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Ethanol fuel is ethyl alcohol, the same type of alcohol found in alcoholic beverages, used as fuel. It
is most often used as a motor fuel, mainly as a biofuel additive for gasoline. The first production car
running entirely on ethanol was the Fiat 147, introduced in 1978 in Brazil by Fiat. Ethanol is
commonly made from biomass such as corn or sugarcane. World ethanol production for transport
fuel tripled between 2000 and 2007 from 17×109 liters (4.5×109 U.S. gal; 3.7×109 imp gal) to more
than 52×109 liters (1.4×1010 U.S. gal; 1.1×1010 imp gal). From 2007 to 2008, the share of ethanol in
global gasoline type fuel use increased from 3.7% to 5.4%.[1] In 2011 worldwide ethanol fuel
production reached 8.46×1010liters (2.23×1010 U.S. gal; 1.86×1010 imp gal) with the United States of
America and Brazil being the top producers, accounting for 62.2% and 25% of global production,
respectively.[2] US ethanol production reached 57.54×109 liters (1.520×1010 U.S. gal;
1.266×1010 imp gal) in 2017-04.
Ethanol fuel has a "gasoline gallon equivalency" (GGE) value of 1.5, i.e. to replace the energy of 1
volume of gasoline, 1.5 times the volume of ethanol is needed.[4][5]
Ethanol-blended fuel is widely used in Brazil, the United States, and Europe (see also Ethanol fuel
by country) Most cars on the road today in the U.S. can run on blends of up to 10% ethanol,[6] and
ethanol represented 10% of the U.S. gasoline fuel supply derived from domestic sources in
2011.[2] Furthermore, many used cars today are flexible-fuel vehicles able to use 100% ethanol fuel.
Since 1976 the Brazilian government has made it mandatory to blend ethanol with gasoline, and
since 2007 the legal blend is around 25% ethanol and 75% gasoline (E25).[7] By December
2011 Brazil had a fleet of 14.8 million flex-fuel automobiles and light trucks[8][9] and 1.5 million flex-
fuel motorcycles[10][11][12] that regularly use neat ethanol fuel (known as E100).
Bioethanol is a form of renewable energy that can be produced from agricultural feedstocks. It can
be made from very common crops such as hemp, sugarcane, potato, cassava and corn. There has
been considerable debate about how useful bioethanol is in replacing gasoline. Concerns about its
production and use relate to increased food prices due to the large amount of arable land required
for crops,[13] as well as the energy and pollution balance of the whole cycle of ethanol production,
especially from corn. Even though this debate has the counterpart of animal agriculture being
already a source of massive arable land, therefore making ethanol a lower resource consumer in
constrast. [14][15] Recent developments with cellulosic ethanol production and commercialization may
allay some of these concerns.[16]
Cellulosic ethanol offers promise because cellulose fibers, a major and universal component in plant
cells walls, can be used to produce ethanol.[17][18] According to the International Energy Agency,
cellulosic ethanol could allow ethanol fuels to play a much bigger role in the future.[19]

During ethanol fermentation, glucose and other sugars in the corn (or sugarcane or other crops) are
converted into ethanol and carbon dioxide.
C6H12O6 → 2 C2H5OH+ 2 CO2 + heat
Ethanol fermentation is not 100% selective with side products such as acetic acid and glycols.
They are mostly removed during ethanol purification. Fermentation takes place in an aqueous
solution. The resulting solution has an ethanol content of around 15%. Ethanol is subsequently
isolated and purified by a combination of adsorption and distillation.
During combustion, ethanol reacts with oxygen to produce carbon dioxide, water, and heat:
C2H5OH + 3 O2 → 2 CO2 + 3 H2O
+ heat
Starch and cellulose molecules are strings of glucose molecules. It is also possible to
generate ethanol out of cellulosic materials. That, however, requires a pretreatment that
splits the cellulose into glucose molecules and other sugars that subsequently can be
fermented. The resulting product is called cellulosic ethanol, indicating its source.
Ethanol is also produced industrially from ethylene by hydration of the double bond in the
presence of a catalyst and high temperature.
C2H4 + H2O → C2H5OH
Most ethanol is produced by fermentation.

In ethanol fermentation, (1) one glucose molecule breaks down into two pyruvates. The energy
from this exothermic reaction is used to bind the inorganic phosphates to ADP and convert NAD+
to NADH. (2) The two pyruvates are then broken down into two acetaldehydes and give off two
CO2 as a by-product. (3) The two acetaldehydes are then converted to two ethanol by using the
H- ions from NADH, converting NADH back into NAD+.


About 5% of the ethanol produced in the world in 2003 was actually a petroleum product.[20] It
is made by the catalytic hydration of ethylene with sulfuric acid as the catalyst. It can also be
obtained via ethylene or acetylene, from calcium carbide, coal, oil gas, and other sources. Two
million short tons (1,786,000 long tons; 1,814,000 t) of petroleum-derived ethanol are produced
annually. The principal suppliers are plants in the United States, Europe, and South
Africa.[21] Petroleum derived ethanol (synthetic ethanol) is chemically identical to bio-ethanol and can
be differentiated only by radiocarbon dating.[22]
Bio-ethanol is usually obtained from the conversion of carbon-based feedstock. Agricultural
feedstocks are considered renewable because they get energy from the sun using photosynthesis,
provided that all minerals required for growth (such as nitrogen and phosphorus) are returned to the
land. Ethanol can be produced from a variety of feedstocks such as sugar
cane, bagasse, miscanthus, sugar beet, sorghum,
grain, switchgrass, barley, hemp, kenaf, potatoes, sweet
potatoes, cassava, sunflower, fruit, molasses, corn, stover, grain, wheat, straw, cotton,
other biomass, as well as many types of cellulose waste and harvesting, whichever has the
best well-to-wheel assessment.
An alternative process to produce bio-ethanol from algae is being developed by the
company Algenol. Rather than grow algae and then harvest and ferment it, the algae grow in
sunlight and produce ethanol directly, which is removed without killing the algae. It is claimed the
process can produce 6,000 U.S. gallons per acre (5,000 imperial gallons per acre; 56,000 liters per
hectare) per year compared with 400 US gallons per acre (330 imp gal/acre; 3,700 L/ha) for corn
Currently, the first generation processes for the production of ethanol from corn use only a small part
of the corn plant: the corn kernels are taken from the corn plant and only the starch, which
represents about 50% of the dry kernel mass, is transformed into ethanol. Two types of second
generation processes are under development. The first type
uses enzymes and yeast fermentation to convert the plant cellulose into ethanol while the second
type uses pyrolysis to convert the whole plant to either a liquid bio-oil or a syngas. Second
generation processes can also be used with plants such as grasses, wood or agricultural waste
material such as straw.

Although there are various ways ethanol fuel can be produced, the most common way is via
The basic steps for large-scale production of ethanol are: microbial (yeast) fermentation of
sugars, distillation, dehydration (requirements vary, see Ethanol fuel mixtures, below),
and denaturing (optional). Prior to fermentation, some crops require saccharification or hydrolysis of
carbohydrates such as cellulose and starch into sugars. Saccharification of cellulose is
called cellulolysis (see cellulosic ethanol). Enzymes are used to convert starch into sugar.[24]

Ethanol is produced by microbial fermentation of the sugar. Microbial fermentation currently only
works directly with sugars. Two major components of plants, starch and cellulose, are both made of
sugars—and can, in principle, be converted to sugars for fermentation. Currently, only the sugar
(e.g., sugar cane) and starch (e.g., corn) portions can be economically converted. There is much
activity in the area of cellulosic ethanol, where the cellulose part of a plant is broken down to sugars
and subsequently converted to ethanol.

For the ethanol to be usable as a fuel, the yeast solids and the majority of the water must be
removed. After fermentation, the mash is heated so that the ethanol evaporates.[25] This process,
known as distillation, separates the ethanol, but its purity is limited to 95–96% due to the formation of
a low-boiling water-ethanol azeotrope with maximum (95.6% m/m (96.5% v/v) ethanol and 4.4%
m/m (3.5% v/v) water). This mixture is called hydrous ethanol and can be used as a fuel alone, but
unlike anhydrous ethanol, hydrous ethanol is not miscible in all ratios with gasoline, so the water
fraction is typically removed in further treatment to burn in combination with gasoline in gasoline

There are three dehydration processes to remove the water from an azeotropic ethanol/water
mixture. The first process, used in many early fuel ethanol plants, is called azeotropic distillation and
consists of adding benzene or cyclohexane to the mixture. When these components are added to
the mixture, it forms a heterogeneous azeotropic mixture in vapor–liquid-liquid equilibrium, which
when distilled produces anhydrous ethanol in the column bottom, and a vapor mixture of water,
ethanol, and cyclohexane/benzene.
When condensed, this becomes a two-phase liquid mixture. The heavier phase, poor in the entrainer
(benzene or cyclohexane), is stripped of the entrainer and recycled to the feed—while the lighter
phase, with condensate from the stripping, is recycled to the second column. Another early method,
called extractive distillation, consists of adding a ternary component that increases ethanol's relative
volatility. When the ternary mixture is distilled, it produces anhydrous ethanol on the top stream of
the column.
With increasing attention being paid to saving energy, many methods have been proposed that
avoid distillation altogether for dehydration. Of these methods, a third method has emerged and has
been adopted by the majority of modern ethanol plants. This new process uses molecular sieves to
remove water from fuel ethanol. In this process, ethanol vapor under pressure passes through a bed
of molecular sieve beads. The bead's pores are sized to allow adsorption of water while excluding
ethanol. After a period of time, the bed is regenerated under vacuum or in the flow of inert
atmosphere (e.g. N2) to remove the adsorbed water. Two beds are often used so that one is
available to adsorb water while the other is being regenerated. This dehydration technology can
account for energy saving of 3,000 btus/gallon (840 kJ/L) compared to earlier azeotropic
Recent research has demonstrated that complete dehydration prior to blending with gasoline is not
always necessary. Instead, the azeotropic mixture can be blended directly with gasoline so that
liquid-liquid phase equilibrium can assist in the elimination of water. A two-stage counter-current
setup of mixer-settler tanks can achieve complete recovery of ethanol into the fuel phase, with
minimal energy consumption.[28]

Post-production water issues

Ethanol is hygroscopic, meaning it absorbs water vapor directly from the atmosphere. Because
absorbed water dilutes the fuel value of the ethanol and may cause phase separation of ethanol-
gasoline blends (which causes engine stall), containers of ethanol fuels must be kept tightly sealed.
This high miscibility with water means that ethanol cannot be efficiently shipped through
modern pipelines, like liquid hydrocarbons, over long distances.[29]
The fraction of water that an ethanol-gasoline fuel can contain without phase separation increases
with the percentage of ethanol.[30] For example, E30 can have up to about 2% water. If there is more
than about 71% ethanol, the remainder can be any proportion of water or gasoline and phase
separation does not occur. The fuel mileage declines with increased water content. The increased
solubility of water with higher ethanol content permits E30 and hydrated ethanol to be put in the
same tank since any combination of them always results in a single phase. Somewhat less water is
tolerated at lower temperatures. For E10 it is about 0.5% v/v at 21° C and decreases to about 0.23%
v/v at −34° C .[3
Efficiency of common crops
As ethanol yields improve or different feedstocks are introduced, ethanol production may become
more economically feasible in the US. Currently, research on improving ethanol yields from each unit
of corn is underway using biotechnology. Also, as long as oil prices remain high, the economical use
of other feedstocks, such as cellulose, become viable. By-products such as straw or wood chips can
be converted to ethanol. Fast growing species like switchgrass can be grown on land not suitable for
other cash crops and yield high levels of ethanol per unit area.[63]

Annual yield Greenhouse-

Crop (Liters/hectare, gas savings Comments
US gal/acre) vs. petrol[a]

Long-season annual grass. Used as feedstock

6800–8000 for most bioethanol produced in Brazil. Newer
Sugar cane L/ha,[38][85][86][87] 87%–96% processing plants burn residues not used for
727–870 g/acre ethanol to generate electricity. Grows only in
tropical and subtropical climates.

Low-input perennial grass. Ethanol production

7300 L/ha,
Miscanthus 37%–73% depends on development of cellulosic
780 g/acre

Low-input perennial grass. Ethanol production

depends on development of cellulosic
3100–7600 L/ha, technology. Breeding efforts underway to
Switchgrass 37%–73%
330–810 g/acre increase yields. Higher biomass production
possible with mixed species of perennial

Fast-growing tree. Ethanol production depends

3700–6000 L/ha, on development of cellulosic technology.
Poplar 51%–100%
400–640 g/acre Completion of genomic sequencing project will
aid breeding efforts to increase yields.

