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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]
Chemistry
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+.
Sources
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
production.[23]
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.
Production
Although there are various ways ethanol fuel can be produced, the most common way is via
fermentation.
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]
Fermentation
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.
Distillation
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
engines
Dehydration
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
distillation.[27]
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]
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]
BIO-METHANE
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.
Production
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]
Composition
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
xO
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
available.[21]
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
4
Nitrogen N 0–10
2
Hydrogen H 0–1
2
H
Hydrogen sulfide 0.1 –0.5
2S
Oxygen O 0–0.5
2
Applications
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
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
present.[27]
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 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 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
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.
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
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]
Economics
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
Production
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
needed]
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
survive.
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
antibodies
Purification
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
resin.
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
support.
Applications
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.
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
pathways.
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
region.
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.
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
transplants
acute rejection of kidney
daclizumab[44] inhibits IL-2 on activated T cells humanized
transplants
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.
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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.
Hepatitis B:
There, the recombinant bacteria use the gene to begin producing human insulin.
Scientists harvest the insulin from the bacteria and…
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].
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
CONCEPTS OF QUALITY CONTROL, QUALITY ASSURANCE AND FOOD
SAFETY
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
safety
(Source Will and Guenther, 2007)
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.
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.
Fig. 2.2 Quality assurance concept in relation to changes in global food safety
standards
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.
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.
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.
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.
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
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
2+
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
pyrite:
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
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.
anaerobic
denitrification 2NO3− + 10e− + 12H+ → N2 + 6H2O 500 ~ 200
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.
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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:
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:
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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.
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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.
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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.
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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.
Tuberculosis:
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.
Subunit vaccines:
Meningitis:
Subunit vaccines:
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.
i. Nasal spray
Humoral immunity:
Cellular immunity:
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.
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.
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.
Cholera:
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:
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.
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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.
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.
Advantages:
2. The virus can replicate in the host cells. This enables the
amplification of the antigens for their action on B-lymphocytes and T-
lymphocytes.
Disadvantages:
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: