Introduction to Biotechnology

Biotechnology is best defined as the exploitation or application of biological

systems (microbial, animal or plant cells or enzymes) to gain a useful product or

service. It depends mainly upon the expertise of biological systems in recognition and

catalysis.

Biotechnology is an art with multi-disciplinary scientific requirements. It needs

the collaboration of several sciences as seen in the following diagram.

The recent milestone development in biotechnology that renders this science

at the frontier of science is tremendous developments in other areas of science

including:

1- Biocatalysis

i- Enzymes (isolation, stabilization and immobilization)

ii- Intact cells (Stabilization and immobilization)

2- Immunology

3- Genetic engineering

4- Fermentation technology

Biotechnology covers a wide range of applications ranging from the production

of beer and cheeses to the production of the new recombinant DNA drugs. The

recent acceptance of biotechnology as a term is an excitement for applied

microbiology and fermentation as an old terms since large-scale industrial application

of microorganisms is not new.

It should be well recognized that the success in this art depends on economic criteria.

Microbiology Biochemistry Genetics

Biotechnology

Biochemical Engineering Chemical Engineering

Electronics and Computer Sciences

Food Technology

Mechanical Engineering

1

Historical background Our ancestors centuries learned to make alcoholic beverages, vinegar and

bread with yeasts and bacteria. Ancient Egyptians were baking and brewing 4000

B.C. All these processes depend on the use of microorganisms for the benefit of

public. However, all these processes were empirical and probably discovered by

accident without any scientific background and without even the realization that

microorganisms played specific roles. th He was Louis Pasteur who in the late 19 century realized that microbes were

responsible for fermentation and showed that different types of products could be

produced by different microbes. He really formed the basis for the future

development of applied (industrial) microbiology and hence much of biotechnology.

His work laid to the subsequent development of industrial processes for the

production of organic solvents (e.g. acetone, ethanol and butanol) and other

chemicals. However, all these processes involved the conversion of plant

carbohydrates to useful chemical products by microbes in absence of oxygen. Under

these conditions, the microbes use the entropy change in the conversion as source

of energy for growth. Other chemicals such as citrate, vinegar and acetate that were

used extensively in food industry, continued to be produced by fermentation, which is

the most economic route.

Substrate Product Microorganism

Specific conditions

Such processes using biomass for making chemicals, constituted the first

phase of modern biotechnology. th In the early decades of the 20 century, large scale processes for the

production of lactic acid, acetone, butanol, ethanol and riboflavin were developed.

Processes for production of amylases and proteases were also developed.

One of the early strategic examples of modern products was the production of

glycerol, which was used for the production of nitroglycerin as an explosive during

World War I. It involves the modification of the yeast ethanolic fermentation to

generate glycerol. A shown in the following figure the addition of bisulfite to the

fermentation broth prevents acetaldehyde from acting as an acceptor for H from

NADH. Dihydroxyacetone phosphate then acts as the acceptor with the consequent

generation of glycerol instead of acetone.

2

Acetaldehyde bisulfite addition complex

Sugar (glucose)

Dihydroxyacetone-P

NADH

Fructose 1,6 diphosphate

NAD

Glycerol

Ethanol Acetaledehyde

Pyruvate

Embeden-Meyerhoff pathway

3-Phosphoglyceraldehyde

Normal route

Sodium bisulfite

The activated sludge process for mineralizing organic wastes was first

developed in 1914 and since then was developed dramatically in size and exploited

worldwide for treatment of sewage. The treatment of sewage by anaerobic digestion

through mixed microflora eventually generating biogas (methane & Co2) has become

an increasingly important process throughout this century.

However, the next milestone in biotechnology took place around the end of the

World War II, when Chain and Flory performed large-scale production of the antibiotic

penicillin after the discovery of its chemotherapeutic properties in 1940. Investment in

antibiotic production was promising. During this process several important factors

were understood such as how to manipulate microbes aseptically, how to achieve

large-scale sterilization of culture media, how to provide adequate oxygen supply

through proper stirring and aeration and how to improve strains by genetic

manipulation.

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Concurrently with the development of antibiotics, the production of amino

acids and nucleotides was successfully accomplished. It was also discovered that

microorganisms could carry out certain difficult chemical reactions required for steroid

synthesis efficiently. The later was used for production steroid hormones.

Recent developments in Biotechnology

Category Examples

1- Medicine - Production of antibiotics, steroids, monoclonal antibodies, vaccines,

gene therapy, recombinant DNA technology drugs and improving

diagnosis by enzymes and enzyme sensors.

2- Agriculture - Plant tissue culture, protoplast fusion, introduction of foreign genes

into plants and nitrogen fixation.

3- Chemicals - Organic acids (citric, gluconic) and mineral extraction.

4- