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Microbial
Biotechnology

Book List
1
...
Galzer
2
...
Clark
3
...
Satyanarayana
4
...
Introduction
Biotechnology:
1
...

3
...


Biotechnology is defined as the use of living systems, organisms or parts of organisms to manipulate
natural processes in order to develop products, systems or environments to benefit people
...

Biotechnology is the science of the production processes based on the action of microorganisms and their
active components and of production processes involving the use of cells and tissues from higher
organisms
...


Examples of biotechnology:
•
•

Old biotechnology – Tissue culture, fermentation technology
...


Genomics: The study of the complete genome of an organism is called genomics
...


2

2
...

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
...

5
...

Microbial production of methane is more expensive than its isolation from the natural gas
...

Being a gaseous fuel, it is quite difficult as well as expensive to store, transport and distribute methane
...
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
...

High cost: To refine biofuels to more efficient energy outputs and to build the necessary manufacturing
plants to increase biofuel quantities, a high initial investment is often required
...

Food shortages: There is concern that using valuable cropland to grow fuel crops could have an impact
on the cost of food and could possibly lead to food shortages
...


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

•

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

7

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
...


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
...


8

5
...


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


Advantages:
1
...

3
...

5
...

7
...


Renewable fuel, obtained from vegetable oils or animal fats
...

Degrades more rapidly than diesel fuel, minimizing the environmental consequences of biofuel spills
...

Lower health risk, due to reduced emissions of carcinogenic substances
...

Higher flash point (1000 C minimum)
...

9
...

10
...

11
...

Disadvantages:
1
...

3
...

5
...

7
...

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
...

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

9

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
...
The actual
composition of biogas will vary depending on the origin of the anaerobic digestion process i
...
the feedstock
...

Use of biogas:
1
...

3
...

5
...


Electricity generation: Biogas can be used to generate electricity in various power plants
...

Burning biogas in domestic gas stoves: Biogas can be used in similar ways as natural gas in gas stoves,
lamps or as fuels for engines
...

But biogas can be used in these engines for electricity generation
...

Direct conversion of biogas to electricity in a fuel cell: In fuel cells the gas is burnt to heat a special electrical
cell
...
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
...

2
...

4
...

6
...


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:
1
...

3
...

5
...

Conversion of biomass to energy: From Ans
...
It may refer to:
•
•

Microbial fuel cells (MFC) which use living microorganisms
...


They are discussed below:

10

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
...
In the anode compartment, fuel is oxidized by microorganisms,
generating CO2, electrons and protons
...
Electrons and
protons are consumed in the cathode compartment, combining with O2 to form H2O
...


2
...
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
...

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)
...
Some bacteria which have pili on their external membrane,
are able to transfer their electron production via these pili
...
) are not particularly well understood
...
This centrifugation is known as a plant microbial fuel cell
...
Given that the power is thus derived from living
plants (in situ energy production), this variant can provide additional ecological advantages
...


2
...


4
...


Power generation: MFCs have a number of potential uses
...
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
...

Education: Soil based MFCs are popular educational tools, as they employ a range of scientific disciplines
(microbiology, geochemistry, electrical engineering etc
...
There are also kits available for classrooms and
hobbyists and research grade kits for scientific laboratories and corporations
...
e
...

Bio-recovery: In 2010, A
...
Microbial electrolysis cells have been demonstrated to produce H
...
The
process is well developed and can handle a high volume of wastewater and reduce pathogens
...
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
...
Enzymatic biofuel cells are
currently confined to research facilities
...
Whereas most fuel cells use
metals like platinum and nickel as catalysts, the enzymatic biofuel cell uses enzymes derived from living cells
...

Enzymes are also specifically designed to process organic compounds such as sugars and alcohols, which
are extremely common in nature
...
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
...
Food, Drink and Biotechnology
Cheese production:
Cheese production is the largest dairy industry in the world
...

Types: Broadly two types:
1
...

2
...
g
...
g
...

