Module – 3

Biobusiness & Entrepreneurship

Ms.Archana Vijay
Topics to be covered:

ENTREPRENEURSHIP OPPORTUNITY IN INDUSTRIAL BIOTECHNOLOGY

Business opportunity, Essential requirement, marketing strategies, schemes, challenges and
scope-with case study-
Pollution monitoring and Bioremediation for Industrial pollutants, Pesticides, Herbicides etc.
Integrated compost production- microbe enriched compost.

Biopesticide/insecticide production. Fermented products-probiotic and prebiotics. Stem cell

production, stem cell bank, contract research. Production of monoclonal/polyclonal antibodies,
Single cell protein and secondary metabolite production
Contact research in microbial genomics

Pollution monitoring and Bioremediation for Industrial pollutants

Industrial pollution is generally referred to the undesirable outcome when factories (or other
industrial plants) emits harmful by-products and waste into the environment such as emissions
to air or water bodies (water pollution), deposition on landfills etc (land pollution) or emission
of toxic chemicals into the atmosphere (air pollution). Hence it is necessary for every industries
to design and set-up a pollution monitoring system.

Objectives of pollution monitoring system:

 To protect the environment from industrial pollution
 To build a robust system that evaluates the industrial pollution continuously and
indicates when there is an increase in the emissions and takes action to control it
 To decrease human interference in monitoring the industrial pollution to reduce
pollution and provide a healthy environment for the workers to work in.
 These improvements can be used to develop and implement its environmental policy
and manage its environmental aspects.

Bioremediation
It is a waste management technique that involves the use of either naturally occurring or
deliberately introduced microorganisms to remove or break down environmental pollutants
from a contaminated site.

With environmental sustainability becoming a critical area of concern across all industrial
sectors, bioremediation solutions present exciting opportunities globally, and in India.
Opportunities are present especially for waste water remediation, across a variety of industrial
sectors that include pharmaceuticals, chemicals, food and more.

Application of bioremedial products:

 Eutraphication of water bodies- The multiple microbial preparation composed of
bacteria can transfer nutritive material harmless and some natural humic acid which can
destroy algae. This also controls the growth of algae in ponds and lakes.
 Aquaculture wastes- in situ removal of nitrogen and other nutrients
 Treating frack water (Water derived from unconventional oil and gas extraction, also
known hydraulic fracturing).
 Soil replenishment
 Water soluble fertilizers
 Understanding the conditions limiting substrate distribution and optimizing microbial
metabolism.
 Bioaccumulation of plants and fungi – Heavy metals are being absorbed by the
organisms and they can be removed easily.

Business opportunities in bioremediation of industrial pollutants

 Water treatment plant
 Supply microbes
 Manufacturing of absorbants, bioremediation products and spill kils.
 Consulting services
 PRP powder (Petroleum remediation product)- oil spill remediation
 Biostimulation reagents to enhance the biodegradative activity of intrinsic
microorganisms.

Advantages of Bioremediation
Bioremediation is natural process and is therefore perceived by the public as an acceptable
waste treatment process for contaminated material such as soil. Microbes able to degrade the
contaminants increase in numbers when the contaminant is present; when the contaminant is
degraded, the bio degradative population declines. The residues for the treatment are usually
harmless products and include carbon dioxide, water and cell biomass. Theoretically,
bioremediation is useful for the complete destruction of a wide variety of contaminants. Many
compounds that are legally considered to be hazardous can be transformed to harmless
products. This eliminates the chance of future liability associated with treatment and disposal
of contaminated material. Instead of transferring contaminants from one environmental
medium to another, for example from land to water or air, the complete destruction of target
pollutants is possible.

• Bioremediation can often be carried out on site, often without causing a major disruption or
normal activities. This also eliminates the need to transport quantities of waste off site and the
potential threats to human health and the environment that can arise during transportation.

• Bioremediation can prove less expensive than other technologies that are used for clean-up
of hazardous waste.

Disadvantages of Bioremediation

Bioremediation is limited to those compounds that are biodegradable, not all compounds are
susceptible to rapid and complete degradation.

• There are some concerns that the products of biodegradation may be more persistent or
toxic than the parent compound

• Biological processes are often highly specific. Important site factors required for successes
include the presence of metabolically capable microbial populations, suitable environmental
growth conditions, and appropriate levels of nutrients and contaminants

• It is difficult to extrapolate from bench and pilot scale studies to full-scale field operations

• Research is needed to develop and engineer bioremediation technologies that are

appropriate for sites with complex mixtures of contaminants that are not evenly dispersed in
the environment. Contaminants may be present as solids, liquids and gases

• Bioremediation often takes longer time than other treatment options, such as excavation
and removal of soil or incineration.

• Regulatory uncertainty remains regarding acceptable performance criteria for

bioremediation. There is no accepted definition of „clean”, evaluating performance of
bioremediation is difficult, and there are no acceptable endpoints for bioremediation
treatments.