Low-input annual grass. Ethanol production

possible using existing technology. Grows in
Sweet 2500–7000 L/ha, tropical and temperate climates, but highest
No data
sorghum 270–750 g/acre ethanol yield estimates assume multiple crops
per year (possible only in tropical climates). Does
not store well.[88][89][90][91]
High-input annual grass. Used as feedstock for
most bioethanol produced in USA. Only kernels
can be processed using available technology;
Corn L/ha,[38][85][86][87] 10%–20%
development of commercial cellulosic technology
330–424 g/acre
would allow stover to be used and increase
ethanol yield by 1,100 – 2,000 litres/ha.

Source (except those indicated): Nature 444 (7 December 2006): 673–676.

[a] – Savings of GHG emissions assuming no land use change (using existing crop lands).

Biogas refers to a mixture of different gases produced by the breakdown of organic matter in the
absence of oxygen. Biogas can be produced from raw materials such as agricultural
waste, manure, municipal waste, plant material, sewage, green waste or food waste. Biogas is
a renewable energy source.
Biogas is produced by anaerobic digestion with methanogen or anaerobic organisms, which digest
material inside a closed system, or fermentation of biodegradable materials.[1] This closed system is
called an anaerobic digester, biodigester or a bioreactor.[2]
Biogas is primarily methane (CH
4) and carbon dioxide (CO
2) and may have small amounts of hydrogen sulfide (H
2S), moisture and siloxanes. The gases methane, hydrogen, and carbon monoxide (CO) can be

combusted or oxidized with oxygen. This energy release allows biogas to be used as a fuel; it can be
used for any heating purpose, such as cooking. It can also be used in a gas engine to convert the
energy in the gas into electricity and heat.[3]
Biogas can be compressed, the same way as natural gas is compressed to CNG, and used to
power motor vehicles. In the United Kingdom, for example, biogas is estimated to have the potential
to replace around 17% of vehicle fuel.[4] It qualifies for renewable energy subsidies in some parts of
the world. Biogas can be cleaned and upgraded to natural gas standards, when it becomes bio-
methane. Biogas is considered to be a renewable resource because its production-and-use cycle is
continuous, and it generates no net carbon dioxide. As the organic material grows, it is converted
and used. It then regrows in a continually repeating cycle. From a carbon perspective, as much
carbon dioxide is absorbed from the atmosphere in the growth of the primary bio-resource as is
released, when the material is ultimately converted to energy.

The biogas is a renewable energy that can be used for heating, electricity, and many other
operations that use a reciprocating internal combustion engine, such as GE
Jenbacher or Caterpillar gas engines.[5] To provide these internal combustion engines with biogas
having ample gas pressure to optimize combustion, within the European
Union ATEX centrifugal fanunits built in accordance with the European
directive 2014/34/EU (previously 94/9/EG) are obligatory. These centrifugal fan units, for
example Combimac, Meidinger AG or Witt & Sohn AG are suitable for use in Zone 1 and 2 .
Other internal combustion engines such as gas turbines are suitable for the conversion of biogas
into both electricity and heat. The digestate is the remaining inorganic matter that was not
transformed into biogas. It can be used as an agricultural fertiliser.
Biogas is produced either;

 As landfill gas (LFG), which is produced by the breakdown of biodegradable waste inside a
landfill due to chemical reactions and microbes, or
 As digested gas, produced inside an anaerobic digester.
Biogas plants
A biogas plant is the name often given to an anaerobic digester that treats farm wastes or energy
crops. It can be produced using anaerobic digesters (air-tight tanks with different configurations).
These plants can be fed with energy crops such as maize silage or biodegradable wastesincluding
sewage sludge and food waste. During the process, the micro-organisms transform biomass waste
into biogas (mainly methane and carbon dioxide) and digestate. Higher quantity of biogas could be
produced when the wastewater is co-fermented with other residual from dairy industry, sugar
industry, brewery industry. For example, while mixing 90% of wastewater from beer factory with 10%
cow whey, the production of biogas is increased by 2.5 times compared to the biogas produced by
wastewater from beer factory only.[

Key processes

There are two key processes: mesophilic and thermophilic digestion which is dependent on
temperature. In experimental work at University of Alaska Fairbanks, a 1000-litre digester
using psychrophiles harvested from "mud from a frozen lake in Alaska" has produced 200–300 liters
of methane per day, about 20%–30% of the output from digesters in warmer climates.[8]


The composition of biogas varies depending upon the substrate composition, as well as the
conditions within the anaerobic reactor (temperature, pH, and substrate concentration).[17] Landfill
gas typically has methane concentrations around 50%. Advanced waste treatment technologies can
produce biogas with 55%–75% methane,[18] which for reactors with free liquids can be increased to
80%–90% methane using in-situ gas purification techniques.[19] As produced, biogas contains water
vapor. The fractional volume of water vapor is a function of biogas temperature; correction of
measured gas volume for water vapour content and thermal expansion is easily done via simple
mathematics[20] which yields the standardized volume of dry biogas.
In some cases, biogas contains siloxanes. They are formed from the anaerobic decomposition of
materials commonly found in soaps and detergents. During combustion of biogas containing
siloxanes, silicon is released and can combine with free oxygen or other elements in the combustion
gas. Deposits are formed containing mostly silica (SiO
2) or silicates (Si

y) and can contain calcium, sulfur, zinc, phosphorus. Such white mineral deposits accumulate to a
surface thickness of several millimeters and must be removed by chemical or mechanical means.
Practical and cost-effective technologies to remove siloxanes and other biogas contaminants are
For 1000 kg (wet weight) of input to a typical biodigester, total solids may be 30% of the wet weight
while volatile suspended solids may be 90% of the total solids. Protein would be 20% of the volatile
solids, carbohydrates would be 70% of the volatile solids, and finally fats would be 10% of the
volatile solids.
Typical composition of biogas

Compound Formula %

Methane CH 50–75

Carbon dioxide CO 25–50


Nitrogen N 0–10

Hydrogen H 0–1

Hydrogen sulfide 0.1 –0.5

Oxygen O 0–0.5

Source: www.kolumbus.fi, 2007[16]


Biogas can be used for electricity production on sewage works,[26] in a CHP gas engine, where
the waste heat from the engine is conveniently used for heating the digester; cooking; space
heating; water heating; and process heating. If compressed, it can replace compressed natural
gas for use in vehicles, where it can fuel an internal combustion engine or fuel cells and is a much
more effective displacer of carbon dioxide than the normal use in on-site CHP plants.[

Biogas upgrading

Raw biogas produced from digestion is roughly 60% methane and 29% CO
2 with trace elements of H

2S: inadequate for use in machinery. The corrosive nature of H

2S alone is enough to destroy the mechanisms.


Methane in biogas can be concentrated via a biogas upgrader to the same standards as
fossil natural gas, which itself has to go through a cleaning process, and becomes biomethane. If the
local gas network allows, the producer of the biogas may use their distribution networks. Gas must
be very clean to reach pipeline quality and must be of the correct composition for the distribution
network to accept. Carbon dioxide, water, hydrogen sulfide, and particulates must be removed if
There are four main methods of upgrading: water washing, pressure swing absorption, selexol
absorption, and amine gas treating.[29] In addition to these, the use of membrane separation
technology for biogas upgrading is increasing, and there are already several plants operating in
Europe and USA.[27][30]
The most prevalent method is water washing where high pressure gas flows into a column where the
carbon dioxide and other trace elements are scrubbed by cascading water running counter-flow to
the gas. This arrangement could deliver 98% methane with manufacturers guaranteeing maximum
2% methane loss in the system. It takes roughly between 3% and 6% of the total energy output in
gas to run a biogas upgrading system.

Biogas gas-grid injection

Gas-grid injection is the injection of biogas into the methane grid (natural gas grid). Until the
breakthrough of micro combined heat and power two-thirds of all the energy produced by biogas
power plants was lost (as heat). Using the grid to transport the gas to customers, the energy can be
used for on-site generation,[31] resulting in a reduction of losses in the transportation of energy.
Typical energy losses in natural gas transmission systems range from 1% to 2%; in electricity
transmission they range from 5% to 8%.[32]
Before being injected in the gas grid, biogas passes a cleaning process, during which it is upgraded
to natural gas quality. During the cleaning process trace components harmful to the gas grid and the
final users are removed.[

Biogas in transport
If concentrated and compressed, it can be used in vehicle transportation. Compressed biogas is
becoming widely used in Sweden, Switzerland, and Germany. A biogas-powered train, named
Biogaståget Amanda (The Biogas Train Amanda), has been in service in Sweden since
2005.[34][35] Biogas powers automobiles. In 1974, a British documentary film titled Sweet as a
Nut detailed the biogas production process from pig manure and showed how it fueled a custom-
adapted combustion engine.[36][37] In 2007, an estimated 12,000 vehicles were being fueled with
upgraded biogas worldwide, mostly in Europe.[38]
Biogas is part of the wet gas and condensing gas (or air) category that includes mist or fog in the
gas stream. The mist or fog is predominately water vapor that condenses on the sides of pipes or
stacks throughout the gas flow. Biogas environments include wastewater digesters, landfills, and
animal feeding operations (covered livestock lagoons).
Ultrasonic flow meters are one of the few devices capable of measuring in a biogas atmosphere.
Most of thermal flow meters are unable to provide reliable data because the moisture causes steady
high flow readings and continuous flow spiking, although there are single-point insertion thermal
mass flow meters capable of accurately monitoring biogas flows with minimal pressure drop. They
can handle moisture variations that occur in the flow stream because of daily and seasonal
temperature fluctuations, and account for the moisture in the flow stream to produce a dry gas value.

Biogas generated heat/electricity

Biogas can be used as the fuel in the system of producing biogas from agricultural wastes and co-
generating heat and electricity in a combined heat and power (CHP) plant. Unlike the other green
energy such as wind and solar, the biogas can be quickly accessed on demand. The global
warming potential can also be greatly reduced when using biogas as the fuel instead of fossil fuel.[39]
However, the acidification and eutrophication potentials produced by biogas are 25 and 12 times
higher respectively than fossil fuel alternative. This impacts can be reduced by using correct
combination of feedstocks, covered storage for digesters and improved technique for retrieving
escaped material. Overall, the results still suggest that using biogas can lead to significant reduction
in most impacts compared to fossil fuel alternative. The balance between environmental damage
and green house gas emission should still be considered while implicating the system.[40]

Biohydrogen is H2 that is produced biologically.[1] Interest is high in this technology because H2 is a
clean fuel and can be readily produced from certain kinds of biomass.[2] Many challenges
characterize this technology, including those intrinsic to H2, such as storage and transportation of a
noncondensible gas. Hydrogen producing organisms are poisoned by O2. Yields of H2 are often low.

Biochemical principles
The main reactions involve fermentation of sugars. Important reactions start with glucose, which is
converted to acetic acid:[3]
C6H12O6 + 2 H2O → 2 CH3CO2H + 2 CO2 + 4 H2
A related reaction gives formate instead of carbon dioxide:
C6H12O6 + 2 H2O → 2 CH3CO2H + 2 HCO2H + 2 H2
These reactions are exergonic by 216 and 209 kcal/mol, respectively.
H2 production is catalyzed by two hydrogenases. One is called [FeFe]-hydrogenase; the
other is called [NiFe]-hydrogenase. Many organisms express these enzymes. Notable
examples are members of the genera Clostridium, Desulfovibrio, Ralstonia, and the
pathogen Helicobacter. E. coli is the workhorse for genetic engineering of hydrogenases.[4]
It has been estimated that 99% of all organisms utilize dihydrogen (H2). Most of these
species are microbes and their ability to use H2 as a metabolite arises from the expression
of H2metalloenzymes known as hydrogenases.[5] Hydrogenases are sub-classified into three
different types based on the active site metal content: iron-iron hydrogenase, nickel-iron
hydrogenase, and iron hydrogenase.

The active site structures of the three types of hydrogenase enzymes.

Production by algae
The biological hydrogen production with algae is a method of photobiological water splitting which
is done in a closed photobioreactor based on the production of hydrogen as a solar
fuel by algae.[6][7] Algae produce hydrogen under certain conditions. In 2000 it was discovered that
if C. reinhardtii algae are deprived of sulfur they will switch from the production of oxygen, as in
normal photosynthesis, to the production of hydrogen

Photosynthesis in cyanobacteria and green algae splits water into hydrogen ions and electrons. The
electrons are transported over ferredoxins.[11] Fe-Fe-hydrogenases (enzymes) combine them into
hydrogen gas. In Chlamydomonas reinhardtii Photosystem II produces in direct conversion of
sunlight 80% of the electrons that end up in the hydrogen gas.[12] Light-harvesting
complex photosystem II light-harvesting protein LHCBM9 promotes efficient light energy
dissipation.[13] The Fe-Fe-hydrogenases need an anaerobic environment as they are inactivated by
oxygen. Fourier transform infrared spectroscopy is used to examine metabolic pathways.[14]

Specialized chlorophyll

The chlorophyll (Chl) antenna size in green algae is minimized, or truncated, to maximize
photobiological solar conversion efficiency and H2 production. The truncated Chl antenna size
minimizes absorption and wasteful dissipation of sunlight by individual cells, resulting in better light
utilization efficiency and greater photosynthetic productivity by the green alga mass culture.[15]


It would take about 25,000 square kilometre algal farming to produce biohydrogen equivalent to the
energy provided by gasoline in the US alone. This area represents approximately 10% of the area
devoted to growing soya in the US

Bioreactor design issues

 Restriction of photosynthetic hydrogen production by accumulation of a proton gradient.

 Competitive inhibition of photosynthetic hydrogen production by carbon dioxide.
 Requirement for bicarbonate binding at photosystem II (PSII) for efficient photosynthetic activity.
 Competitive drainage of electrons by oxygen in algal hydrogen production.
 Economics must reach competitive price to other sources of energy and the economics are
dependent on several parameters.
 A major technical obstacle is the efficiency in converting solar energy into chemical energy
stored in molecular hydrogen.
Monoclonal antibody
Monoclonal antibodies (mAb or moAb) are antibodies that are made by identical immune
cells that are all clones of a unique parent cell. Monoclonal antibodies can have monovalent affinity,
in that they bind to the same epitope (the part of an antigen that is recognized by the antibody). In
contrast, polyclonal antibodies bind to multiple epitopes and are usually made by several
different plasma cell (antibody secreting immune cell) lineages. Bispecific monoclonal antibodies can
also be engineered, by increasing the therapeutic targets of one single monoclonal antibody to two
Given almost any substance, it is possible to produce monoclonal antibodies that specifically bind to
that substance; they can then serve to detect or purify that substance. This has become an
important tool in biochemistry, molecular biology, and medicine. When used as medications, non-
proprietary drug names end in -mab (see "Nomenclature of monoclonal antibodies") and
many immunotherapy specialists use the word mab anacronymically.

Much of the work behind production of monoclonal antibodies is rooted in the production of
hybridomas, which involves identifying antigen-specific plasma/plasmablast cells (ASPCs) that
produce antibodies specific to an antigen of interest and fusing these cells with myeloma cells.[citation
Rabbit B-cells can be used to form a rabbit hybridoma. Polyethylene glycol is used to fuse
adjacent plasma membranes,[6] but the success rate is low, so a selective medium in which only
fused cells can grow is used. This is possible because myeloma cells have lost the ability to
synthesize hypoxanthine-guanine-phosphoribosyl transferase (HGPRT), an enzyme necessary
for the salvage synthesis of nucleic acids. The absence of HGPRT is not a problem for these cells
unless the de novo purine synthesis pathway is also disrupted. Exposing cells to aminopterin (a folic
acidanalogue, which inhibits dihydrofolate reductase, DHFR), makes them unable to use the de
novo pathway and become fully auxotrophic for nucleic acids, thus requiring supplementation to
The selective culture medium is called HAT medium because it contains hypoxanthine, aminopterin
and thymidine. This medium is selective for fused (hybridoma) cells. Unfused myeloma cells cannot
grow because they lack HGPRT and thus cannot replicate their DNA. Unfused spleen cells cannot
grow indefinitely because of their limited life span. Only fused hybrid cells, referred to as
hybridomas, are able to grow indefinitely in the medium because the spleen cell partner supplies
HGPRT and the myeloma partner has traits that make it immortal (similar to a cancer cell).
This mixture of cells is then diluted and clones are grown from single parent cells on microtitre wells.
The antibodies secreted by the different clones are then assayed for their ability to bind to the
antigen (with a test such as ELISA or Antigen Microarray Assay) or immuno-dot blot. The most
productive and stable clone is then selected for future use.
The hybridomas can be grown indefinitely in a suitable cell culture medium. They can also be
injected into mice (in the peritoneal cavity, surrounding the gut). There, they produce tumors
secreting an antibody-rich fluid called ascites fluid.
The medium must be enriched during in vitro selection to further favour hybridoma growth. This can
be achieved by the use of a layer of feeder fibrocyte cells or supplement medium such as briclone.
Culture-media conditioned by macrophages can be used. Production in cell culture is usually
preferred as the ascites technique is painful to the animal. Where alternate techniques exist, ascites
is considered unethical
A general representation of the method used to produce monoclonal
After obtaining either a media sample of cultured hybridomas or a sample of ascites fluid, the
desired antibodies must be extracted. Cell culture sample contaminants consist primarily of media
components such as growth factors, hormones and transferrins. In contrast, the in vivo sample is
likely to have host antibodies, proteases, nucleases, nucleic acids and viruses. In both cases, other
secretions by the hybridomas such as cytokinesmay be present. There may also be bacterial
contamination and, as a result, endotoxins that are secreted by the bacteria. Depending on the
complexity of the media required in cell culture and thus the contaminants, one or the other method
(in vivo or in vitro) may be preferable.
The sample is first conditioned, or prepared for purification. Cells, cell debris, lipids and clotted
material are first removed, typically by centrifugation followed by filtration with a 0.45 µm filter. These
large particles can cause a phenomenon called membrane fouling in later purification steps. In
addition, the concentration of product in the sample may not be sufficient, especially in cases where
the desired antibody is produced by a low-secreting cell line. The sample is therefore concentrated
by ultrafiltration or dialysis.
Most of the charged impurities are usually anions such as nucleic acids and endotoxins. These can
be separated by ion exchange chromatography.[14] Either cation exchange chromatography is used at
a low enough pH that the desired antibody binds to the column while anions flow through, or anion
exchange chromatography is used at a high enough pH that the desired antibody flows through the
column while anions bind to it. Various proteins can also be separated along with the anions based
on their isoelectric point (pI). In proteins, the isoelectric point (pI) is defined as the pH at which a
protein has no net charge. When the pH >pI, a protein has a net negative charge, and when the pH
<pI, a protein has a net positive charge. For example, albumin has a pI of 4.8, which is significantly
lower than that of most monoclonal antibodies, which have a pI of 6.1. Thus, at a pH between 4.8
and 6.1, the average charge of albumin molecules is likely to be more negative, while mAbs
molecules are positively charged and hence it is possible to separate them. Transferrin, on the other
hand, has a pI of 5.9, so it cannot be easily separated by this method. A difference in pI of at least 1
is necessary for a good separation.
Transferrin can instead be removed by size exclusion chromatography. This method is one of the
more reliable chromatography techniques. Since we are dealing with proteins, properties such as
charge and affinity are not consistent and vary with pH as molecules are protonated and
deprotonated, while size stays relatively constant. Nonetheless, it has drawbacks such as low
resolution, low capacity and low elution times.
A much quicker, single-step method of separation is protein A/G affinity chromatography. The
antibody selectively binds to protein A/G, so a high level of purity (generally >80%) is obtained.
However, this method may be problematic for antibodies that are easily damaged, as harsh
conditions are generally used. A low pH can break the bonds to remove the antibody from the
column. In addition to possibly affecting the product, low pH can cause protein A/G itself to leak off
the column and appear in the eluted sample. Gentle elution buffer systems that employ high salt
concentrations are available to avoid exposing sensitive antibodies to low pH. Cost is also an
important consideration with this method because immobilized protein A/G is a more expensive
To achieve maximum purity in a single step, affinity purification can be performed, using the antigen
to provide specificity for the antibody. In this method, the antigen used to generate the antibody is
covalently attached to an agarose support. If the antigen is a peptide, it is commonly synthesized
with a terminal cysteine, which allows selective attachment to a carrier protein, such as KLH during
development and to support purification. The antibody-containing medium is then incubated with the
immobilized antigen, either in batch or as the antibody is passed through a column, where it
selectively binds and can be retained while impurities are washed away. An elution with a low pH
buffer or a more gentle, high salt elution buffer is then used to recover purified antibody from the

Diagnostic tests
Once monoclonal antibodies for a given substance have been produced, they can be used to detect
the presence of this substance. The Western blot test and immuno dot blot tests detect the protein
on a membrane. They are also very useful in immunohistochemistry, which detect antigen in fixed
tissue sections and immunofluorescence test, which detect the substance in a frozen tissue section
or in live cells.

Analytic and chemical uses

Antibodies can also be used to purify their target compounds from mixtures, using the method
of immunoprecipitation.

Therapeutic uses
Therapeutic monoclonal antibodies act through multiple mechanisms, such as blocking of targeted
molecule functions, inducing apoptosis in cells which express the target, or by modulating signalling
Cancer treatment
One possible treatment for cancer involves monoclonal antibodies that bind only to cancer cell-
specific antigens and induce an immune response against the target cancer cell. Such mAbs can be
modified for delivery of a toxin, radioisotope, cytokine or other active conjugate or to
design bispecific antibodies that can bind with their Fab regions both to target antigen and to a
conjugate or effector cell. Every intact antibody can bind to cell receptors or other proteins with its Fc
Monoclonal antibodies for cancer. ADEPT, antibody directed enzyme prodrug therapy; ADCC: antibody
dependent cell-mediated cytotoxicity; CDC: complement-dependent cytotoxicity; MAb: monoclonal antibody;
scFv, single-chain Fv fragment.[42]MAbs approved by the FDA (for cancer) as of 2005 include:[43]

 Alemtuzumab
 Bevacizumab
 Cetuximab
 Gemtuzumabozogamicin
 Ipilimumab
 Ofatumumab
 Panitumumab
 Pembrolizumab
 Ranibizumab
 Rituximab
 Trastuzumab

Autoimmune diseases

Monoclonal antibodies used for autoimmune diseases include infliximab and adalimumab, which are
effective in rheumatoid arthritis, Crohn's disease, ulcerative colitis and ankylosing spondylitis by their
ability to bind to and inhibit TNF-α.[44] Basiliximab and daclizumab inhibit IL-2 on activated T cells and
thereby help prevent acute rejection of kidney transplants.[44] Omalizumab inhibits
human immunoglobulin E (IgE) and is useful in treating moderate-to-severe allergic asthma.

Examples of therapeutic monoclonal antibodies

 rheumatoid arthritis
 Crohn's disease
infliximab[44] inhibits TNF-α chimeric
 ulcerative colitis
 ankylosing spondylitis
 rheumatoid arthritis
 Crohn's disease
adalimumab inhibits TNF-α human
 ulcerative colitis
 ankylosing spondylitis
 acute rejection of kidney
basiliximab[44] inhibits IL-2 on activated T cells chimeric
 acute rejection of kidney
daclizumab[44] inhibits IL-2 on activated T cells humanized
 moderate-to-severe inhibits human immunoglobulin
omalizumab humanized
allergic asthma E (IgE)
Recombinant Vaccines—General:
Recombinant DNA technology in recent years has become a boon to
produce new generation vaccines. By this approach, some of the
limitations (listed above) of traditional vaccine production could be
overcome. In addition, several new strategies, involving gene
manipulation are being tried to create novel recombinant vaccines.

Types of Recombinant Vaccines:

The recombinant vaccines may be broadly categorized into
three groups:


1. Subunit recombinant vaccines:

These are the components of the pathogenic organisms. Subunit

vaccines include proteins, peptides and DNA.

2. Attenuated recombinant vaccines:

These are the genetically modified pathogenic organisms (bacteria or

viruses) that are made non-pathogenic and used as vaccines.


3. Vector recombinant vaccines:

These are the genetically modified viral vectors that can be used as
vaccines against certain pathogens. Some of the developments made in
the production of recombinant vaccines against certain diseases are
briefly described.

Type # 1. Subunit Vaccines:

As already stated, subunit recombinant vaccines are the components
(proteins, peptides, DNAs) of the pathogenic organisms. The
advantages of these vaccines include their purity in preparation,
stability and safe use. The disadvantages are — high cost factor and
possible alteration in native conformation. Scientists carefully evaluate
the pros and cons of subunit vaccines for each disease, and proceed on
the considered merits.

Hepatitis B:

Hepatitis B is a widespread disease in man. It primarily affects liver

causing chronic hepatitis, cirrhosis and liver cancer. Hepatitis B virus
is a 42 nm particle, called Dane particle. It consists of a core
containing a viral genome (DNA) surrounded by a phospholipid
envelope carrying surface antigens (Fig. 16.1 A).

Infection with hepatitis B virus produced Dane particles and 22 nm

sized particles. The latter contain surface antigens which are more
immunogenic. It is however, very difficult to grow hepatitis B virus in
mammalian cell culture and produce surface antigens.

How did they make insulin from recombinant DNA?

Recombinant DNA is a technology scientists developed that made it possible to insert a
human gene into the genetic material of a common bacterium. This “recombinant” micro-
organism could now produce the protein encoded by the human gene.
Scientists build the human insulin gene in the laboratory. Then they remove a loop of
bacterial DNA known as a plasmid and…

insert the human insulin gene into the plasmid.

Researchers return the plasmid to the bacteria and…
put the “recombinant” bacteria in large fermentation tanks.

There, the recombinant bacteria use the gene to begin producing human insulin.
Scientists harvest the insulin from the bacteria and…

purify the substance for use as a medicine for people.

The rapid increase in the number of diabetic patients globally and exploration of
alternate insulin delivery methods such as inhalation or oral route that rely on
higher doses, is bound to escalate the demand for recombinant insulin in near
future. Current manufacturing technologies would be unable to meet the growing
demand of affordable insulin due to limitation in production capacity and high
production cost. Manufacturing of therapeutic recombinant proteins require an
appropriate host organism with efficient machinery for posttranslational
modifications and protein refolding. Recombinant human insulin has been
produced predominantly using E. coli and Saccharomyces cerevisiae for
therapeutic use in human. E. coli expression system for production of insulin
E. coli is a preferred microorganism for large-scale production of recombinant proteins.
However, several disadvantages limit its use for production of recombinant biopharmaceuticals.
Various post-translational modifications (PTMs) such as glycosylation, phosphorylation,
proteolytic processing and formations of disulfide bonds which are very crucial for biological
activity, do not occur in E. coli[39],[40]. N-linked glycosylation is the most common
posttranslational modification of proteins in eukaryotes. It has been discovered that the
bacterium Campylobacter jejuni possess the capability to glycosylate the proteins and it was also
shown that a functionally active N-glycosylation pathway could be transferred to E. coli[41].
Although the structure of bacterial N-glycan is different from that observed in eukaryotes,
engineering of Campylobacter N-linked glycosylation pathway into E. coli, provides an
opportunity to express heterologous proteins in glycosylated form in E. coli. Expression of
Pglboligosaccharyltransferase or (OTase) from C. jejuni in E. coli showed a significant increase
in glycopepetide yield [42],[43]. Recently efforts has been made to produce glycosylated
proteins with substrates other than native and non-native to E. coli and C.jejuni[44]-[48].

The codon usage of the heterologous protein also plays a major role in determining the
expression level of recombinant protein. If the codon usage of heterologous protein differs
significantly from the average codon usage of the E. coli host, it could result in very low
expression. Usually, the frequency of the codon usage reflects the abundance of their
corresponding tRNA. Therefore, significant differences in codon usage could result in premature
termination of translation, misincorporation of aminoacids and inhibition of protein synthesis
[49]. Expression of heterologous proteins in E. coli can be improved by replacing codons that are
rarely found in highly expressed E. coli genes with more favorable major codons. Similarly, co-
expression of the genes encoding for a number of the tRNA for rare codon, may enhance the
expression of heterologous proteins in E. coli. There are some commercial E. coli strains
available that encodes for tRNA for rare codons such as BL21 (DE3) CodonPlus-RIL, BL21
(DE3) CodonPlus-RP (Stratagene, USA) and Rosetta (DE3). BL21 (DE3) CodonPlus-RIL
harbors tRNA genes for rare codons like AGG, AGA (arginine), AUA (isoleucine) and CUA
(leucine). Similarly, Rosetta (DE3) strain harbors tRNA genes for rare codons like AGG, AGA
(arginine), CGG (arginine), AUA (isoleucine), CUA (leucine), CCC (proline) and GGA
(glycine). These rare codons have been associated with low expression of proteins in E.
coli, hence application of these genetically engineered E. coli host strains may improve the
expression level of heterologous proteins and thus might result in higher yield of desired protein
[50]-[52]. The use of protease-deficient E. coli strains, which carry mutations that eliminate the
production of proteases may also improve the yield of recombinant protein by reducing
proteolytic degradation. E. coli strain BL-21, is deficient in two proteases encoded by
the lon (cytoplasmic) and ompT (periplasmic) genes. Rather than the external parameters,
targeted methods such as modifications in protease or secretion pathways can provide the insight
into biology of recombinant proteins [53]. In E. coli, complex and large therapeutic proteins can
be secreted in periplasm as it provides an oxidizing environment and help in forming disulphide
bonds, which facilitate the proper folding of recombinant proteins and likely to yield reliable N-
terminus of expressed protein [54]. Periplasm has advantages over cytoplasm in less protein
concentration and proteolytic activity, improve the production titer [55], and enhance the
solubility of recombinant protein. Altogether, with these advanced modifications and
developments ease the process of target protein production thus accelerating the drug
development [56].

Heterologous proteins generally accumulate in E. coli as inclusion bodies, which comprise of

insoluble misfolded aggregates of proteins. Use of molecular chaperones may increase the
protein solubility and assist in proper folding of recombinant protein. Some of the chaperones
prevent aggregation of protein and some assist in refolding and solubilization of misfolded
proteins. The most important chaperones in E. coli are GroEL, GroES, DnaK, DnaJ, GrpE and
Trigger factor. These chaperones may be used singly, or in combination to enhance the protein
solubility in E. coli[57],[58].

Recombinant human insulin was first produced in E. coli by Genentech in 1978, using a
approach that required the expression of chemically synthesized cDNA encoding for the insulin
A and B chains separately in E. coli[59]. After expressing independently, the two chains are
purified and co-incubated under optimum reaction conditions that promoted the generation of
intact and bioactive insulin by disulphide bond formation. The first commercial recombinant
insulin was developed for therapeutic use in human by this two-chain combination procedure
[60]. Another approach involves the expression of a single chemically synthesized cDNA
encoding for human proinsulin in E. coli followed by purification and subsequent excision of C-
peptide by proteolytic digestion. This approach was more efficient and convenient for large scale
production of therapeutic insulin as compared to the two chain combination approach and has
been used commercially since 1986 [60]. Eli Lilly followed this technology to produce Humulin,
the first recombinant insulin approved in 1982, for the treatment of diabetic patients. These first
generation recombinant insulins have an amino acid sequence identical to native human insulin
and are preferred over animal derived insulin products [14]. However, advancement in the field
of genetic engineering and development of technology to chemically synthesize genes with
altered nucleotide sequence, facilitated the development of insulin analogues with altered amino
acid sequence. It had been observed that native insulin in commercial preparations usually exist
in oligomeric form, as zinc-containing hexamer due to very high concentration, but in blood,
biologically active insulin is in monomeric form [61]. Hence, this oligomeric complex should
dissociate so that insulin can be absorbed from the site of injection into the blood. Due to this,
subcutaneously injected recombinant insulin usually have a slow onset with peak plasma
concentration after 2 hours of injection and longer duration of action that last for 6-8 hours [62].
Hence, in order to develop a fast- acting insulin analogue, it was required to modify the amino
acids residues whose side chains are involved in dimer or oligomer formation. It has been shown
that amino acids residues in insulin B-chain particularly B8, 9,12, 13, 16 and 23-28 play critical
role in oligomerization [63],[64]. Lispro, developed by Eli Lilly, was the first fast acting insulin
analogue to obtain regulatory approval in 1996, for therapeutic use [60]. Insulin Lispro is
engineered in such a way that it has similar amino acid sequence as the native insulin but has an
inversion of proline-lysine sequence at position 28 and 29 of the B-chain, which resulted in
reduced hydrophobic interactions and thus prevented dimer formations. For commercial
production of insulin Lispro, a synthetic cDNA encoding for Lys B28- Pro B29 human proinsulin
was expressed in E. coli and insulin Lispro was excised proteolytically from the proinsulin by
treating with trypsin and carboxypeptidase. Another rapid-acting insulin analogue, produced
in E. coli is Glulisine (Apidra) which was developed by Aventis Pharmaceuticals and approved
by US regulatory authorities in 2004. Insulin Glulisine have been generated by replacing B3
asparagine by a lysine and B29 lysine replaced by glutamic acid [14].

To avoid multiple injection, long-acting insulin analogues with prolonged duration of actions
have also generated. Insulin Glargine is one of such long-acting insulin analogues, which was
developed by Aventis Pharmaceuticals and approved by regulatory authorities of USA and EU in
2000. Insulin Glargine was generated by replacing the C-terminal asparagine of the A-chain with
a glycine residue and the C-terminal of the B- chain was modified by adding two arginine
residues. These modifications resulted in increase of the isoelectric point (pI) from 5.4 to neutral
values. Glargine was produced as proinsulin and expressed in E. coli and was finally formulated
at pH 4 in soluble form. However, after subcutaneous administration, it precipitated due to
neutral pH in the subcutaneous tissue. Resolubilization of insulin occur slowly, resulting in
longer duration for its release in the blood [14].

Lesson 2

2.1 Introduction
With the rising liberalization of agro-industrial markets and thus the world-wide
integration of food supply chains, the assurance of food quality and safety has become
a major concern. Following serious and repeated incidents such as mad cow disease
(Bovine Spongiform Encephalitis–BSE), Dioxin, Aflatoxin and most recently, Sudan
Red consumer protection has become a priority in policy making in the large
consumer markets. The recent occurrence of serious food scares and food
contamination events – such asSalmonella contagion of peanut butter in the US,
melamine contamination of milk in China and high pesticide content of aerated drinks
manufactured in India – has significantly enhanced the concern for food safety and its
impact on health, marketing and foreign trade. Protecting consumer health from food
borne hazards has become a compelling duty for policy makers across the globe.
Consequently, regulatory frameworks and standards are being developed wherein
trade and health issues are being addressed by prioritizing consumer protection over
freedom of trade. Thus, it has become imperative for the Indian industry and policy
makers to adopt strong practices of food safety so as to remain sustainably
competitive both in domestic and export markets. In this context, it is essential to have
a close look at the recent changes in food safety regulations adopted in India which if
effectively implemented will not only protect domestic consumers from food
contamination hazards, but also become instrumental in making India meet
international standards of food safety.
Hence, legal requirements for quality assurance systems and food control along the
entire food chain, from seed and agricultural production, through food processing and
the distribution system, up to the consumers’ table, are increasing considerably. Major
prerequisite for ensuring food quality and safety is that all stakeholders in the food
supply chain recognize that primary responsibility lies with those who produce,
process and trade food and that public control should be based on scientific risk
assessment (Fig. 2.1).

Fig. 2.1 Food supply chain and operators’ responsibility for food quality and
(Source Will and Guenther, 2007)

GAP = Good Agricultural Practices

GDP = Good Distribution Practices
GMP = Good Manufacturing Practices
GHP = Good Hygienic Practices

Operators’ responsibilities cover the whole food supply and marketing chain from
primary production to final consumption and encompass all actors in exporting and
importing countries. Public and private standards are subject to continuous changes as
a result of on-going process of liberalization of the world trade to establish cost-
effective supplier-buyer linkages and to gain a competitive edge. Globalization of the
food supply chain, the increasing importance of the Codex Alimentarius Commission
and the obligations emerging from the World Trade Organization (WTO) agreements
have resulted in unprecedented interest in the development of food standards and
regulations and the strengthening of food control infrastructure at the country level.

2.2 Food Safety, Quality and Consumer Protection

Food safety provides an assurance that food will not cause harm to the consumer
when it is prepared and/or eaten according to its intended use. Safety is a component
of quality. In fact, many experts have argued that safety is the most important
component of quality since a lack of safety can result in serious injury and even death
for the consumer of the product. Safety differs from many other quality attributes
since it is a quality attribute that is difficult to observe. A product can appear to be of
high quality, i.e. well colored, appetizing, flavorful, etc. and yet be unsafe because it
is contaminated with undetected pathogenic organisms, toxic chemicals, or physical
hazards. On the other hand, product that seems to lack many of the visible quality
attributes can be safe. Obvious quality defects can result in consumer rejection and
lower sales, while safety hazards may be hidden and go undetected until the product is
consumed. Since assuring safety is vital to public health, achieving safety must always
take precedence over achieving high levels of other quality attributes. Food safety is
not limited to microbiological safety. As recent history has demonstrated with bovine
spongiform encephalitis (BSE) and Variant Creutzfeldt-Jakob disease (vCJD),
anaphylactic shock from eating peanuts, dioxins entering the human food chain via
animal feedstuffs, benzene in mineral water and glass fragments in baby food, food
safety also includes chemical contamination and foreign bodies. Prions, the cause of
BSE and vCJD, are an entirely new source of food-borne disease. Food-borne viruses
are becoming recognized as significant to public health. As the examples of benzene
and dioxins demonstrate, food safety is not necessarily about real risk to public health,
but also about perceived risk.