Production process
All cheese are irreversibly made from casein of milk that is produced after separating the whey (liquid protein of
milk)
...
g
...


The production process of cheese is discussed hereunder:
1
...


3
...


Acidification of milk: By employing lactic acid bacteria (Streptococcus lactis, Lactobacillus lactis) the sugar of
milk (lactose) can be converted to lactic acid
...
6 and thus acidifies milk
...
e
...
Casein mainly consists of three components – insoluble α and ꞵ
caseins and a κ casein that keeps them in soluble state
...

Consequently, α and ꞵ caseins and the degraded products of κ casein combine to form a coagulum (curd)
...

Separation of curd from whey: When the temperature of the coagulum is raised to around 400C, the
coagulum (curd) and whey (fluid portion) get separated
...

Ripening of cheese: The flavour of raw cheese (with rubber texture) such as cheddar is bland
...
The procedures adopted for ripening (or
maturation) are highly variable depending on the type of cheese to be prepared
...
Alternatively, they may be inoculated
with certain fungi (e
...
Penicillium roquefortii)
...
Mild hydrolysis of fats
(or cheese), usually carried out by lipases or Aspergillus niger or Mucor maihai results in butyric acid
formation with characteristic flavour
...
While L
...

thermophilus results in the formation of lactic acid to give acid flavour
...
Yogurt is very delicious and in fact, frozen
yoghurt is becoming popular as an alternative to ice cream
...
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
...


Microorganisms and substrates used for production of SCP:

14

The most commonly used raw materials can be grouped in the following categories:
1
...

3
...


High energy sources: e
...
alkanes, methane, methanol, ethanol, gas oil
...
g
...

Agricultural and foresty sources: e
...
cellulose, lignin
...


Reformulation: Products can be developed again (reformulated), often changing or improving the end product
...

2
...


Reducing saturated fat, sugar or salt of a product
...

Improving the taste of a product
...

These include:
1
...

3
...

5
...

Possible negative health impacts of substitutes, replacements or additions
...

The cost of reformulation to the food industry
...


Reformulation strategies: Examples

15

1
...


3
...


Reducing fat and sugar: Foods high in saturated fats can be reformulated using ingredients with lower
saturated fat content
...
There are limits to how
far these nutrients can be reduced in different products without adversely affecting the safety or structure
of food or compromising taste
...
Microparticulation uses
special production techniques in conjugation with fat replacers to create a more appealing texture
...

Microparticulation: Whey proteins are used in a wide variety of food products for their functional
properties and high nutritional value
...

Whey protein concentrate undergoes a process resulting in uniform protein particles
...

Fat mimetics: Fat mimetics (replacers) can be carbohydrates or proteins and are used to replace fat in food
because of their textual or organoleptic properties
...
They are totally digestible and have a caloric value from 1 to 4 kcal/g
(4 to 16 KJ per gram)
...
There were exceptions in the following:
•
•
•

Ready meals
White sliced bread
Some fast food meals

The portion size of these have increased and contributed significantly to intake of salt, saturated fat and overall
energy intake
...
Though it is not a reformulation
of the nutrient profile, this approach reformulates the size of the foods offered
...

Several studies show that reducing energy density at the same time decreasing portion size has a greater impact
than what could be achieved by just doing one or the other
...

According to the WHO, genetically modified organisms can be defined as organisms (i
...
plants, animals or
microorganisms) in which the genetic material (DNA) has been altered in a way that does not occur naturally by
mating and/or natural recombination
...
Foods produced from or using GM
organisms are often referred to as GM foods
...
With the discovery of DNA in the early 1900s it became possible
to directly alter DNA and genes within food
...
Genetically
modified microbial enzymes were the first application of genetically modified organisms in food production
...
Developed by Calgene,
it was engineered to have a longer shelf life by inserting an antisense gene that delayed ripening
...


16

By 2010, 29 countries had planned commercialized biotech crops and a further 31 countries had granted regulatory
approval for transgenic crops to be imported
...