Biopesticides production
 • Pesticides are substances meant for attracting ,seducing and then destroying any pest
• The most common use of pesticides is as plant protection products (also known as crop
protection products),which in general protect plants from damaging influences such
as weeds ,fungi, or insects
Types of pesticides
• There are two types
1.Chemical pesticide: eg: organophosphates ,carbamates , pyrethroids , sulfonylureas
2.Biopesticide:
• Biopesticides or biological pesticides baesd on pathogenic microorganisms specific to a
target pest offer an ecologically sound and effective solution to pest problems
• They pose less threat to the environment and to the human health
• The most commonly used biopesticides are living organisms , which are pathogenic for
the pest of interest . These include biofungicides (Tricoderma),
bioherbicides(phytopthora) and bioinsectides(Bacillus thuringenesis)
• Biopesticide fall into three major categories
1. Microbial pesticide
2. Plant pesticide
3. Biochemical pesticide
Microbial pesticide
• Contain a microorganism (bacterium, fungus, protozoan or algae) as the active
ingredient
• Microbial pesticides can control many different kinds of pests, although each separate
active ingredients is relatively specific for its target pests
The most widely known microbial pesticides are varieties of the bacterium Bacillus
thuringiensis, or Bt which can control certain insects in cabbage, potatoes and other crops . Bt
produces a protein that is harmful to specific insect pests .
Plant Incorporated Pesticides

• Plant pesticides are pesticidal substances that plants produce from genetic material that
has been added to the plant.

• For eg; scientists can take the gene from the Bt pesticidal protein and introduce the
gene into the plants own genetic material . Then the plant , instead of the Bt bacterium
manufactures the substance that destroys the pest. Both the protein and its genetic
material are regulated

Biochemical pesticide

• naturally occuring substances that control pests by non toxic mechanisms.

• Biochemical pesticides include substances that interfere with the growth or mating ,
such as plant growth regulators or substances that repel or attract pests, such as
pheromones .
Neem extracts doesn’t directly kill insects on the crop . It act as an antifeedant

Biopesticide production process

Advantages

• Inherently less harmful and less environmental load

• Designed to affect only one specific pest or , in some cases , a few target organisms.

• Often effective in very small quantities and often decompose quickly , thereby
resulting in lower exposures and largely avoiding the pollution problems.

Disadvantages

• Slow effect

• Lack persistence and wide spectrum activity

• Rapidly degraded by UV lights so residual action is slow.

• They are not available easily

Pesticides & Herbicides

Pesticides are substances that are meant to control pests, including weeds.[1] The term
pesticide includes all of the following: herbicide, insecticides (which may include insect growth
regulators,
termiticides, etc.) nematicide, molluscicide, piscicide, avicide, rodenticide, bactericide, insect
repellent, animal repellent, antimicrobial, fungicide, disinfectant(antimicrobial), and sanitizer.
[2]
The most common of these are herbicides which account for approximately 80% of all
pesticide use.[3] Most pesticides are intended to serve as plant protection products (also known
as crop protection products), which in general, protect plants from weeds, fungi, or insects.

In general, a pesticide is a chemical or biological agent (such as a virus, bacterium, or fungus)

that deters, incapacitates, kills, or otherwise discourages pests. Target pests can include insects,
plant pathogens, weeds, molluscs, birds, mammals, fish, nematodes (roundworms),
and microbes that destroy property, cause nuisance, or spread disease, or are disease vectors.
Although pesticides have benefits, some also have drawbacks, such as potential toxicity to
humans and other species.
Uses
Pesticides are used to control organisms that are considered to be harmful. For example, they
are used to kill mosquitoes that can transmit potentially deadly diseases like West Nile
virus, yellow fever, and malaria. They can also kill bees, wasps or ants that can cause allergic
reactions. Insecticides can protect animals from illnesses that can be caused by parasites such
as fleas. Pesticides can prevent sickness in humans that could be caused by moldy food or
diseased produce. Herbicides can be used to clear roadside weeds, trees, and brush. They can
also kill invasive weeds that may cause environmental damage.
Herbicides are commonly applied in ponds and lakes to control algae and plants such as water
grasses that can interfere with activities like swimming and fishing and cause the water to look
or smell unpleasant. Uncontrolled pests such as termites and mold can damage structures such
as houses. Pesticides are used in grocery stores and food storage facilities to
manage rodents and insects that infest food such as grain. Each use of a pesticide carries some
associated risk. Proper pesticide use decreases these associated risks to a level deemed
acceptable by pesticide regulatory agencies such as the United States Environmental Protection
Agency (EPA) and the Pest Management Regulatory Agency (PMRA) of Canada.
DDT, sprayed on the walls of houses, is an organochlorine that has been used to
fight malaria since the 1950s. Recent policy statements by the World Health Organization have
given stronger support to this approach. However, DDT and other organochlorine pesticides
have been banned in most countries worldwide because of their persistence in the
environment and human toxicity. DDT use is not always effective, as resistance to DDT was
identified in Africa as early as 1955, and by 1972 nineteen species of mosquito worldwide were
resistant to DDT.
Benefits
Pesticides can save farmers' money by preventing crop losses to insects and other pests; in the
U.S., farmers get an estimated fourfold return on money they spend on pesticides.One study
found that not using pesticides reduced crop yields by about 10%. Another study, conducted in
1999, found that a ban on pesticides in the United States may result in a rise of food prices, loss
of jobs, and an increase in world hunger.
There are two levels of benefits for pesticide use, primary and secondary. Primary benefits are
direct gains from the use of pesticides and secondary benefits are effects that are more long-
term.
Primary benefits
Controlling pests and plant disease vectors

 Improved crop/livestock yields

 Improved crop/livestock quality
 Invasive species controlled
Controlling human/livestock disease vectors and nuisance organisms

 Human lives saved and disease reduced. Diseases controlled include malaria, with millions
of lives having been saved or enhanced with the use of DDT alone.
 Animal lives saved and disease reduced
Controlling organisms that harm other human activities and structures

 Drivers view unobstructed

 Tree/brush/leaf hazards prevented
 Wooden structures protected

Integrated Compost Production – Microbe enriched compost

Compost is a mixture of decayed organic materials decomposed by microorganism in a warm,
moist, and aerobic environment, releasing nutrients into readily available forms for plant use.

