Methane production by gasification of coal is more economical than its production from biomass
...


40

5
...
This is because it is very difficult to convert the gaseous
methane into liquid state
...
This is due to the fact that the biomass used for methane generation is renewable, in
contrast to the permanent depletion of naturally produced methane (in the gas and oil fields)
...

These fuels are generally in the form of alcohols, esters, ethers and other chemicals produced from biomass
...
Bioethanol fuel is mainly produced
by the sugar fermentation process, although it can also be manufactured by the chemical process of reacting ethylene
with steam
...
These crops are
grown specially for energy use and include corn, maize and wheat crops
...
Ethanol burns to produce CO2 and water
...
Biodiesel can be produced from straight vegetable
oil, animal oil/fats, tallow and waste cooking oil
...


Why biofuels?
Biofuels production and consumption ensures that the natural carbon cycle to be 100% achieved which
completely eliminates the continuous increase in CO2 rates in the atmosphere which in turns will have the
greatest effect on the environment and a way to end global warming
...


Types of biofuels: On the basis of generation, biofuels are of the following types:
1
...

2
...
Example: Cellulosic ethanol
...

2
...


4
...

Aircraft: Recent testing has shown to viability of biofuel use in the aviation industry and use of biofuels to
power aircraft is expected to substantially increase in the next decade
...

Small engines: Small engines like those found in lawn and mowers and chainsaws, can use ethanol blend up
to 10% without problems
...

2
...


Biodegradable: It is biodegradable and non-toxic
...

Economic stimulation: Because biofuels are produced locally, biofuel manufacturing plants can employ
hundreds of workers, creating new jobs in rural areas
...
Easy to recycle: Whereas oil is a limited resource that comes from specific materials, biofuels can be
manufactured from a wide range of material including crop waste, manure and other byproducts
...

5
...

6
...

7
...

8
...

9
...

10
...
It can be used purely or blended in any ratio with petroleum diesel
...

2
...

4
...


Produce carbon emissions: Several studies have been conducted that the process to produce fuel including the
machinery necessary to cultivate the crops and the plants has hefty carbon emissions
...

Food prices: As demand for food crops such as corn grows for biofuel production, it could also raise prices for
necessary staple food crops
...

Water use: Massive quantities of water are required for proper irrigation of biofuel crops as well as
manufacture the fuel, which could strain local and regional water crisis
...

2
...

4
...

A suitable climate is needed to grow most crops
...

There is not enough food waste to produce large amounts of biodiesel
...

Cellulosic biomass derived from non- food sources such as trees and grasses, is also being developed as a feedstock for
ethanol production
...
It is one of the widely used alternative
automotive fuels in the world (Brazil and USA are the largest ethanol producers)
...

Sources: Ethanol is produced from sugar crops, starches and cellulose
...

Applications of bioethanol:
Ethanol can be used:
• as a transport fuel to replace gasoline
• as a fuel for power generation by thermal combustion
• as a fuel for fuel cells by thermochemical reaction

42

•
•

as a fuel in cogeneration systems
as a feedstock in the chemicals industry

Advantages of bioethanol:
1
...

3
...

5
...


It burns more cleanly as a result of more complete combustion
...

It is carbon natural
...

It is a renewable energy source
...


Disadvantages of bioethanol:
1
...

3
...

5
...


Large amount of arable land is required to grow crops
...

Due to lucrative prices of bioethanol, some farmers may sacrifice food crops for biofuels which will increase
food prices around the world
...

It is not as efficient as petroleum
...

There is a difficulty in transportation of bioethanol
...
It operates in compression ignition engines like petroleum diesel, thereby requiring no essential engine
modification
...

Sources: Biodiesel is produced from soybean oils, seed and fats
...
Biodiesel is also produced from cooking oils, methane (anaerobic
digestion) and ethanol (from wood waste)
...

2
...

4
...
The process is discussed
hereunder:
The first step is to mix the alcohol for reaction with the catalyst, typically a strong base such as NaOH or KOH
...

CH3OH + Na+ + OH— → Na+ + O—CH3 + H2O
↓
Once the catalyst is prepared, the triglyceride will react with 3 moles of methanol, so excess methanol has to
be used in the reaction to ensure complete the reaction
...

With 100 lbs
...
of alcohol and 1 lbs
...
of biodiesel
and 10 lbs
...
The reaction typically takes place in between 40-650 C
...

↓
Both the glycerol and biodiesel need to have alcohol removed and recycled
...

↓
The wash water is separated out similar to solvent extraction and the trace water is evaporated out of the
biodiesel
...

Future of biodiesel:
1
...

3
...

5
...


Should be considered for use as an alternative and not a primary fuel
...

Storage issues with stability and transportations issues with high cost of delivered fuel compared to fossil fuels
...

Lack of understanding of environmental impact; no emissions
...


Advantages:
1
...

3
...

5
...

7
...

9
...

11
...

Low toxicity, in comparison with diesel fuel
...

Lower emissions of contaminants: CO, particulate matter, polycyclic aromatic hydrocarbons, aldehydes
...