The first genetically modified animal to be approved for food use was AquAdvantage salmon in 2015
...

In 2016, a white button mushroom (Agaricus bisporus) modified using the CRISPR technique received de facto
approval in the US
...
Chemistry and Biotechnology
Production of solvents
The commercially important organic solvents are:

1
...

3
...


Ethanol
Acetone
Butanol and
Glycerol

Ethanol
Biosynthesis of ethanol

Production process of ethanol

18

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 CO 2 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
...


Production of organic acids
The major organic acids of commercial importance produced by fermentation are:
1
...

3
...

5
...


Citric acid
Gluconic acid
Lactic acid
Acetic acid
Ascorbic acid and
Itaconic acid

Citric acid
Applications:
1
...
g
...
Besides brightening the color, citric
acid acts as an antioxidant and preserves the flavors of foods
...

3
...


5
...
In metal industry,
pure metals are complexed with citrate and produced as metal citrates
...
Citric acid is also used
for preservation of ointments and cosmetic preparations
...

Citric acid can be utilized as an agent for stabilization of fats, oils or ascorbic acid
...
Citric acid is also used as stabilized of
emulsions in the preparation of cheese
...


Production process:

There two processes of citric acid production
...
Thus the surface
process has supported growth systems
...
There are two types of submerged fermenters (bioreactors) – stirred bioreactors and airlift
bioreactors
...
Surface process
1
...
It is
done by the following steps:
i
...

ii
...

iii
...
niger is sprayed as layers (3-6 cm thickness)
and inoculated at 280 C
...

The growth of the organism can be accelerated by addition of α amylase
...

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:
i
...

ii
...
The temperature is maintained
around 300 C
...

As the spores germinate (that occurs within 24 hours of inoculation), a layer of mycelium is
formed over the medium
...

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 fermentation is stopped after 7-15 days
...

The mycelium and nutrient solution are separated
...

vii
...


21

viii
...
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:

Gluconic acid
Application:
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:

22

5
...
These include
mineral formation, mineral degradation, sedimentation and geochemical cycling
...
Broadly speaking, biohydrometallugry deals with the application of biotechnology in
mining industry
...
g
...
Mining with microbes is both economical and environmental friendly
...
Some of the metals, however, are relatively soft, malleable and ductile e
...
sulfur
...


Majority of microorganisms can interact with metals
...

2
...

Biosorption: It deals with the microbial cell surface adsorption of metals from the mine wastes or dilute
mixtures
...

Although recovery of metals (e
...
copper) from the drainage water of mines has been known for centuries, the
involvement of microbes in this process was recognized about 40 years ago
...

2
...

Bacteria produce certain substance such as sulfuric acid and ferric iron which extract the metal (indirect
action)
...


Organisms for bioleaching:
Bacteria:
1
...

3
...


Thiobacillus ferrooxidans
Thiobacillus thiooxidans
Sulfolobus acidocaldarious
Sulfolobus brierlevi

Fungi:
1
...


Aspergillus niger: Extracts copper and nickel
...


Mechanism of bioleaching:

23

It can occur by the following two processes:
1
...
For instance, certain bacteria e
...
T
...
That is, these organisms
can obtain energy from the oxidation of Fe2+ to Fe3+ or from the oxidation of sulfur and reduced sulfur
compounds to sulfate as illustrated below:
FeSO4 + H2SO4 + O2 → Fe2(SO4)3 + H2O
S 0 + O2 + H 2 O → H 2 O
FeS2 + O2 + H2O → FeSO4 + H2SO4
As is evident from the third reaction given above, iron is extracted in the soluble form the iron ore pyrite
(FeS2)

2
...
Ferric iron or sulfuric acid, being
powerful oxidizing agents reacts with metals and extract them
...
It is possible
to continuously maintain acidic environment by the oxidation of iron, sulfur, metal sulfides or by
dissolution of carbonate ions
...
ENVIRONMENT AND BIOTECHNOLOGY
Bio-deterioration: Deterioration means destruction, impairment
...

Biodegradation: Biodegradation is biochemically catalyzed transformation of a compound to one or more
metabolites of lower molecular weight
...

Biotransformation: The conversion of a substrate to particular product or products by cells by isolated enzymes is
called biotransformation
...


Biodegradation is the biological process in
which a material is biologically degraded
through metabolic or enzymatic process
...


Biodeterioration is the biological means in
which material are physically, chemically and
biochemically damaged
...


It means degradation of useful substance by
microbial activity
...


It means the degradation
substance into a simple one
...


It is a negative aspect
...


It is a positive aspect
...


It indicates any undesirable change
...


It indicates desirable changes
...


It helps in element recycling but it is
economically harmful
...


It helps in element recycling but it is
economically important
...


Biodegradation can be occurred both in
aerobic and anaerobic condition
...


Biodeterioration can be occurred in aerobic
conditions
...


Biodegradation can be both beneficial and
harmful
...


Biodeterioration is always harmful
...


2
...


Slaughter: It is done by lipolytic and proteolytic bacteria
...

Soaking: Bacteria e
...
Bacillus megaterium, B
...
pumilis and Pseudomonas aeruginosa affects
perforations in goat and cattle hides
...

Tanning: After soaking, limiting and de-limiting hides are tanned
...

Temperature can be reduced or fungicides may be used
...

• Semi chrome tanning: Semi chrome tanning is intermediate in its resistance seldom supporting
slight growth of mold, but is never completely resistant
...
Leather can also be attacked by insects, notably by the dermestid
beetles – the hide (skin) and larder (Bacon) beetles during storage
...
Physical control
B
...
Biological method
They are discussed below:
A
...

• Complete sterilization at -1210 C for a minimum of 15 minutes using a pressurized autoclave at
15lb per inch2
...

B
...
Fumigants: Gaseous sterilization of fumigation is done
...
on the addition of toxic chemicals
...
Example: Ethylene oxide and propylene oxide
...
Preservatives: Chemical preservatives are variously referred to as biocides, bactericides,
fungicides, fungistats and material protectants
...
Genetics and biotechnology

27

8
...

During the production of alcohol, the gas bubbles of CO 2 appear at the surface of the boiling liquid
...

Bioprocess technology is a more recent usage to replace fermentation technology
...

Some workers prefer to use bioprocess technology for industrial use of higher plant and animal cells while
fermentation technology is confined to microbial use
...

Bioreactors or fermenters: The heart of fermentation (or bioprocessing) technology is the fermenter or bioreactor
...
It is a containment system designed to give right environment for the optimal growth and metabolic
activity of the organism
...

Traditional fermenters are open vats made up of wood or slate
...
A high quality stainless steel that does not corrode or leak toxic metals into the growth medium is used
...

Types of bioreactors: Based on the designs, bioreactors are of the following types:
1
...

3
...

5
...


Continuous stirred tank bioreactors
Bubble column bioreactors
Airlift bioreactors
Fluidized bed bioreactors
Packed bed bioreactors
Photobioreactors

In all types of bioreactors, the ultimate aim is to ensure that all parts of the system are subjected to the same
conditions
...
The
end products include antibiotics, amino acids, vitamins, organic acids, industrial enzymes and vaccines
...
DSP is as complex and important as fermentation process
...

In the present day biotechnology, the fermentation and downstream processing are considered as an integrated
system
...
The product
recovery yield in general is expected to be higher, if the number of steps in DSP is lower
...

2
...


Intracellular metabolites: These products are located within the cells e
...
vitamins and enzymes
...
g
...

Both intracellular and extracellular: Examples include vitamin B12, flavomycin
...
g
...
The
product recovery as a biomass is simpler
...


Stages in downstream processing
1
...

3
...

5
...