Why use compost?

• There is a need for sustainable production through integrated nutrient management

• Compost produces less methane than uncomposted rice straw when incorporated in the soil
• Solves problem of declining yield.

• Corrects micronutrient problems like zinc deficiency. Benefits of compost

• Big savings, increased farmer self-reliance

• Increases yields

• Improves soil tilth and structure

• Increases water holding capacity of the soil

• Improves aeration

• Provides humus or organic matter, vitamins, hormones, and plant enzymes which are not
supplied by chemical fertilizers.

• Acts as buffer to changes in soil pH

• Kills pathogenic organisms, weeds, and other unwanted seeds when temperatures of over
60oC is reached

• Mature compost quickly comes into equilibrium with the soil

• Different materials can be blended or mixed together which can increase the nutrient content
of the compost fertilizer.

2 Recommended fertilizer rate

The GINTONG ANI program recommends basal application of 6-8 bags inorganic fertilizer and 8
bags organic fertilizer per hectare by composting all the rice straw after harvest, this
requirement is adequately met, and one does not need to buy commercial organic fertilizers. *
enriched with animal manure, nitrogen rich farm residues like legumes, and acted upon by
microorganisms like fungus Trichoderma sp. and nitrogen fixing bacteria, Azotobacter sp.

Three Methods of Compost Formation

Traditional Method This is a slow process, requiring 3-4 months before farm wastes are fully
decomposed and ready for use as compost fertilizer. This means that the fertilizer can only be
used after one planting season. This also requires a bigger composting area. However, this
method involves only eight steps, and it is inexpensive to produce, requiring no extensive
inputs except labor.

Rapid Method With the aid of fungus activator Trichoderma harzianum, decomposition of farm
wastes is accelerated to just 3-4 weeks! This means that the compost can be used in the next
planting season. This involves ten steps.

Bio-Enriched Method Employing both a fungus activator and a nitrogen-fixing bacteria, farm
wastes are first decomposed by Trichoderma sp. for 2-3 weeks, after which the resulting
compost is inoculated with live N-fixing bacteria Azotobacter sp. Incubation for 1 week
produces a nitrogen-enriched compost that can supply a rice crop’s total N requirement,
depending on the material used, soil condition, and planting season. This involves 10 steps.

Guide For Compost Production

Most of the steps are common to the three methods of composting. Step 4 or the addition of
fungus activator, however, does not apply to the traditional method. Step 8 or the addition of
bacteria inocula, on the other hand, applies only to the Bio-Enriched method of composting.

Step 1 Gather materials Gather rice straw, weeds, sugarcane bagasse, corn stalks and stovers,
leguminous materials such as ipil-ipil, azolla, sesbania, mungbean, cowpea, soybean crop residues, and
animal manure. Soak rice straw for 6-12 hours before piling. Chop materials for easier decomposition.
Ideal proportion of composting materials is 3 parts rice straw and 1 part mixture of animal manure and
leguminous plant residues. Less than this proportion prolongs the decomposition process

Step 2 Prepare Compost Area Choose a shaded and well-drained area. To compost 5 tons of rice
straw, we need a volume of 90 m3 . A plot size of 2m x 6m x 1.5m can acccomodate 1 ton of rice straw.
Make 5 plots. If you want smaller plots, a plot size of 2m x 3m x 1.5m can accommodate 500 kg of rice
straw materials. Make 10 small plots to be able to compost 5 tons rice straw.

Step 3 Pile materials

Traditional Method Make six layers of compost materials, each layer about 25 cm thick. A layer of
compost material consists of three parts rice straw, one part manure, soil, and ash or lime spread on top
of each other. Stack the layers until the compost heap 1.5 high. Insert several perforated bamboo poles
into compost bed to serve as breathers.

Rapid method (Trichoderma) To provide aeration at the bottom, construct platfrom or use available
materials such as coconut leaf, kakawate, banana trunk, and bamboo. Make six layers of compost
materials, each layer about 25 cm thick. a layer of compost material consists of three parts rice straw,
one part mixture of animal manure and leuminous materials, annd a thin layer of fungus activator,
known as Compost Fungal Activator (CFA). There is no need to put ash/lime or bamboo breathers.

Bio-Enriched Method (Trichoderma and Azotobacter) Mix all the rice straw, animal manure, and
leguminous materials into 3:1 proportion. Apply 2.5 kg of the fungus activator, known as BIO-QUICK, for
every ton of composing material. Spread evenly on top of the first layer. Place 2-3 perforated bamboo
poles horizontal across the first layer before adding the next layer. Make three layers.

Step 4 Spread Fungus activator

Spread evenly 5-10 kg of Trichoderma fungal activator for every ton of composting material.

Step 5 Water compost heap

Water each layer of compost heap until it is sufficiently moist.

Step 6 Cover Compost Heap

Cover with plastic sheet, used sacks, banana and coconut leaves to increase temperature and
prevent too much water into the compost heap which could leach the nutrients.

Step 7 Turn Compost Heap

Traditional Method Turn or mix compost heap after 3 weeks, then agein after 5 weeks. 1st turnover
after 3 weeks after 5 weeks 2nd turnover after 2 weeks 11

Rapid Method (Trichoderma) Turn compost heap from top to bottom after 2 weeks. This step, however,
is optional.

Bio-Enriched Method (Trichoderma and Azotobacter) Remove cover after 2-3 weeks or when the
compost heap has decomposed. Separate undecomposed materials for further composting.

Step 8 Harvest Compost

Traditional Method Harvest 4 weeks after the second turnover of the compost heap. The N content of
the compost is now 1.5%. Use 2 tons of compost per hectare.

Rapid method (Trichoderma) Harvest 1-2 weeks after turning over the compost heap. The N content of
the ripe compost varies from 1.0% - 3.0% depending on the amount of manure and nitrogenous plant
materials used as substrates. Use all the compost produced in the field which could be about 2.0 tons
per half commercial organic fertilizer produced through the rapid composting method is used, mix 8-10
bags per ha.

Bio-Enriched method (Trichoderma and Azotobacter) After 1 week of incubation of the bacteria inocula,
the compost is ready for use. N content of the compost ranges from 1.5% to 3%. You need only to apply
250- 500 kg or 5-10 bags compost per hectare. Presence of live N-fixing bacteria in the compost will
boost total N in the soil.

Step 9 Apply Compost

Broadcast compost as basal fertilizer before final harrowing during land preparation.

Prebiotics and Probiotics

PREBIOTICS
Prebiotics are classified as the non-digestible food ingredients that probiotics can feed off. They
are selectively utlilised in the gut to increase healthy bacteria, aid digestion and enhance the
production of valuable vitamins. Good bacteria play a significant role in regulating your immune
system, inhibiting the growth of pathogens (disease causing bacteria) and digesting food.
Galactooligosaccharides (GOS) are the most advanced form of prebiotics which belong to a
group of particular nutrient fibres that feed and encourage the growth of good bacteria in the
gut.
Source of Prebiotics
The major source of prebiotics is dietary fibre. They occur naturally in fruits and vegetables, but
you can also take them in the form of nutritional supplements for maximum health benefits.
The soluble dietary fibre inulin, for example, is found in garlic, asparagus, onion, Jerusalem
artichokes, and leeks. However, you’d have to eat a huge amount of these natural prebiotics —
preferably raw — to gain any benefit from them.

While all prebiotics are fibres, not all fibres are prebiotics. The common forms of dietary fibre
present in the majority of plant based foods and grains are less selectively fermented by the
bacteria in the gut and lack some of the health benefits demonstrated by prebiotics. However,
they are still of benefit to our health and their consumption is to be encouraged as they help
maintain regular toilet habits as well as promoting the health of the gut itself.

Prebiotic Foods
It is possible to find prebiotics in some everyday foods including onions, garlic, leeks, Jerusalem
artichokes, asparagus and bananas. However, in order to experience any meaningful health
benefits from these sources, you would have to consume unrealistically large portions.

Health Benefits Using Prebiotics

Scientific Research has shown that prebiotic supplements work naturally in your gut to feed
existing good bacteria, helping them to grow and multiply. Important benefits can include the
following:

 Boosting overall digestive health

 Improving the barrier function of the gut
 Strengthening the immune system
 Reducing the inflammation and symptoms associated with Irritable Bowel Syndrome
(IBS)
 Minimising the risk of developing diarrhoea
 Enhancing your body’s nutrition by allowing better mineral and nutrient uptake
 Contributing positively to your mental health
 Increasing absorption of calcium to improve bone density
 Lowering some risk factors for cardiovascular disease
Probiotics

Probiotics are live bacteria and yeasts that are good for your health, especially your digestive
system. We usually think of these as germs that cause diseases. But your body is full of bacteria,
both good and bad. Probiotics are often called "good" or "helpful" bacteria because they help keep
your gut healthy.

Types of Probiotics

Many types of bacteria are classified as probiotics. They all have different benefits, but most come
from two groups.

Lactobacillus. This may be the most common probiotic. It's the one you'll find in yogurt and other
fermented foods. Different strains can help with diarrhea and may help with people who can't
digest lactose, the sugar in milk.

Bifidobacterium. You can also find it in some dairy products. It may help ease the symptoms of
irritable bowel syndrome (IBS) and some other conditions.

Saccharomyces boulardii is a yeast found in probiotics. It appears to help fight diarrhea and other
digestive problems.

What Do They Do?