No SO2 emissions
...

May be blended with diesel fuel at any proportion; both fuels may be mixed during the fuel supply to vehicles
...

It is the only alternative fuel that can be used in a conventional diesel engine, without modifications
...


Disadvantages:
1
...

3
...

5
...

Slightly higher fuel consumption due to the lower calorific value of biodiesel
...

Higher freezing point than diesel fuel
...

It is less stable than diesel fuel and therefore, long term storage (more than six months) of biodiesel is not
recommended
...

7
...

It dissolves the deposits of sediments and other contaminants from diesel fuel in storage tanks and fuel lines,
which then are flushed away by biofuel into the engine, where they can cause problems in the valves and
injection systems
...


It must be noted that these disadvantages are significantly reduced when biodiesel is used in blends with diesel fuel
...

2
...


Coal
Natural gas and
Oil

Approximately 93% of fossil fuel consumed throughout the world is for energy production, with only 7% being used
by industry for the production of solvents, plastics and a host of other organic chemicals
...
It is principally a mixture of CH 4 and CO2 along with other trace gases
...

Typical composition of biogas:
Compounds

%

Methane

50-75

CO2

25-50

Nitrogen

0-10

Hydrogen

0-1

H2S

0-3

O2

0

What is biogas made from?
Biogas is a commonly used biofuel in around the world and is generated through the process of anaerobic digestion or
the fermentation of biodegradable materials such as biomass, manure, sewage, municipal waste, rubbish dumps, septic
tank, green waste and energy crops
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The actual composition of
biogas will vary depending on the origin of the anaerobic digestion process i
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the feedstock
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Use of biogas:
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In transport sector to power vehicles: Just like natural gas, biogas can be easily compressed and used to power
automobiles
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As fuel for portable electricity generator: Portable electricity generators are now a days use natural gas
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In gas cylinders: Biogas can be compressed to use to use in gas cylinders instead of compressed natural gas
(CNG)
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Direct conversion of biogas to electricity in a fuel cell: In fuel cells the gas is burnt to heat a special electrical
cell
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This process requires very clean
gas and expensive fuel cells but there is a lot of potential in this technology now a days as well as for the future
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Free source of energy
Reduction of indoor air pollution and deforestation (if firewood or coal was previously used)
Reduces workload in collecting firewood and in cooking (if firewood or coal was previously used)
Little operation skills or maintenance required
Contributing to reducing the emission of gases that contribute to global warming
Deforestation and soil erosion can be reduced
Cooking on biogas is quicker and easier than cooking with firewood

Disadvantages:
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Conversion of biomass to energy: From Ans
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It may refer to:
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Microbial fuel cells (MFC) which use living microorganisms
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They are discussed below:
Microbial fuel cell (MFC):
Definition: A microbial fuel cell is a device that converts chemical energy to electrical energy by the catalytic reactions
of microorganisms
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In the anode compartment, fuel is oxidized by microorganisms, generating CO 2,
electrons and protons
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Electrons and protons are consumed in
the cathode compartment, combining with O2 to form H2O
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The electrons transfers
from microbial cells to the electrode is facilitated by mediators such as thionine, methyl viologen, methyl blue,
humic acid, neutral red etc
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Mediator free microbial fuel cell: Mediator free microbial fuel cells do not require a mediator but use
electrochemically active bacteria to transfer electrons to the electrode (electrons are carried directly from the
bacterial respiratory enzyme to electrode)
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Some bacteria which have pili on their external membrane, are able to
transfer their electron production via these pili
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) are not particularly well understood
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This centrifugation is known as a plant microbial fuel cell
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Given that the power is thus derived from living plants (in situ
energy production), this variant can provide additional ecological advantages
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Power generation: MFCs have a number of potential uses
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The use of MFCs is attractive for applications that require only low power
but where replacing batteries may be time consuming and expensive such as wireless sensor networks
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Education: Soil based MFCs are popular educational tools, as they employ a range of scientific disciplines
(microbiology, geochemistry, electrical engineering etc
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There are also kits available for classrooms and hobbyists and
research grade kits for scientific laboratories and corporations
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e
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Bio-recovery: In 2010, A
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Microbial electrolysis cells have been demonstrated to produce H
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The process is well
developed and can handle a high volume of wastewater and reduce pathogens
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The challenge is
that it is difficult to scale up the MFCs for practical wastewater treatment because of the power output
challenges of a larger surface area MFC
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Enzymatic biofuel cells are currently confined
to research facilities
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Whereas most fuel cells use metals
like platinum and nickel as catalysts, the enzymatic biofuel cell uses enzymes derived from living cells
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Enzymes are also specifically designed to process organic compounds such as sugars and alcohols, which are
extremely common in nature
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What is most
significant is that the enzymes that allow the fuel cell to operate must be immobilized near the anode and
cathode in order to work properly; if not mobilized, the enzymes will diffuse into the cell’s fuel and most of
the liberated electrons will not reach the electrodes, reducing its effectiveness

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