Solid-liquid separation: The first step in product recovery is the separation of whole cells (cell biomass)
and other insoluble ingredients from the culture broth
...
Some authors use the term
harvesting of microbial cells from the culture medium
...
The cells and other
solid particles get adsorbed on gas bubbles
...
The presence of certain substances, referred to as collector substances,
facilitates stable foam formation e
...
long chain fatty acids, amines
...
The process of flocculation depends on the nature of cells and the ionic constituents of
the medium
...

Filtration: Filtration is the most commonly used technique for separating the biomass and culture
filtrate
...
The
particles are trapped within the matrix and the fluid passes out
...

Absolute filters: These filters are with specific pore sizes that are smaller than the particles to be removed
...

Rotary drum vacuum filters: These filters are frequently used for separation of broth containing 10-40%
solids (by volume) and particles in the size of 0
...
Rotary drum vacuum filters have been
successfully used for filtration of yeast cells and filamentous fungi
...
However,
clogging of filters is a major limitation
...

i
...

iii
...
Thus centrifugation is mostly used for separating solid
particles from liquid phase (fluid/particle separation)
...
Tubular blow centrifuge can be operated at a high centrifugal speed, and can be run in
both batch and continuous mode
...

• Disc centrifuge: It consists of several discs that separate the bowl into settling zones
...
The clarified fluid moves upwards while the solids
settle at the lower surface
...

It consists of several chambers connected in such a way that the feed flows in a zigzag fashion
...
The force is much higher in
the periphery chambers, as a result smallest particles settle down in the outermost chamber
...
The decanter is generally used to concentrate fluids with high solid concentration
(biomass content 5-80%)
...


2
...
The microorganisms and
other cells can be disintegrated or disrupted by physical, chemical or enzymatic methods
...
For instance, Gram negative bacteria and filamentous fungi can be more easily
broken compared to Gram positive bacteria and yeasts
...


Concentration: The filtrate that is free from suspended particles (cells, cell debris etc
...
The desired product is a very minor constituent
...
The commonly used techniques for concentrating biological products are:
i
...

Liquid-liquid extraction
iii
...

Precipitation and
v
...


ii
...
The evaporators,
in general, have a heating device for supply of steam and unit for the separation of concentrated product
and vapour, a condenser for condensing vapour, accessories and control equipment
...

Types:
• Plate evaporators: The liquid to be concentrated flows over plates
...

• Falling film evaporators: In this case, the liquid flows down long tubes which gets distributed
as a thin film over the heating surface
...

• Forced film evaporators: This liquid films are mechanically driven and these devices are
suitable for producing dry product concentrates
...
In these evaporators,
a centrifugal force is used to pass on the liquid over heated plates or conical surfaces for
instantaneous evaporation
...

Types:
• Extraction of low molecular weight products: By using organic solvents, the lipophilic
compounds can be conveniently extracted
...
Extraction of lipophilic products can be done by the following techniques:
o Physical extraction: The compound gets itself distributed between two liquid phases
based on the physical properties
...

o Dissociation extraction: This technique is suitable for the extraction of ionisable
compounds
...


31

Reactive extraction: In this case, the desired product is made to react with a carrier
molecule (e
...
phosphorous compounds, aliphatic amine) and extracted into organic
solvent
...
g
...

o Supercritical fluid extraction (SCF): This techniques differs from the above procedures,
since the materials used for extraction are supercritical fluids (SCFs)
...
Supercritical CO2, with a low critical temperature and pressure
is commonly used in the extraction
...
(SCF has been used for the extraction of caffeine from coffee
beans, and pigments and ingredients from biological materials)
...
Organic solvents cannot be
used for protein extraction as they lose their biological activities
...

4
...
It is basically an analytical
technique dealing with the separation of closely related compounds from a mixture
...

The stationary phase is the porous solid matrix packed in a column (equilibrated with a suitable solvent)
on to which the mixture of compounds to be separated is located
...
A single mobile phase may be used continuously or it may be changed appropriately to facilitate
the release of desired compounds
...
g
...