Among other things, probiotics help send food through your gut by affecting nerves that control gut
movement. Researchers are still trying to figure out which are best for certain health problems.
Some common conditions they treat are:

 Irritable bowel syndrome

 Inflammatory bowel disease (IBD)
 Infectious diarrhea (caused by viruses, bacteria, or parasites)
  Antibiotic-related diarrhea

Stem Cell Production

Stem cells are biological cells that can differentiate into other types of cells and can divide to
produce more of the same type of stem cells. They are found in multicellular organisms.
In mammals, there are two broad types of stem cells: embryonic stem cells, which are isolated
from the inner cell mass of blastocysts, and adult stem cells, which are found in various tissues.
In adult organisms, stem cells and progenitor cells act as a repair system for the body,
replenishing adult tissues. In a developing embryo, stem cells can differentiate into all the
specialized cells—ectoderm, endoderm and mesoderm (see induced pluripotent stem cells)—
but also maintain the normal turnover of regenerative organs, such as blood, skin, or intestinal
tissues.
There are three known accessible sources of autologous adult stem cells in humans:

1. Bone marrow, which requires extraction by harvesting, that is, drilling into bone
(typically the femur or iliac crest).
2. Adipose tissue (fat cells), which requires extraction by liposuction.
3. Blood, which requires extraction through apheresis, wherein blood is drawn from the
donor (similar to a blood donation), and passed through a machine that extracts the
stem cells and returns other portions of the blood to the donor.
Stem cells can also be taken from umbilical cord blood just after birth. Of all stem cell types,
autologous harvesting involves the least risk. By definition, autologous cells are obtained from
one's own body, just as one may bank his or her own blood for elective surgical procedures.
Adult stem cells are frequently used in various medical therapies (e.g., bone marrow
transplantation). Stem cells can now be artificially grown and transformed (differentiated) into
specialized cell types with characteristics consistent with cells of various tissues such as muscles
or nerves. Embryonic cell lines and autologous embryonic stem cells generated through somatic
cell nuclear transfer or dedifferentiation have also been proposed as promising candidates for
future therapies.
Treatments
Stem cell therapy is the use of stem cells to treat or prevent a disease or condition. Bone
marrow transplant is a form of stem cell therapy that has been used for many years without
controversy. No stem cell therapies other than bone marrow transplant are widely used.
Advantages[
Stem cell treatments may lower symptoms of the disease or condition that is being treated. The
lowering of symptoms may allow patients to reduce the drug intake of the disease or condition.
Stem cell treatment may also provide knowledge for society to further stem cell understanding
and future treatments.
Disadvantages
Stem cell treatments may require immunosuppression because of a requirement for radiation
before the transplant to remove the person's previous cells, or because the patient's immune
system may target the stem cells. One approach to avoid the second possibility is to use stem
cells from the same patient who is being treated.
Pluripotency in certain stem cells could also make it difficult to obtain a specific cell type. It is
also difficult to obtain the exact cell type needed, because not all cells in a population
differentiate uniformly. Undifferentiated cells can create tissues other than desired types.
Some stem cells form tumors after transplantation; pluripotency is linked to tumor formation
especially in embryonic stem cells, fetal proper stem cells, induced pluripotent stem cells. Fetal
proper stem cells form tumors despite multipotency.
Some of the fundamental patents covering human embryonic stem cells are owned by
the Wisconsin Alumni Research Foundation (WARF) – they are patents 5,843,780, 6,200,806,
and 7,029,913 invented by James A. Thomson. WARF does not enforce these patents against
academic scientists, but does enforce them against companies.
Treatment

Diseases and conditions where stem cell treatment is being investigated.

Diseases and conditions where stem cell treatment is being investigated include:

 Diabetes
 Rheumatoid arthritis
 Parkinson's disease
 Alzheimer's disease
 Osteoarthritis
 Stroke and traumatic brain injury repair
 Learning disability due to congenital disorder
 Spinal cord injury repair
 Heart infarction
 Anti-cancer treatments
 Baldness reversal
 Replace missing teeth
 Repair hearing
 Restore vision and repair damage to the cornea
 Amyotrophic lateral sclerosis
 Crohn's disease
 Wound healing
 Male infertility due to absence of spermatogonial stem cells
Research is underway to develop various sources for stem cells, and to apply stem cell
treatments for neurodegenerative diseases and conditions, diabetes, heart disease, and other
conditions. Research is also underway in generating organoids using stem cells, which would
allow for further understanding of human development, organogenesis, and modeling of
human diseases.
In more recent years, with the ability of scientists to isolate and culture embryonic stem cells,
and with scientists' growing ability to create stem cells using somatic cell nuclear transfer and
techniques to create induced pluripotent stem cells, controversy has crept in, both related
to abortion politics and to human cloning.
Hepatotoxicity and drug-induced liver injury account for a substantial number of failures of new
drugs in development and market withdrawal, highlighting the need for screening assays such
as stem cell-derived hepatocyte-like cells, that are capable of detecting toxicity early in the drug
development process.
Stem Cell bank
An amniotic stem cell bank is a facility that stores stem cells derived from amniotic fluid for
future use. Stem cell samples in private (or family) banks are stored specifically for use by the
individual person from whom such cells have been collected and the banking costs are paid by
such person. The sample can later be retrieved only by that individual and for the use by such
individual or, in many cases, by her or his first-degree blood relatives. In case of amniotic fluid
stem cell banking, the mother providing the donation initially has ownership of the stem cells
and financial responsibility for its storage. When the child from that pregnancy reaches legal
age, the ownership and responsibility for the sample may be transferred. The first private
amniotic stem cell bank in the US was opened by Biocell Center in October 2009 in Medford,
Massachusetts
Collection Process
Amniotic stem cells are collected from amniotic fluid extracted during a genetic amniocentesis,
a prenatal diagnosis procedure typically performed during the 2nd trimester of pregnancy. For
the purpose of stem cell preservation, a small amount of the fluid withdrawn for genetic
analysis is saved in a collection container for transport to a processing and storage facility.
Collection of the sample causes no change to the standard amniocentesis procedure and
therefore adds no additional risk to the mother. In the United States, the Food and Drug
Administration regulates amniotic stem cells under the category of “Human Cells, Tissues, and
Cellular and Tissue Based-Products.”
Cryopreservation
After the collection, the amniotic fluid sample containing the stem cells is shipped to a lab for
processing, cryopreservation and storage. The processed sample is exposed to a gradual
freezing process which is important because it keeps the cells alive during the cryopreservation
process. After freezing, the sample is transferred to a liquid nitrogen storage tank. The
protocols used for the cryopreservation process have largely been adapted from those
originally designed for bone marrow haematopoietic stem cells.