2.2.1 New quality and food safety approaches

The aspects of liberalization of the global trade and the fact that the consumers in the
industrialized countries are more and more demanding food to be not only
economical, but also healthy, tasty, safe and sound in respect to animal welfare and
the environment, are changing the so far quantity-oriented food production,
guaranteeing the nutrient supply for a nation, into an international quality-oriented
food market where commodities, production areas, production chains and brands
compete with one another. The competitiveness of food production will soon be more
dependent on the reliability of the safety and the quality of the food and acceptability
of the production procedures than on quantity and price. In contrast to the quantity-
oriented markets that are often subsidized and producers can always sell everything
they produce, quality-oriented markets are market-driven or demand. Thus, apart from
the steady increase of the national and international standards for food safety and
public health, there is a growing influence of the consumer's demands on the
production, its allied industries, advisers, consultants and marketing bodies. All of this
means that the agricultural supply of food production is facing remarkable changes in
the years to come, which is both challenge and opportunity for food producers,
packing plants and processors as well as for the dairy and food profession.

2.2.2 Necessities for new approach

There are five major reasons for this need:

1. Despite the generally recognized achievements in making food safer over the
decades with the mandatory inspection and the principles of food hygiene being
the most successful means in protecting the consumer against food-borne health
risks, there are still deaths due to food-borne disease in man. Furthermore, the
consumer's confidence in the safety of food is getting sceptical;
2. Modern agriculture is contributing to the increase of drug-resistant pathogens in
humans, and, thus often being attacked by the medical society and
consequently by the public;
3. Food safety issues can easily become non-tariff trade barriers and are
increasingly used as marketing tools, nationally and internationally;
4. The consumer has the tendency to ask more and more for fresh and naturally
raised products;
5. The traditional mandatory inspection still is indispensable, but unable to control
and prevent the emerging food-borne pathogens that nowadays pose risks to
human health.

2.3 Quality Control and Quality Assurance Concepts

Quality is defined by the International Organization for Standardization (ISO) as
―the totality of features and characteristics of a product that bear on its ability to
satisfy stated or implied needs/ or the operational techniques and activities that are
used to satisfy quality requirements. A food industry quality management system is an
integrated set of documented food quality and food safety activities with clearly
established inter relationships among the various activities. The objective of a quality
system is to provide a food company with the capability to produce a food that fulfills
all quality and safety requirements (Fig 2.2).

Fig. 2.2 Quality assurance concept in relation to changes in global food safety
2.3.1 Quality control
Quality control is the evaluation of a final product prior to its marketing, i.e. it is
based on quality checks at the end of a production chain aiming at assigning the final
product to quality categories such as "high quality", "regular quality", "low quality"
and "non-marketable". Since, at the end of the production chain, there is no way to
correct production failures or upgrade the quality of the final product, the low-quality
products can only be sold at lower prices and the non-marketable products have to be
discarded. Their production costs, however, had been as high as those of the high and
regular quality products. Thus, quality control has only a limited potential to increase
the quality and efficiency of a multi-step production procedure. Rules of quality control

1. The dominant raw material(s) are selected for priority of attention

2. The selected raw materials are tested in relation to their contribution to product
3. The raw materials tested are released from the stores only after the test results
have been properly recorded.
4. Process control must relate the processing results to the raw materials test.
5. Define the critical points in the process and concentrate on these.
6. Finished product inspection should be reduced to the minimum level
compatible with the confidence justified by the raw materials and process
7. Quality control is effective in proportion to its degree of integration into the
overall organization of the factory.
2.3.2 Quality assurance
The ISO definition reads: "the assembly of all planned and systematic actions
necessary to provide adequate confidence that a product, process, or service will
satisfy given quality requirements." Quality Assurance, in contrast to quality control
(Table 2.1), is the implementation of quality checks and procedures to immediately
correct any failure and mistake that is able to reduce the quality of the interim
products at every production step. Thus, the desired high quality of the final product is
planned and obtained.

2.3.3 Quality control versus quality assurance

Table 2.1 Comparison between quality control and quality assurance


3.1 Introduction

With India being a member of the Codex Alimentarius Committee since 1970, the
Ministry of Health and Family Welfare (FSSAI acting as the National Codex Contact
Point), has the primary responsibility for determination of government policy
relating to food standards and enforcement of food control including national
position on various issues relating to Codex. With the global food industry looking
towards India as a food hot-spot, it is about time the national food legislation is
aligned with Codex, encouraging innovation and facilitating trade without
compromising consumer safety. Whilst formulating and implementing a single
unified standard is a prodigious task, one of the major concerns of the industry which
needs to be addressed by the government while finalizing the Food Safety and
Standards Regulations, 2010 is tuning of international best practices with the
domestic ground realities. Both the domestic and international industry is looking
forward to FSSAI for the harmonization of Indian food standards for all food
categories with the Codex Alimentarius Commission (CAC) standards. CAC is
regarded as the ‘World Authority’ on food standards (Joint FAO/ WHO Food Standard
Programme). Codex’s focused objectives of (1) protecting consumers and (2)
facilitating trade are shared by member countries and its standards based on
scientific evidence and risk analysis principles are followed and/or adopted partially
or in totality by countries around the world. The WTO in its Sanitary and
Phytosanitary (SPS) Agreement recognizes the Codex standards as the global
reference standards for consumers, food producers, processors, national food
control agencies and all others involved in international food trade. The Agreement
on the Application of SPS Measures and the Agreement on Technical Barriers to
Trade (TBT) also encourage the international harmonization of food standards.
Codex standards have thus become the benchmarks against which national food
control measures and regulations are evaluated under the relevant provisions of the
WTO Agreements.

3.4 Codex Alimentarius Commission

The CAC is a body of United Nations (UN) established by FAO in 1961 and is an inter-
governmental organisation that coordinates food standards at the international
level. Its main objectives are to protect the health of consumers and ensure fair
practices in food trade. The Codex Food Code (CFC) attempts to create harmonized
standards. Prior to the SPS Agreement, the CFC could be adopted, applied and/ or
ignored at the discretion of a government. However, the CFC has now been adopted
within the SPS Agreement as the benchmark. The CAC has proved to be most
successful in achieving international harmonization in food quality and safety
requirements. It has formulated international standards for a wide range of food
products and specific requirements covering pesticide residues, food additives,
veterinary drug residues, hygiene, food contaminants, labelling, etc. These codex
recommendations are used by governments to determine and refine policies and
programmes under their national food control system. More recently, Codex has
embarked on a series of activities based on risk assessment to address
microbiological hazards in foods, an area previously unattended. Codex work has
created worldwide awareness of food safety, quality and consumer protection
issues, and has achieved international consensus on how to deal with them
scientifically, through a risk-based approach. As a result, there has been a continuous
appraisal of the principles of food safety and quality at the international level. There
is increasing pressure for the adoption of these principles at the national level.
Quality assurance systems have become a focal point for inclusion in the work of
Codex. As an example, the CAC has recently adopted guidelines for the application of
the HACCP system. The HACCP approach, along with the use of GMPs, is strongly
recognized and recommended by Codex. The principal consideration behind the
development of any Codex standard, guideline or other recommendation is the
protection of consumer’s health.

3.5 HACCP System

HACCP stands for Hazard Analysis Critical Control Point. HACCP is a systematic
approach to the identification, evaluation, and control of food safety hazards. It is a
proactive strategy where hazards are identified and assessed, and control measures
are developed to prevent, reduce, or eliminate a hazard.
The HACCP system, which is science based and systematic, identifies specific hazards
and measures for their control to ensure the safety of food. HACCP is a tool to assess
hazards and establish control systems that focus on prevention rather than relying
mainly on end-product testing. Any HACCP system is capable of accommodating
change, such as advances in equipment design, processing procedures or
technological developments.

HACCP can be applied throughout the food chain from primary production to final
consumption and its implementation should be guided by scientific evidence of risks
to human health. As well as enhancing food safety, implementation of HACCP can
provide other significant benefits. In addition, the application of HACCP systems can
aid inspection by regulatory authorities and promote international trade by
increasing confidence in food safety. The successful application of HACCP requires
the full commitment and involvement of management and the work force. It also
requires a multidisciplinary approach; this multidisciplinary approach should include,
when appropriate, expertise in agronomy, veterinary health, production,
microbiology, medicine, public health, food technology, environmental health,
chemistry and engineering, according to the particular study. The application of
HACCP is compatible with the implementation of quality management systems, such
as the ISO 9000 series, and is the system of choice in the management of food safety
within such systems.

3.6 Management Systems for Quality and Food Safety

Excellence in food quality and safety has taken a tangible form with the advent of ISO
9000 Quality Management System and HACCP standards. ISO 9000 encompasses all
the activities of a company to ensure that it meets its quality objectives, while HACCP
is directed towards ensuring food safety. The ISO 9000 standards were brought by
the International Organization for Standardization (ISO) and the HACCP standards by
the CAC. These standards have assumed importance worldwide both as an essential
requirement to tap the market potential and as a marketable feature of the
company. Since the global market has become more demanding in terms of quality,
safety and timely delivery, installation of the ISO 9000 Quality Management System
and HACCP by the food industry is essential for getting a competitive international
edge. Food Safety Programs may need to be implemented to meet regulatory
requirements, retailer requirements or manufacturer’s requirements.

ISO 9000 Quality Management Systems: The ISO 9000 system is looked at as a
system with minimum quality requirements. It builds a baseline system for managing
quality. The focus, therefore, is on designing a total quality management system, one
that complies with external standards, but includes the specific requirement of
industry and integrates elements of competitiveness.

Biomining is a technique of extracting metals from ores and other solid materials typically
using prokaryotes or fungi. These organisms secrete different organic compounds
that chelate metals from the environment and bring it back to the cell where they are typically used
to coordinate electrons. It was discovered in the mid 1900s that microorganisms use metals in the
cell. Some microbes can use stable metals such as iron, copper, zinc, and gold as well as unstable
atoms such as uranium and thorium. Companies can now grow large chemostats of microbes that
are leaching metals from their media, these vats of culture can then be transformed into many
marketable metal compounds. Biomining is an environmentally friendly technique compared to
typical mining. Mining releases many pollutants while the only chemicals released from biomining is
any metabolites or gasses that the bacteria secrete. The same concept can be used
for bioremediation models. Bacteria can be inoculated into environments contaminated with metals,
oils, or other toxic compounds. The bacteria can clean the environment by absorbing these toxic
compounds to create energy in the cell. Microbes can achieve things at a chemical level that could
never be done by humans. Bacteria can mine for metals, clean oil spills, purify gold, and use
radioactive elements for energy.