Types:

5
...
The formulation of low molecular weight
products (solvents, organic acids) can be achieved by concentrating them with removal of most of the
water
...


32

Proteins are highly susceptible to loss of biological activity, hence their formulation requires special care
...
The stabilizers of protein
formulation include the following:
• Sugars e
...
sucrose, lactose
• Salts e
...
NaCl, ammonium sulfate
...
g
...
g
...


33

9
...
e
...
Such
use of enzymes is wasteful, particularly for industrial processes, since enzymes are not stable, and they cannot be
recovered for reuse
...
By employing this technique, enzymes are made more
efficient and cost effective for their industrial use
...

Examples of immobilized enzymes: Penicillin G acyclase, lipase, proteases, invertase etc
...

2
...

4
...

6
...

8
...
uses immobilized
enzymes or whole cells
...
Immobilized enzymes can be used to overcome inborn metabolic disorders by the supply of
immobilized enzymes
...

Food industry: Enzymes like pectinases and cellulases immobilized on suitable carriers are successfully
used in the production of jams, jellies and syrups from fruits and vegetables
...
The use of immobilized enzymes allows
researchers to increase the efficiency of different enzymes such as Horse Radish Peroxidase (HRP) in
blotting experiments and different Proteases for cell or organelle lysis
...

Waste water management: Treatment of sewage and industrial effluents
...

Detergent industry: Immobilization of lipase enzyme for effective dirt removal from cloths
...

2
...

4
...

6
...

8
...

10
...


Increased functional efficiency of the enzyme
...

Continuous use of enzyme
...

Saving in capital cost and investment of the process
...

Less chance to contamination in products
...

Stable supply of products in the market
...

High enzyme to substrate ratio
...

2
...

4
...


The possibility of loss of biological activity of an enzyme during immobilization or while it is in use
...

Industrial applications are limited
...

Some enzymes become unstable after immobilization
...

Methods of immobilization
Physical methods:

34

1
...

3
...


Covalent binding
• Support and
• Cross linking

They are discussed below:
Physical methods:
1
...
The support materials may be inorganic (e
...
alumina, silica gel, calcium phosphate gel, glass) or
organic (starch, carboxymethyl cellulose, DEAE cellulose, DEAE sephadex)
...
Therefore, the adsorbed enzymes can be easily removed by minor changes in pH,
ionic strength or temperature
...
g
...

• Organic support e
...
starch
...


Methods of adsorption:
a
...

b
...

c
...

d
...

Advantages:
i
...

ii
...

iii
...

iv
...

v
...

vi
...

Disadvantages:
i
...

2
...

Efficiency is less
...
Bonds
involved in stabilizing the enzyme to the matrix may be covalent or non-covalent
...


Enzyme inclusion in gels: This is an entrapment of enzymes inside the gels
...

c
...

Enzyme inclusion in microcapsules: In this case, the enzymes are trapped inside a microcapsule
matrix
...

Advantages:
i
...

iii
...

v
...


It is fast method for immobilization
...

Easy to practice at small scale
...

Less chance of conformational changes in enzyme
...


Disadvantages:
i
...

iii
...


Leakage of enzyme
...

Chance of microbial containment
...


Microencapsulation: Microencapsulation is a type of entrapment
...
The
membrane may be polymeric, lipoidal, lipoprotein based or non-ionic in nature
...

i
...

iii
...

Formation of emulsions
...


Microencapsulation is recently being used for immobilization of enzymes and mammalian cells
...
Hybridoma
cells have also been immobilized successfully by this technique
...


Encapsulation: This type of immobilization is done by enclosing the enzymes in a membrane capsule
...
In this method, the
effectiveness depends upon the stability of enzymes inside the capsule
...

ii
...

Large quantity of enzymes can be immobilized by this method
...

ii
...


Pore size limitation
...