Stem cells and regenerative medicine

Regenerative medicine is a field of medical research developing treatments to repair or re-grow
specific tissue in the body. Because a person’s own (autologous) amniotic stem cells can be
safely infused back into that individual without being rejected by the body’s immune system -
and because they have unique characteristics compared to other sources of stem cells - they
are an increasing focus of regenerative medicine research. Research in this area has the
potential to revolutionize medicine. It is advancing so rapidly that it is even difficult for medical
professionals to supply the most up-to-date information for their patients. Possible future uses
in the field of regenerative medicine include repairing heart tissue, birth defects, and other
damaged tissues.
Production of monoclonal/polyclonal antibodies, Single cell protein and
secondary metabolite production
 Monoclonal antibodies (mAb) are antibodies that are identical because they were produced
by one type of immune cell, all clones of a single parent cell. Given (almost) any substance,
it is possible to create 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.

 All current therapeutic antibodies are of the IgG class.

 When the objective of antibody therapy is to directly kill the target cell, the isotype of
choice is IgG1, since this isotype is optimal for complement fixation.

 Fab = Fragment, antigen binding

 Fc = Fragment, crystalline

 The Fc fragment specifies other biological activities of the molecule. For example, the Fc
fragment may determine whether the antibody simply prevents signaling through a
receptor, or alternatively, causes the cell’s destruction through complement fixation or
targeting immune effector cells.

 The antibody alone may not be sufficient to destroy a foreign ‘object’.

 For example, a common test for HIV is the presence of anti-HIV antibodies in the blood.
Obviously, those antibodies alone are not sufficient to protect the host from the virus

History of Mab Development

 1890 Von Behring and Kitasato discovered the serum of vaccinated persons contained
certain substances, termed antibodies

 1900 Ehrlich proposed the “ side-chain theory”

 1955 Jerne postulated natural selection theory. Frank Macfarlane Burnet expended.

 Almost the same time, Porter isolated fragment of antigen binding (Fab) and fragment
crystalline (Fc) from rabbit y-globulin.

 1964 Littlefield developed a way to isolate hybrid cells from 2 parent cell lines using the
hypoxanthine-aminopterin-thymidine (HAT) selection media.

 1975 Kohler and Milstein provided the most outstanding proof of the clonal selection
theory by fusion of normal and malignant cells. This resulted in the first monoclonal
antibodies, for which they received the Nobel Prize in 1984.

Diseases to Target

 Cancer cells express a variety of antigens that are attractive targets for monoclonal
antibody-based therapy.

 The development of monoclonal antibodies against specific targets has been largely
accomplished by immunizing mice against human tumor cells and screening the
hybridomas for antibodies of interest

Types of Mab

A. Murine source mAbs: rodent mAbs with excellent affinities and specificities, generated
using conventional hydrioma technology. Clinical efficacy compromised by
HAMA(human anti murine antibody) response, which lead to allergic or immune
complex herpersensitivities.

B. Chimeric mAbs: chimers combine the human constant regions with the intact rodent
variable regions. Affinity and specificity unchanged. Also cause human antichimeric
antibody response (30% murine resource)

C. Humanized mAbs: contained only the CDRs of the rodent variable region grafted onto
human variable region framework
Common Chemotherapy in treatment of Cancer

A. Nature of cytotoxin

B. Lack of in vivo selectivity

C. The mechanism of anti-proliferation on cells cycle, rather than specific toxicity directed
towards particular cancer cell

D. Host toxicity: treatment discontinued, most of them had bad side-effects, such as no
appetites, omit, lose hair

Monoclonal Antibodies for Cancer Treatment

Three mechanisms that could be responsible for the cancer treatment.

A. mAbs act directly when binding to a cancer specific antigens and induce immunological
response to cancer cells. Such as inducing cancer cell apoptosis, inhibiting growth, or
interfering with a key function.

B. mAbs was modified for delivery of a toxin, radioisotope, cytokine or other active
conjugates.

C. it is also possible to design bispecific antibodies that can bind with their Fab regions
both to target antigen and to a conjugate or effector cell .

Polyclonal Antibodies
The blood contains two types of white blood cell or leukocyte

Phagocytes ingest bacteria by endocytosis

Lymphocytes produce antibodies

A lymphocyte can produce only one type of antibody so a huge number of different types are
needed

Each lymphocyte has some of its antibody on its surface…

The antigens of a pathogen bind to the antibodies in the surface membrane of a lymphocyte
This activates the lymphocyte.

The active lymphocyte divides by mitosis to produce a clone of many identical cells
The clone of cells starts to produce large quantities of the same antibody… … the same antibody
needed to defend against the pathogen!

Most microbes have more than one antigen on their surface, so… …they stimulate more than

one type of lymphocyte …resulting in the production of many different antibodies.

These are called polyclonal antibodies

Microbial Genomics

• An E. coli strain, lets say K12 was infected with a bacteriophage grown on E. coli strain B
and only a fraction of the expected plaques was observed !

• One plaque was picked and a new phage suspension prepared which in turn was used to
infect E. coli strain K12. This time the correct number of plaques was observed.

• E. coli K12 has a restriction modification system which is not found in E. coli B.

• The restriction modification system consists of two components: a site (sequence)

specific endonuclease and a corresponding methylase.

• The methylase methylates the recognition site and the endonuclease only cuts the DNA
at non-methylated sites. So the E. coli DNA is protected by methylation.

• The bacteriophage DNA is not methylated and when it is injected into the bacteria cell it
is cut up into pieces by the endonuclease.

• The system is not 100% perfect and a few bacteriophage manage to compete a
successful infection. These bacteriphage are methylated in the correct sites and the
phage can now infect E. Coli K12 without being cut up.

Plasmid Vectors for construction of Recombinant Molecules

Plasmids pBR322, pACYC184, and pUC18 contain several unique restriction enzyme recognition
sequences (shaded) as well as resistance determinants for ampicillin (AmpR), tetracycline (TcR),
and chloramphenicol (CmR). Plasmid pUC18 has a multiple cloning site with recognition
sequence for 13 different restriction enzymes. In the case of pUC18, to screen for inserted DNA,
the plasmids must be transformed into special E. coli strains that carry the gene for the non-
lacZ' portion of β-galactosidase; the two portions of the protein form the active enzyme.
Artificial formation of an active enzyme from two fragments (called complementation) allows
screening for insertions by colony color in gel containing X-gal, the chromogenic indicator of β-
galactosidase. Bacterial colonies with intact plasmid have a blue appearance; colonies with
plasmids that carry inserts are white.

How are these methods used in microbiological Research

Bottom up approach:

The organism was mutated using transposon insertional mutagenesis and a very large number
of mutants were screened for strains in which the observed phenotype was affected.

So the insertion of a transposon knocked out a gene which was important for the phenotype
under investigation. Make a genomic library, find the clone containing the transposon, find the
gene that was knocked out and sequence it.

Gene identified !

Repeat the procedure for all the mutants and isolate all the genes affecting the phenotype
under investigation.

Carry out complementation analysis to verify that the correct gene has been isolated.

Time consuming but immensely productive and the basis of modern molecular biology.

Whole Genome shotgun sequencing

Determination of a microbial genome sequence. (A) Construction of a random library of DNA

fragments in a cloning vector. (B) Random sequencing of clones. Short sequences are obtained
from each end of the cloned DNA, and thousands of clones are sequenced

Identification of gene and elucidation of gene function

From nucleotide sequence to potential genes: bacterial genes have well defined features
which makes it possible to identify genes be computational analysis. The features are;
promotor regions 5’ to the gene, a DNA dependent RNA polymerase binding site, a start codon,
an open reading frame in units of 3 nucleotides, a stop codon, a transcription termination
sequence.

From open reading frames to amino acid sequences: translate codons to corresponding amino
acid sequences.

From amino acid sequences to protein function: this is the tricky part. Compare the amino acid
sequence for sequence similarity to all known prokaryote proteins. Computational analysis
using Basic local alignment tool = BLAST.
Interpretation of the results: At the one extreme, the function of the protein is well known in
other species and the sequences are very similar = function assignment. At the other extreme,
there are no proteins in the databases which resemble our sequence.

Reality: The function of about 40% of the proteins in most bacteria is unknown.

Transcription Analysis

• DNA > RNA > protein

• When the genome sequence is known a set of DNA molecules which correspond to each
gene can be synthesized.

• These a spotted on a glass slide and this is called a DNA micro-array because the spots
are very small and can only be seen under a microscope.

• Total RNA is isolated and reverse transcribed into cDNA. The cDNA is labeled using
fluorochromes and hybridize to the array. Positive signals means that the gene is
expressed.

• Change the growth conditions and isolate a new RNA. Repeat as above and compare.

• In practice the two different RNA preparations (cDNA) are labeled with two different
fluorochromes and the comparative levels of transcription from the two different
growth conditions are compared.

Single Cell Protein

The term single cell protein was introduced in the 1960s to describe protein-rich foods
manufactured from yeasts that served as dietary supplements for livestock and humans. Single
cell protein was viewed as a product category that might address food shortages at a time
when it seemed unlikely that agricultural production could keep pace with the skyrocketing
human population. Interest in food yeast declined as improvements in plant breeding and
agricultural practices led to the contemporary boom in global food production. Saccharomyces
cerevisiae produced in stirred fermenters on molasses is an example of single cell protein that is
manufactured today. The yeast produced in this fashion is not consumed directly, but is used
for baking. Marmite is a savoury spread made from yeast extract that has been popular in the
United Kingdom for more than a century. Spent yeast from beer brewing is used to produce this
sticky dark brown paste. Vegemite is a similar product made in Australia.
Another fungal protein product, called Quorn, is a very successful meat substitute
manufactured by a single strain of a filamentous saprotrophic ascomycete, Fusarium
venenatum. Because Quorn is produced from a multi-cellular, filamentous fungus, the term
single cell protein is inaccurate and mycoprotein is the preferred name. The use of a
filamentous fungus makes it possible to produce a meat-like consistency that cannot be
replicated with a single-cell protein. The fungus is grown in pairs of 50 m tall air-lift fermenter
vessels that contain 230 tonnes of broth . The vessels are connected at the top and the bottom
to form a continuous loop. Compressed air and ammonia are pumped into the bottom of the
first vessel, called the ‘riser’, oxygenating the culture and circulating the liquid containing the
fungus toward the top. Carbon dioxide from the respiring cells is released through a vent at the
top of the system, and the liquid falls through the second ‘downcomer’ vessel and is infused
with fresh nutrient solution (glucose plus vitamins and minerals). A heat exchanger at the
bottom of the system maintains the temperature at 30 °C and the culture is harvested at a rate
of 30 tonnes per hour. The dense mycelium of branched hyphae harvested from the fermenter
has a very high RNA content. This is problematic for a food product because its consumption
could raise uric acid levels in the blood and lead to gout and other illnesses. This is addressed by
heating the mycelium at 68 °C for 20 min, which allows endogenous enzymes to destroy much
of the RNA without reducing its protein content. The heated mycoprotein is then dried and
bound with egg white. Further processing creates the meaty texture and adds flavourings and
colourings.

Secondary Metabolism

Secondary metabolism produces a large number of specialized compounds (estimated

200,000) that do not aid in the growth and development of plants but are required for the plant
to survive in its environment. Secondary metabolism is connected to primary metabolism by
using building blocks and biosynthetic enzymes derived from primary metabolism. Primary
metabolism governs all basic physiological processes that allow a plant to grow and set seeds,
by translating the genetic code into proteins, carbohydrates, and amino acids. Specialized
compounds from secondary metabolism are essential for communicating with other organisms
in mutualistic (e.g. attraction of beneficial organisms such as pollinators) or antagonistic
interactions (e.g. deterrent against herbivores and pathogens). They further assist in coping
with abiotic stress such as increased UV-radiation. The broad functional spectrum of specialized
metabolism is still not fully understood. In any case, a good balance between products of
primary and secondary metabolism is best for a plant’s optimal growth and development as
well as for its effective coping with often changing environmental conditions. Well known
specialized compounds include alkaloids, polyphenols including flavonoids, and terpenoids.
Humans use quite a lot of these compounds, or the plants from which they originate, for
medicinal and nutraceutical purposes.
Primary Vs Secondary Plant Metabolism

Primary metabolism in a plant comprises all metabolic pathways that are essential to the plant's
survival. Primary metabolites are compounds that are directly involved in the growth and
development of a plant whereas secondary metabolites are compounds produced in other
metabolic pathways that, although important, are not essential to the functioning of the plant.
However, secondary plant metabolites are useful in the long term, often for defense purposes,
and give plants characteristics such as color. Secondary plant metabolites are also used in
signalling and regulation of primary metabolic pathways. Plant hormones, which are secondary
metabolites, are often used to regulate the metabolic activity within cells and oversee the
overall development of the plant. As mentioned above in the History tab, secondary plant
metabolites help the plant maintain an intricate balance with the environment, often adapting
to match the environmental needs. Plant metabolites that color the plant are a good example
of this, as the coloring of a plant can attract pollinators and also defend against attack by
animals.

Contract Research

A contract research organization (CRO) is an organization that provides support to

the pharmaceutical, biotechnology, and medical device industries in the form of research
services outsourced on a contract basis. A CRO may provide such services as biopharmaceutical
development, biologic assay development, commercialization, preclinical research, clinical
research, clinical trials management, and pharmacovigilance. CROs provide a more affordable
outlet for companies to pursue new medicines, and a cost-effective solution to develop drugs
for even niche markets. Working with CROs, entry into drug development has become
immensely simplified, as the need for large pharma companies to do everything ‘in house’ is
now redundant. CROs also support foundations, research institutions, and universities, in
addition to governmental organizations (such as the NIH, EMA, etc.).
Many CROs specifically provide clinical-study and clinical-trial support for drugs and/or medical
devices. CROs range from large, international full-service organizations to small, niche specialty
groups.
CROs that specialize in clinical-trials services can offer their clients the expertise of moving a
new drug or device from its conception to FDA/EMA marketing approval, without the drug
sponsor having to maintain a staff for these services.

Thank You