The development of industrial mineral processing has been established now in several countries
including South Africa, Brazil and Australia. Iron-and sulfur-oxidizing microorganisms are used to
release occluded copper, gold and uranium from mineral sulfides. Most industrial plants for
biooxidation of gold-bearing concentrates have been operated at 40 °C with mixed cultures
of mesophilic bacteria of the genera Acidithiobacillus or Leptospirillumferrooxidans. In subsequent
studies the dissimulatory iron-
reducing archaea Pyrococcusfuriosus and Pyrobaculumislandicum were shown to reduce gold
chloride to insoluble gold.
Using Bacteria such as Acidithiobacillusferrooxidans to leach copper from mine tailings has
improved recovery rates and reduced operating costs. Moreover, it permits extraction from low grade
ores - an important consideration in the face of the depletion of high grade ores.[8]
Some examples of past projects in biotechnology include a biologically assisted in situ mining
program, biodegradation methods, passive bioremediation of acid rock drainage, and bioleaching of
ores and concentrates. This research often results in technology implementation for greater
efficiency and productivity or novel solutions to complex problems. Additional capabilities include the
bioleaching of metals from sulfide materials, phosphate ore bioprocessing, and the bioconcentration
of metals from solutions. One project recently under investigation is the use of biological methods for
the reduction of sulfur in coal-cleaning applications. From in situ mining to mineral processing and
treatment technology, biotechnology provides innovative and cost-effective industry solutions.
The potential of thermophilic sulfide-oxidizing archaea in copper extraction has attracted interest due
to the efficient extraction of metals from sulfide ores that are recalcitrant to dissolution.
Microbial leaching is especially useful for copper ores because copper sulfate, as formed during the
oxidation of copper sulfide ores, is very water-soluble. Approximately 25% of all copper mined
worldwide is now obtained from leaching processes.
The acidophilic archaea Sulfolobusmetallicus and Metallosphaerasedula tolerate up to 4% of copper
and have been exploited for mineral biomining. Between 40 and 60% copper extraction was
achieved in primary reactors and more than 90% extraction in secondary reactors with overall
residence times of about 6 days.
The oxidation of the ferrous ion (Fe2+) to the ferric ion (Fe3+) is an energy producing reaction for some
microorganisms. As only a small amount of energy is obtained, large amounts of (Fe2+) have to be
oxidized. Furthermore, (Fe3+) forms the insoluble Fe(OH)
3 precipitate in H2O. Many Fe oxidizing microorganisms also oxidize sulfur and are thus obligate

acidophiles that further acidify the environment by the production of H2SO4. This is due in part to the
fact that at neutral pH Fe2+ is rapidly oxidized chemically in contact with the air. In these conditions
there is not enough Fe2+ to allow significant growth. At low pH, however, Fe2+ is much more stable.
This explains why most of the Fe2+ oxidizing microorganisms are only found in acidic environments
and are obligate acidophiles.
The best studied Fe2+ oxidizing bacterium is Acidithiobacillusferrooxidans, an acidophililic
chemolithotroph. The microbiological oxidation of Fe2+ is an important aspect of the development of
acidic pH’s in mines, and constitutes a serious ecological problem. However, this process can also
be usefully exploited when controlled. The sulfur containing ore pyrite (FeS2) is at the start of this
process. Pyrite is an insoluble crystalline structure that is abundant in coal- and mineral ores. It is
produced by the following reaction:
S + FeS → FeS2
Normally pyrite is shielded from contact with oxygen and not accessible for microorganisms.
Upon exploitation of the mine, however, pyrite is brought into contact with air (oxygen) and
microorganisms and oxidation will start. This oxidation relies on a combination of chemically and
microbiologically catalyzed processes. Two electron acceptors can influence this process:
O2 and Fe3+ ions. The latter will only be present in significant amounts in acidic conditions (pH <
2.5). First a slow chemical process with O2 as electron acceptor will initiate the oxidation of
FeS2 + 7/2 O2 + H2O → Fe2+ + 2 SO42− + 2 H+
This reaction acidifies the environment and the Fe2+ will be formed is rather stable. In such
an environment Acidithiobacillusferrooxidans will be able to grow rapidly. Upon further
acidification Ferroplasma will also develop and further acidify. As a consequence of the
microbial activity (energy producing reaction):
Fe2+ → Fe3+
This Fe3+ that remains soluble at low pH reacts spontaneously with the pyrite:
FeS2 + 14 Fe3+ + 8 H2O → 15 Fe2+ + 2 SO42− + 16 H+
The produced Fe2+ can again be used by the microorganisms and thus a cascade reaction
will be initiated

Processing methods
In the industrial microbial leaching process popularly known as bioleaching, low grade ore is dumped
in a large pile (the leach dump) and a dilute sulfuric acid solution (pH 2) is percolated down through
the pile.[8] The liquid coming out at the bottom of the pile, rich in the mineral is collected and
transported to a precipitation plant where the metal is reprecipitated and purified. The liquid is then
pumped back to the top of the pile and the cycle is repeated.
Acidithiobacillusferrooxidans is able to oxidize the Fe2+ in to Fe3+.
Chemical oxidation of the copper ore with ferric (Fe3+) ions formed by the microbial oxidation of
ferrous ions (derived from the oxidation of pyrite). Three possible reactions for the oxidation of
copper ore are:
Cu2S + 1/2 O2 + 2 H+ → CuS + Cu2+ + H2O
CuS + 2 O2 → Cu2+ + SO42−
CuS + 8 Fe3+ + 4 H2O → Cu2+ + 8 Fe2+ + SO42− + 8 H+
The copper metal is then recovered by using Fe0 from steel cans:
Fe0 + Cu2+ → Cu0 + Fe2+
The temperature inside the leach dump often rises spontaneously as a result of
microbial activities. Thus, thermophilic iron-oxidizing chemolithotrophs such as
thermophilic Acidithiobacillus species and Leptospirillum and at even higher
temperatures the thermoacidophilic
archaeon Sulfolobus (Metallosphaerasedula) may become important in the leaching
process above 40 °C. Similarly to copper, Acidithiobacillusferrooxidans can oxidize
U4+ to U6+ with O2 as electron acceptor. However, it is likely that the uranium
leaching process depends more on the chemical oxidation of uranium by Fe3+,
with At. ferrooxidans contributing mainly through the reoxidation of Fe2+ to Fe3+ as
described above.
UO2 + Fe(SO4)3 → UO2SO4 + 2 FeSO4
Gold is frequently found in nature associated with minerals
containing arsenic and pyrite. In the microbial leaching process At.
ferrooxidans and relatives are able to attack and solubilize the arsenopyrite
minerals, and in the process, releasing the trapped gold (Au):
2 FeAsS[Au] + 7 O2 + 2 H2O + H2SO4 → Fe(SO4)3 + 2 H3AsO4 + [Au]
Biohydrometallurgy is an emerging trend in biomining in which commercial
mining plants operate continuously stirred tank reactor (STR) and the airlift
reactor (ALR) or pneumatic reactor (PR) of the Pachuca type to extract the
low concentration mineral resources efficiently.[8]
The development of industrial mineral processing using microorganisms
has been established now in several countries including South Africa, Brazil
and Australia. Iron-and sulfur-oxidizing microorganisms are used to release
copper, gold and uranium from minerals. Electrons are pulled off ofsulfur
metal through oxidation and then put onto iron, producing reducing
equivalents in the cell in the process. This is shown in this figure.[5] These
reducing equivalents then go on to produce adenosine triphosphate in the
cel through the electron transport chain. Most industrial plants for
biooxidation of gold-bearing concentrates have been operated at 40 °C with
mixed cultures of mesophilic bacteria of the
genera Acidithiobacillus or Leptospirillumferrooxidans.[6] In other studies the
iron-reducing archaea Pyrococcusfuriosus were shown to produce
hydrogen gas which can then be used as fuel.[7] Using Bacteria such as
Acidithiobacillusferrooxidans to leach copper from mine tailings has
improved recovery rates and reduced operating costs. Moreover, it permits
extraction from low grade ores - an important consideration in the face of
the depletion of high grade ores.
The acidophilic
archaea Sulfolobus metallicus and Metallosphaerasedula can tolerate up to
4% of copper and have been exploited for mineral biomining. Between 40
and 60% copper extraction was achieved in primary reactors and more than
90% extraction in secondary reactors with overall residence times of about
6 days. All of these microbes are gaining energy by oxidizing these metals.
Oxidation means increasing the number of bonds between an atom to
oxygen. Microbes will oxidize sulfur. The resulting electrons will reduce iron,
releasing energy that can be used by the cell.

Bioremediation is the process of using microbial systems to restore the environment to a healthy
state. Certain microorganisms can survive in metal rich environments where they can then leach
metallic cations for use in the cell. These microbes can be used to remove metals from the soil or
water. These metal extractions can be performed in situ or ex situ where in situ is preferred since it
is less expensive to excavate the substrate.[9]
Bioremediation is not specific to metals. In 2010 there was a massive oil spill in the Gulf of Mexico.
Populations of bacteria and archaea were used to rejuvenate the coast after the oil spill. These
microorganisms over time have developed metabolic networks that can utilize hydrocarbons such as
oil and petroleum as a source of carbon and energy.[10] Microbial bioremediation is a very effective
modern technique for restoring natural systems by removing toxins from the environment.
Bioremediation is a process used to treat contaminated media, including water, soil and subsurface
material, by altering environmental conditions to stimulate growth of microorganisms and degrade
the target pollutants. In many cases, bioremediation is less expensive and more sustainable than
other remediation alternatives.[1] Biological treatment is a similar approach used to treat wastes
including wastewater, industrial waste and solid waste.
Most bioremediation processes involve oxidation-reduction reactions where either an electron
acceptor (commonly oxygen) is added to stimulate oxidation of a reduced pollutant (e.g.
hydrocarbons) or an electron donor (commonly an organic substrate) is added to reduce oxidized
pollutants (nitrate, perchlorate, oxidized metals, chlorinated solvents, explosives and
propellants).[2] In both these approaches, additional nutrients, vitamins, minerals, and pH buffers may
be added to optimize conditions for the microorganisms. In some cases, specialized microbial
cultures are added (bioaugmentation) to further enhance biodegradation. Some examples of
bioremediation related technologies
are phytoremediation, mycoremediation, bioventing, bioleaching, landfarming, bioreactor, compostin
g, bioaugmentation, rhizofiltration, and biostimulation.
Most bioremediation processes involve oxidation-reduction (Redox) reactions where a chemical
species donates an electron (electron donor) to a different species that accepts the electron
(electron acceptor). During this process, the electron donor is said to be oxidized while the electron
acceptor is reduced. Common electron acceptors in bioremediation processes
include oxygen, nitrate, manganese (III and IV), iron (III), sulfate, carbon dioxide and some pollutants
(chlorinated solvents, explosives, oxidized metals, and radionuclides). Electron donors include
sugars, fats, alcohols, natural organic material, fuel hydrocarbons and a variety of reduced organic
pollutants. The redox potential for common biotransformation reactions is shown in the table.

Process Reaction Redox potential (Eh in mV)

aerobic O2 + 4e− + 4H+ → 2H2O 600 ~ 400

denitrification 2NO3− + 10e− + 12H+ → N2 + 6H2O 500 ~ 200

manganese IV reduction MnO2 + 2e− + 4H+ → Mn2+ + 2H2O 400 ~ 200

iron III reduction Fe(OH)3 + e− + 3H+ → Fe2+ + 3H2O 300 ~ 100

sulfate reduction SO42− + 8e− +10 H+ → H2S + 4H2O 0 ~ −150

fermentation 2CH2O → CO2 + CH4 −150 ~ −220

The use of genetic engineering to create organisms specifically designed for bioremediation is under
preliminary research.[23] Two category of genes can be inserted in the organism: degradative genes
which encode proteins required for the degradation of pollutants, and reporter genes that are able to
monitor pollution levels.[24] Numerous members of Pseudomonashave also been modified with the lux
gene, but for the detection of the polyaromatic hydrocarbon naphthalene. A field test for the release
of the modified organism has been successful on a moderately large scale.[25]
There are concerns surrounding release and containment of genetically modified organisms into the
environment due to the potential of horizontal gene transfer.[26] Genetically modified organisms are
classified and controlled under the Toxic Substances Control Act of 1976 under United States
Environmental Protection Agency.[27] Measures have been created to address these concerns.
Organisms can be modified such that they can only survive and grow under specific sets of
environmental conditions.[26] In addition, the tracking of modified organisms can be made easier with
the insertion of bioluminescence genes for visual identification.[28]
This article throws light upon the three types of
recombinant vaccines. The three types are: (1) Subunit
Vaccines (2) Attenuated Recombinant Vaccines and (3)
Vector Recombinant Vaccines.

Recombinant Vaccines—General:
Recombinant DNA technology in recent years has become a boon to
produce new generation vaccines. By this approach, some of the
limitations (listed above) of traditional vaccine production could be
overcome. In addition, several new strategies, involving gene
manipulation are being tried to create novel recombinant vaccines.

Types of Recombinant Vaccines:

The recombinant vaccines may be broadly categorized into
three groups:


1. Subunit recombinant vaccines:

These are the components of the pathogenic organisms. Subunit

vaccines include proteins, peptides and DNA.

2. Attenuated recombinant vaccines:

These are the genetically modified pathogenic organisms (bacteria or

viruses) that are made non-pathogenic and used as vaccines.


3. Vector recombinant vaccines:

These are the genetically modified viral vectors that can be used as
vaccines against certain pathogens. Some of the developments made in
the production of recombinant vaccines against certain diseases are
briefly described.
Type # 1. Subunit Vaccines:
As already stated, subunit recombinant vaccines are the components
(proteins, peptides, DNAs) of the pathogenic organisms. The
advantages of these vaccines include their purity in preparation,
stability and safe use. The disadvantages are — high cost factor and
possible alteration in native conformation. Scientists carefully evaluate
the pros and cons of subunit vaccines for each disease, and proceed on
the considered merits.

Hepatitis B:

Hepatitis B is a widespread disease in man. It primarily affects liver

causing chronic hepatitis, cirrhosis and liver cancer. Hepatitis B virus
is a 42 nm particle, called Dane particle. It consists of a core
containing a viral genome (DNA) surrounded by a phospholipid
envelope carrying surface antigens (Fig. 16.1 A).

Infection with hepatitis B virus produced Dane particles and 22 nm

sized particles. The latter contain surface antigens which are more
immunogenic. It is however, very difficult to grow hepatitis B virus in
mammalian cell culture and produce surface antigens.

The gene encoding for hepatitis B surface antigen (HBsAg) has been
identified. Recombinant hepatitis B vaccine as a subunit vaccine, is
produced by cloning HbsAg gene in yeast cells. Saccharomyces
cerevisiae, a harmless baking and brewing yeast, is used for this
purpose (Fig. 16.1B). The gene for HBsAg is inserted (pMA 56) which
is linked to the alcohol dehydrogenase promoter. These plasmids are
then transferred 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
2nm 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.
Hepatitis B vaccine-the first synthetic vaccine:

In 1987, the recombinant vaccine for hepatitis B (i.e. HBsAg) became

the first synthetic vaccine for public use. It was marketed by trade
names Recombivax and Engerix-B. Hepatitis B vaccine is safe to use,
very effective and produces no allergic reactions. For these reasons,
this recombinant vaccine has been in use since 1987.


The individuals must be administered three doses over a period of six

months. Immunization against hepatitis B is strongly recommended to
anyone coming in contact with blood or body secretions. All the health
professionals—physicians, surgeons, medical laboratory technicians,
nurses, dentists, besides police officers, firefighters etc., must get
vaccinated against hepatitis B.

Hepatitis B vaccine in India:

India is the fourth country (after USA, France and Belgium) in the
world to develop an indigenous hepatitis B vaccine. It was launched in
1997, and is now being used.

Hepatitis B vaccine tomato?


Biotechnologists have been successful in inserting hepatitis B gene

into the cells of the tomato plant. These genetically engineered plants
produce hepatitis B antigens. The day may not be far off to get
immunized against hepatitis B by having a tomato with lunch!

Foot and Mouth Disease:

Foot and mouth disease (FMD) is a highly contagious disease affecting

cattle and pigs. A formalin killed foot and mouth disease virus
(FMDV) was previously used to vaccinate against this disease. The
genome of FMDV is composed of a single— stranded RNA, covered by
four viral proteins (VP1, VP2, VP3 and VP4). Among these, VPI is
immunogenic. The nucleotide sequence encoding VPI was identified in
the FMDV genome. A double- stranded complementary DNA (cDNA)
from the single-stranded viral RNA (genome) was synthesized.

This cDNA was then digested with restriction enzymes and the
fragments were cloned by using plasmid pBR322 in E. coli. The
recombinant vaccine for FMDV in the form of viral protein 1 was used
to vaccinate animals. However, VPI vaccination was found to be less
effective than that of the whole virus in protecting FMD. Further,
studies are being pursued to improve the efficiency of subunit vaccine.

The concept of peptide vaccines:


Theoretically, it is expected that only small portions of a given protein

(i.e., domains) are immunogenic and bind to antibodies. Logically, it is
possible to use short peptides that are immunogenic as vaccines.
These are referred to as peptide vaccines.

Peptide vaccines for foot and mouth disease:

Some details on the FMD are described above. The domains of viral
protein I (VPI) of FMDV were chemically synthesized. From the C-
terminal end of VPI, amino acids 141 to 160, 151 to 160 and 200 to 213,
and from N-terminal end, amino acids 9 to 24, 17 to 32 and 25 to 41
were synthesized.


Each one of these short peptides (domains) was bound to the surface
of a carrier protein (Fig. 16.2) and used as a vaccine. Among the
peptides used, the one corresponding to amino acids 141 to 160 was
found to be effective in immunizing guinea pigs against FMD. In
addition, when two peptides were joined together (amino acids 141 to
158, and amino acids 200 to 213), they served as more efficient
recombinant vaccines.

The success so far to use recombinant peptides as vaccines has been

very limited. This is mainly because a short peptide usually is not
enough to be sufficiently immunogenic, since it may not have the same
conformation as that of the original viral particle. However, scientists
continue their search for specific, inexpensive and safe synthetic
peptide vaccines for various diseases.

Herpes Simplex Virus:

Herpes simplex virus (HSV) is an oncogenic (cancer-causing) virus. In

addition, it also causes sexually transmitted diseases, encephalitis and
severe eye infections. Attempts have been made to produce subunit
vaccines against HSV. An envelope glycoprotein D (gD) of HSV that
can elicit antibody production has been identified. This is a membrane
bound protein, and difficult to isolate and purify. The glycoprotein D
was modified by deleting the trans membrane portion of the protein
(Fig. 16.3) and the gene was modified.

This gene for gD was cloned in a mammalian vector and expressed in

Chinese hamster ovary (CHO). The advantage here is that the protein
can get glycosylated (unlike in E. coli system). In the experimental
trials, the modified form of gD was found to be effective against HSV.

Tuberculosis is caused by the bacterium Mycobacterium tuberculosis.
It is often fatal, and as per some estimates nearly 3 million deaths
occur every year due to this highly infectious disease. Antibiotics are
used to treat tuberculosis. However, drug-resistant M. tuberculosis
strains have been developed making the drug therapy sometimes
ineffective. Vaccination for tuberculosis is therefore, advocated.

Bacillus Calmette-Guerin (BCG) vaccine:

In some countries, particularly the developing ones, BCG vaccine is

widely used to protect against tuberculosis. However, countries like
United States have not approved BCG vaccination for various reasons.
BCG vaccine itself causes tuberculosis in some individuals (AIDS
victims) and the vaccinated people respond positively for laboratory
diagnosis of tuberculosis.

Subunit vaccines:

The secretory (extracellular proteins of M. tuberculosis have been

purified and used for immuno-protection against tuberculosis. Of
about 100 such proteins, six were found to be useful (either
individually or in combination) to immunize guinea pigs. Attempts are
underway to develop recombinant subunit vaccine against


Group B strain of meningococci, namely Neisseria meningitidis causes

meningitis in adolescents and young adults. Meningitis is
characterized by inflammation of the membranes covering brain and
spinal cord. The symptoms include headache, photophobia,
irritability, and neck stiffness.

Pizza et al (2000) made a novel approach to develop a vaccine against

meningitis. They identified 350 proteins (potential protective
antigens) and the entire sequence of genome coding for these proteins
in N. meningitidis. All the 350 candidate antigens were expressed in E.
coli, purified and used to immunize mice. A good bactericidal antibody
response was observed in these mice.

AIDS (Acquired Immunodeficiency Syndrome):

AIDS is a retroviral disease caused by human immunodeficiency virus

(HIV). This disease is characterized by immunosuppression, neo-
plasma and neurological manifestations. AIDS is invariably fatal, since
as of now there is no cure. Development of a vaccine against AIDS is a
top priority by DNA technologists world over. In fact, vaccines are
being continuously developed and field tested, although there has
been no success so far.

Subunit vaccines:

The development of two subunit vaccines, specifically the

glycoproteins of HIV envelope is described here. The functions of
gp120 and gp41 of HIV are illustrated in Fig. 16.4A. The glycoprotein
gp120 projects out of the HIV envelope while the other glycoprotein
gp41 lies beneath gp120.

On entering the body, the HIV binds to the host cells (T-lymphocytes)
by attaching gp120 to the CD4 receptor sites on the cell surface. This
attachment uncovers gp41 molecules and the viral envelope. Now gp41
binds to the host cell surface and opens a passage for the entry of the
virus into the cell.

Biotechnologists have isolated the genes for gp120 and gp41 and
inserted them into the bacterium E. coli. These bacterial cells produce
gp120 and gp41 that can be used as recombinant vaccines against
AIDS. The action of gp120 and gp41 in immunizing host T-
lymphocytes is depicted in Fig. 16.4B.

The gp120 molecules stimulate the host immune system to produce

anti- gp120 antibodies. These antibodies bind to gp120 and prevent its
attachment to CD4. In a comparable manner, gp41 molecules also
result in the production of anti-gp41 antibodies. These antibodies also
bind to gp41 and block the virus- host cell union. The net result of
using gp120 and gp41 vaccines is that the entry of HIV into the host
cells is prevented.

DNA Vaccines (Genetic Immunization):

Genetic immunization by using DNA vaccines is a novel approach that

came into being in 1990. The immune response of the body is
stimulated by a DNA molecule. A DNA vaccine consists of a gene
encoding an antigenic protein, inserted onto a plasmid, and then
incorporated into the cells in a target animal.

The plasmid carrying DNA vaccine normally contains a promoter site,

cloning site for the DNA vaccine gene, origin of replication, a
selectable marker sequence (e.g. a gene for ampicillin resistance) and a
terminator sequence (a poly—A tail).

DNA vaccine—plasmids can be administered to the animals by one of

the following delivery methods.

i. Nasal spray

ii. Intramuscular injection

iii. Intravenous injection

iv. Intradermal injection

v. Gene gun or biolistic delivery (involves pressure delivery of DNA-

coated gold beads).

DNA Vaccine and Immunity:

An illustration of a DNA vaccine and the mechanism of its action in

developing immunity is given in Fig. 16.5. The plasmid vaccine
carrying the DNA (gene) for antigenic protein enters the nucleus of the
inoculated target cell of the host. This DNA produces RNA, and in turn
the specific antigenic protein. The antigen can act directly for
developing humoral immunity or as fragments in association with
major histocompatibility class (MHC) molecules for developing
cellular immunity.

Humoral immunity:

As the antigen molecules bind to B- lymphocytes, they trigger the

production of antibodies which can destroy the pathogens. Some of
the B-lymphocytes become memory cells that can protect the host
against future infections.

Cellular immunity:

The protein fragments of the antigen bound to MHC molecules can

activate the cytotoxic T-lymphocytes. They are capable of destroying
the infected pathogenic cells. Some of the activated T-lymphocytes
become memory cells which can kill the future infecting pathogens.
Complementary DNA vaccines:

For genetic immunization, complementary DNA (cDNA) vaccines can

also be used. Some workers have successfully used cDNA as vaccines
e.g. immunization of mice against influenza.

DNA vaccines for production of antigens and antibodies:

A novel approach for the production of antigens as well as antibodies

by DNA vaccine was developed in 1997. In the experiments conducted
in mice, researchers injected plasmids containing the genes for
malarial parasite and also the genes for the antibodies against malarial
parasite. The B-lymphocytes 4 of these mice performed a double duty,
and produced antigens for and antibodies against malarial parasite.

The antigens stimulate to produce more and more antibodies. The

antibodies so produced react with malarial parasite. The generation of
antigens and antibodies by using a DNA vaccine is a recent
development in immunology, and is referred to as antigenic antibody
approach of DNA vaccine.

Screening of pathogenic genome for selecting DNA vaccines:

The ultimate goal of scientists is to choose the right DNA fragment

from the pathogen to serve as a vaccine for the strongest immune
response against the invading pathogen. For this purpose, the
pathogen’s DNA can be broken into fragments and a large number of
vaccines DNA—plasmids can be prepared.

The immune response for each one of the DNA vaccines can be studied
by injecting the pathogen. By screening the DNA fragments of the
pathogenic genome, it is possible to choose one or few DNA vaccines
that can offer maximal immune protection.

Advantages of DNA vaccines:

There are several advantages of using DNA vaccines in

1. The tedious and costly procedures of purifying antigens or creating
recombinant vaccines are not necessary.

2. DNA vaccines are very specific in producing the target proteins

(antigens or antibodies). Thus, they trigger immune response only
against the specific pathogen.

3. In general, DNA vaccines elicit much higher immune response

compared to other kinds of vaccines.

4. DNA vaccines are more stable for temperature variations (low or

high) than the conventional vaccines. Thus, the storage and transport
problems associated with vaccines are minimal.
5. The delivery methods to the host are simpler for DNA vaccines.

Disadvantages of DNA vaccines:

1. The fate of the DNA vaccine in the host cells is not yet clear. There is
a possibility of this DNA getting integrated into the host genome and
this may interrupt the normal functions.

2. There also exists a danger of cancer due to DNA vaccines.

3. The post-translational modification of the gene (DNA vaccine)

product in host cells may not be the same as that found in the native

Present status of DNA vaccines:

Since 1990, several groups of workers world-over have been trying to

develop DNA vaccines against various diseases in experimental
animals. Genetic immunization has been done against a number of
pathogenic organisms. These include influenza A virus, rabies virus,
hepatitis B virus, bovine herpes virus, HIV type I, and Plasmodium
species (malarial parasite). It must be noted that DNA vaccines have
not been tried in humans for obvious reasons. The most important
being the unknown risks of these foreign DNAs in human subjects.
RNA Vaccines:

Several workers are trying to use RNA molecules as vaccines. These

RNAs can readily synthesize the antigenic proteins and offer
immunity. But unfortunately, RNAs are less stable than DNAs. This
poses a big problem for RNA vaccine manufacture and distribution.
Therefore, the progress in the development of RNA vaccines has been
rather slow compared to DNA vaccines.