Covalent binding: Immobilization of enzymes can be achieved by creation of covalent bonds between the
chemical groups of enzymes and the chemical groups of the support
...

However, covalent binding is often associated with loss of some enzyme activity
...

Methods:
a
...

b
...
They, in turn, bind covalently to tyrosyl or histidyl groups of enzymes
...


Peptide bond formation: Enzyme immobilization can also be achieved by the formation of
peptide bonds between the amino (or carboxyl groups) of the support and the carboxyl (or amino)
groups of the enzymes
...

d
...
g
...

Carriers or supports commonly used for covalent bonding are:
•
•
•
•
•

Carbohydrates e
...
cellulose, DEAE cellulose, agarose
...
g
...
g
...

Amino group bearing carriers e
...
amino benzyl cellulose
...


Advantages:
i
...

iii
...

v
...

No leakage or desorption problem
...

A variety of support with different functional groups are available
...


Disadvantages:
i
...


5
...

Enzyme inactivation by changes in the confirmation when undergoes reactions at the active
site
...


Cross linking: The absence of a solid support is a characteristic feature of immobilization of enzymes by
cross-linking
...
These reagents in fact, react with the enzyme molecules and create
bridges which form the backbone to hold enzyme molecules
...
These are:
i
...

Diazobenzidine
iii
...

Toluene diisothiocyanate

Immobilization of cells: Immobilization of whole cells is an alternative to enzyme immobilization and it is a welldeveloped method for the utilization of enzymes from microbes
...
The greatest advantage of whole cell immobilization is that here the
enzymes will be active and stable for long period of time since they are in their natural environment
...
Bacteria or yeast cells are immobilized by adsorbing it on
woodchips
...

Advantages of whole cell immobilization:
1
...

3
...

5
...


Multiple enzymes can be introduced to a single step
...

Enzymes are stable for long time
...

Cell organelles like mitochondria and chloroplasts can be immobilized
...


Disadvantages:

37

1
...

3
...

Unwanted enzymes and products are often produced
...


Methods of immobilization of whole cells: Same as enzyme immobilization
...


Preparation

Simple

Difficult

Difficult

Simple

2
...


Binding force

Variable

Strong

Weak

Strong

4
...


Applicability

Wide

Selective

Wide

Very wide

6
...


Matrix effects

Yes

Yes

Yes

No

8
...


Microbial protection

No

No

Yes

Yes

Limitations of enzyme immobilization:
1
...

3
...

5
...


Cost of carriers and immobilization
...

Mass transfer limitations
...

Problems with multi-enzyme systems
...


38

10
...
Thus, culturing is a process of growing cells artificially
...

Terminology in cell culture
The term tissue culture is commonly used to include both organ culture and cell culture
...
e
...

Cell culture: This refers to the culture of dispersed or disaggregated cells obtained from the original tissue or from
a cell line
...

Organotypic culture: This culture technique involves the recombination of different cell types to form a more
defined tissue or an organ
...
These cells are heterogenous and slow growing
...

Cell line: The sub-culturing of the primary culture gives rise to cell lines
...
On the other hand, finite cell lines represent the death
of cells after several subcultures
...

2
...

4
...


Clean and quite sterile area
Preparation facilities
Animal house
Microbiology laboratory
Storage facilities for glassware, chemicals, liquids, small equipment
...

2
...

4
...

6
...

8
...

10
...

12
...

14
...

16
...

18
...

20
...

22
...
Hence, there is a lot of importance attached to the nature of the materials used and the quality
of the culture vessels
...
On the other
hand, some cells undergo transformation, and become anchorage independent
...

2
...

This is mainly because of the availability of more suitable and alternate substrate
...
The most commonly used plastics are polystyrene,
polyvinyl chloride (PVC), polycarbonate, metinex and thermonex (TPX)
...

2
...

4
...

2
...

4
...


The way cells grow in culture – monolayer or suspension
...

The frequency of sampling for the desired work
...

The cost factor
...
The
flasks are usually employed for this purpose
...
It is necessary to slowly and continuously
agitate the suspended cells in the vessel
...
The following measures are suggested for minimizing contamination and
maintenance of aseptic conditions:
1
...

3
...

5
...


Strict adherence to standard sterile techniques and code of practices
...

Checking of cultures by eyes and microscopes (phase contrast) every time they are used
...

Maintenance of clean and tidy conditions at work places
...


Stem cell culture
Stem cell: Stem cells are unspecialized cells that develop into the specialized cells that make up the different types
of tissue in the human body
...
They are vital to the development, growth,
maintenance and repair of our brains, bones, muscles, nerves, blood, skin and other organs
...
They are obtained from human or animal tissues and can replicate for long periods of time in vitro (in an
artificial environment)
...
Once stem cells have been allowed to divide and propagate in a controlled culture, the collection of
healthy, dividing and undifferentiated cells is called a stem cell line
...
On the basis of potency, stem cells are classified as follows:
1
...
Examples are the zygote formed at
egg fertilization and the first few cells that result from the division of the zygote
...
Pluripotent: The ability to differentiate into almost all cell types
...

3
...
Examples include
hematopoietic (adult) stem cells that can become red and white blood cells or platelets
...
Oligopotent: The ability to differentiate into a few cells
...

5
...
Examples include (adult) muscle stem cells
...
The more cell types a cell can
differentiate into, the greater its potency
...
On the basis of their sources, stem cells are of the following types:
1
...
They are derived from embryos at a developmental stage before the time of
implantation would normally occur in the uterus
...

2
...
The primary roles of adult stem cells in a living
organism are to maintain and repair the tissue in which they are found
...

3
...
Scientists have engineered these induced pluripotent stem cells by
manipulating the expression of certain genes – reprogramming somatic cells back to a pluripotent
state
...

↓
Totipotent

Pluripotent

Multipotent

Relative potency

High

Medium

Low

Cell types capable of
generating

Differentiate into any
cell type

Differentiate into cells
from any of the three
germ layers

Differentiate into a
limited range of cell
types

Terminology

Toti = Whole

Pluri = Many

Multi = Several

Examples

Zygote, early morula

Embryonic stem cells,
Induced pluripotent stem
cells

Haematopoietic stem
cells, neural stem cells,
mesenchymal stem cells

Found

Early cells of fertilised
egg

Inner mass cells of the
blastocyst

In many tissues

Expression of
pluripotency genes

+++

++

+

Expression of lineagespecific genes

+

++

+++

Pros of use in research

Easy to isolate and grow

Easy to isolate and grow

Less ethical issues, less
chance of immune
rejection if taken from
same patient

Cons of use in research

Ethical issues

Ethical issues, teratoma
formation

Hard to isolate, limited
differentiation, scarce

The cells divide and spread over the surface of the dish
...

↓
The process of re-planting or sub-culturing the cells is repeated many times and for many months
...

↓
Once the cell line is established, the original cells yield millions of embryonic stem cells
...

↓
At any stage in the process, batches of cells can be frozen and shipped to other laboratories for further culture and
experiments
...
It is a blue dye,
itself weekly fluorescent until it is irreversibly reduced to the pink colored and highly red fluorescent resorufin
...

Material required:
1
...


Milk sample
Resazurin color or solution 0
...

2
...

4
...


All purpose lovibond comparator
One resazurin color disk from blue to white
Water path, thermostatically controlled to maintain temperature of 37 0 C
...


Procedure:
The sample is milked thoroughly by inverting from one to another container
↓
10 ml of milk sample is poured into previously sterilized test tube
↓
1 ml of resazurin solution is added quickly in the test tube
↓
The milk is mixed and then dyed thoroughly by inverting 2-3 times
↓
Test tubes are placed into water bath at the temperature of 37
...
If color falls between two disc numbers half value is recorded
...

Example: Kefir
...

Production process of kefir: This drink can be made by a method called immobilization

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