Plants as Edible Subunit Vaccines:

Plants serve as a cheap and safe production systems for subunit

vaccines. The edible vaccines can be easily ingested by eating plants.
This eliminates the processing and purification procedures that are
otherwise needed. Transgenic plants (tomato, potato) have been
developed for expressing antigens derived from animal viruses (rabies
virus, herpes virus). A selected list of recombinant vaccines against
animal viruses produced in plants is given in Table 16.2.

Edible vaccine production and use:

The production of vaccine potatoes is illustrated in Fig. 16.6. The

bacterium, Agrobacterium tumefaciens is commonly used to deliver
the DNA (genetic material) for bacterial or viral antigens. A plasmid
carrying the antigen gene and an antibiotic resistance gene are
incorporated into the bacterial cells (A. tumefaciens)

The cut pieces of potato leaves are exposed to an antibiotic which can
kill the cells that lack the new genes. The surviving cells (i.e., gene
altered ones) can multiply and form a callus (clump of cells). This
callus is allowed to sprout shoots and roots, which are grown in soil to
form plants.

In about three weeks, the plants bear potatoes with antigen vaccines.
The first clinical trials in humans, using a plant- derived vaccine were
conducted in 1997. This involved the ingestion of transgenic potatoes
with a toxin of E. coli causing diarrhea.
Type # 2. Attenuated Recombinant Vaccines:
In the early years of vaccine research, attenuated strains of some
pathogenic organisms were prepared by prolonged cultivation —
weeks, months or even years. Although the reasons are not known, the
infectious organism would lose its ability to cause disease but retains
its capability to act as an immunizing agent. This type of approach is
almost outdated now.

It is now possible to genetically engineer the organisms (bacteria or

viruses) and use them as live vaccines, and such vaccines are referred
to as attenuated recombinant vaccines. The genetic manipulations for
the production of these vaccines are broadly of two types:

1. Deletion or modification of virulence genes of pathogenic


2. Genetic manipulation of non-pathogenic organisms to carry and

express antigen determinants from pathogenic organisms.

The advantage with attenuated vaccines is that the native

conformation of the immunogenic determinants is preserved; hence
the immune response is substantially high. This is in contrast to
purified antigens which often elicit poor immunological response.

Some of the important attenuated vaccines developed by genetic

manipulations are briefly described.


Cholera is an intestinal disease characterized by diarrhea,

dehydration, abdominal pain and fever. It is caused by the bacterium,
Vibrio cholera. This pathogenic organism is transmitted by drinking
water contaminated with fecal matter. Cholera epidemics are
frequently seen in developing countries where the water purification
and sewage disposal systems are not well developed.
On entering the small intestine, V. cholera colonizes and starts
producing large amounts of a toxic protein, a hexameric enterotoxin.
This enterotoxin stimulates the cells lining intestinal walls to release
sodium, bicarbonate and other ions. Water accompanies these ions
leading to severe diarrhea, dehydration, and even death.

The currently used cholera vaccine is composed of phenol-killed V.

cholera. The immuno-protection, lasting for 3-6 months is just
moderate. Attempts are being made to develop better vaccines. The
DNA technologists have identified the gene encoding enterotoxin
(toxic protein). Enterotoxin, an hexamer, consists of one A subunit
and five identical B subunits. The A subunit has two functional
domains-the A1 peptide which possesses the toxic activity and
A2 peptide that joins A subunit to B subunits.

By genetic engineering, it was possible to delete the DNA sequence

encoding A1peptide and create a new strain of V. cholera. This strain is
non-pathogenic, since it cannot produce enterotoxin. The genetically
engineered V. cholera is a good candidate to serve as an attenuated

Creating a new strain of V. cholera:

The development of a new strain of Vibrio cholera that can effectively

serve as an attenuated recombinant vaccine is depicted in Fig. 16.7,
and briefly described below

1. A tetracycline resistance gene was inserted into the A1 peptide

sequence of V. cholera chromosome. This destroys the DNA sequence
encoding for A1 peptide, besides making the strain resistant to
tetracycline. Unfortunately, the tetracycline resistant gene is easily lost
and the enterotoxin activity is restored. Because of this, the new strain
of V. cholera as such cannot be used as a vaccine.
2. The DNA sequence of A1 peptide is incorporated into a plasmid,
cloned and digested with restriction enzymes (Clal and Xbal). In this
manner, the A1 peptide coding sequence is deleted (the DNA encoding
for 183 of the 194 amino acids of the A1 peptide is actually removed).
By using T4 DNA ligase, the plasmid is re-circularized. This plasmid
contains a small portion of A1 peptide coding sequence.

3. The plasmid, containing the deleted A1 peptide sequence is

transferred by conjugation into the V. cholera strain carrying a
tetracycline resistance gene.

4. Recombination can occur between the plasmid (containing a small

portion of peptide A1 coding sequence) and the chromosome of V.
cholera (carrying tetracycline resistance gene). The result of this
double crossover is the formation of V. cholera containing a
chromosomal DNA lacking A1 peptide DNA sequence. As the
bacterium, V. cholera multiplies, the plasmids are lost in the next few

5. The V. cholera cells defective in A1 peptide are selected, based on

tetracycline sensitivity. It may be noted that this new strain lacks
tetracycline resistance gene.

The genetically engineered V. cholera cells with deleted A1 peptide

DNA sequence are quite stable. They cannot produce active
enterotoxin but possess all other biochemical functions of the
pathogen. This new strain of V. cholera is undergoing trials for its
efficiency as a vaccine. Preliminary results indicate that this
attenuated vaccine can protect about 90% of the volunteers against
cholera. But there are some side effects. Scientists continue their work
to develop a better vaccine against cholera.

Potato as a vehicle for cholera vaccine:

A group workers have developed a gene altered potato containing

attenuated cholera vaccine. These potatoes when fed to mice induced
immunity against cholera.
Salmonella Species:

The different strains of Salmonella genus are responsible for causing

typhoid, enteric fever, food poisoning and infant death.
Immunoprotection against Salmonella pathogens is really required.
Some workers have been successful in deleting aro genes and pur
genes in Salmonella.

Aro genes encode for the enzymes responsible for the biosynthesis of
aromatic compounds, while pur genes encode for enzymes of purine
metabolism. The new strains of Salmonella can be grown in vitro on a
complete medium.

The doubly deleted strains have a very restricted growth in vivo, while
they can stimulate immunological response. The genetically
engineered attenuated vaccines of Salmonella have been shown to be
effective as oral vaccines in experimental animals (mice, cattle, sheep,
and chickens). Some workers claim that the new strain of Salmonella
offers immunoprotection in humans also.

Leishmania Species:

Leishmania species are flagellated protozoan parasites and are

responsible for the disease leishmaniasis. This disease is characterized
by cutaneous, visceral and mucosal leisons. Leishmaniasis is
transmitted by sand flies.

An attenuated strain of leishmania has been created and successfully

used in mice to offer immunoprotection against leishmaniasis. In
Leishmania major, the genes encoding dihydrofolate reductase-
thymidylate synthase can be replaced by the genes encoding resistance
to antibiotics G-418 and hygromycin.

This new strain of L. major invariably requires thymidine in the

medium for its growth and multiplication. The attenuated strain of L.
major can survive only a few days when administered to mice. This
short period is enough to induce immunity in mice against the lesions
of leishmania. However, more experiments on animals have to be
carried out before the leishmania attenuated vaccine goes for human

Type # 3. Vector Recombinant Vaccines:

Some of vectors can be genetically modified and employed as vaccines
against pathogens.

Vaccines against Viruses- Vaccinia Virus:

Vaccinia viruses is basically the vaccine that was originally used by

Jenner for the eradication of smallpox. The molecular biology of this
virus has been clearly worked out. Vaccinia virus contains a double-
stranded DNA (187 kb) that encodes about 200 different proteins. The
genome of this virus can accommodate stretches of foreign DNA which
can be expressed along with the viral genes.

The vaccinia virus can replicate in the host cell cytoplasm (of the
infected cells) rather than the nucleus. This is possible since the
vaccinia virus possesses the machinery for DNA replication,
transcription-DNA polymerase, RNA polymerase etc. The foreign
genes inserted into the vaccinia virus can also be expressed along with
the viral genome. Thus, the foreign DNA is under the control of the
virus, and is expressed independently from the host cell genome.

The vaccinia viruses are generally harmless, relatively easy to cultivate

and stable for years after lyophilization (freeze-drying). All these
features make the vaccinia virus strong candidates for vector vaccine.
The cloned foreign genes (from a pathogenic organism) can be
inserted into vaccinia virus genome for encoding antigens which in
turn produces antibodies against the specific disease- causing agent.

The advantage with vector vaccine is that it stimulates B-lymphocytes

(to produce antibodies) and T-lymphocytes (to kill virus infected
cells). This is in contrast to a subunit vaccine which can stimulate only
B-lymphocytes. Thus, vaccinia virus can provide a high level of
immunoprotection against pathogenic organisms. Another advantage
of vaccinia virus is the possibility of vaccinating individuals against
different diseases simultaneously. This can be done by a recombinant
vaccinia viruses which carries genes encoding different antigens.

Antigen genes for certain diseases have been successfully incorporated

into vaccinia virus genome and expressed. Thus, vector vaccines have
been developed against hepatitis, influenza, herpes simplex virus,
rabies, angular stomatitis virus and malaria. However, none of these
vaccines has been licensed for human use due to fear of safety. It is
argued that recombinant vaccinia virus might create life threatening
complications in humans.

Production of recombinant vaccinia viruses:

The development of recombinant vaccinia virus is carried out by a

two-step procedure (Fig. 16.8).1. Assembly of plasmid insertion


Fresh vaccinia (cow pox) viruses are processed to release their DNAs.
Now genes from hepatitis B virus, herpes simplex virus and influenza
virus are added one after another and inserted into vaccinia virus
genome. These DNA clusters are cloned in E. coli for increasing their
number and to produce plasmid insertion vectors. The plasmid
contains the foreign subunit genes, the natural vaccinia genes,
including the promoter genes. The recombinant plasmids are isolated
and purified and serve as plasmid insertion vectors.

2. Production of recombinant vaccinia viruses:

The animal cells are infected with plasmid insertion vectors and
normal vaccinia viruses. As the viral replication occurs, the plasmids
are taken up to produce recombinant vaccinia viruses. The plasmid
insertion vector incorporates its genes into vaccinia virus genome at a
place that encodes for the enzyme thymidine kinase (TK).
Thus the recombinant viruses have lost their ability to produce TK.
There are two advantages of loss of TK gene. One is that it is easy to
select recombined vaccinia viruses that lack TK gene and the second is
that these viruses are less infectious than the normal viruses. The
recombinant vaccinia viruses, released from the cultured animal cells,
can be successfully used as vaccines. These live viral vaccines have
some advantages over the killed or subunit vaccines.


1. Authenticated antigens that closely resemble natural antigens can be


2. The virus can replicate in the host cells. This enables the
amplification of the antigens for their action on B-lymphocytes and T-

3. There is a possibility of vaccinating several diseases with one

recombinant vaccinia virus.


1. The most important limitation is the yet unknown risks of using

these vaccines in humans.

2. There may be serious complications of using recombinant viral

vaccines in immunosuppressed individuals such as AIDS patients.

Other viral recombinant vaccines:

Most of the work on the development of live viral vaccines has been
carried out on vaccinia virus. Other viruses such as adenovirus,
poliovirus and varicella-zoster virus are also being tried as
recombinant vaccines. Scientists are attracted to develop a
recombinant poliovirus as it can be orally administered. It might take
many more years for the recombinant viral vaccines to become a
reality for human use.
Delivery of Antigens by Bacteria:

It is known that the antigens located on the surface of a bacterial cell

are more immunogenic than the antigens in the cytoplasm. Based on
this observation, scientists have developed strategies to coat the
surfaces of non-pathogenic organisms with antigens of pathogenic

Flagellin is a protein present in the fragella (thread like filaments) of

Salmonella. A synthetic oligonucleotide encoding the epitope of
cholera toxin B subunit was inserted into Salmonella flagellin gene.
This epitope was in fact found on the flagellum surface. These flagella-
engineered bacteria, when administered to mice, raised antibodies
against the cholera toxin B subunit peptide. It may be possible in
future to incorporate multiple epitopes (2 or 3) into the flagellin gene
to create multivalent bacterial vaccines.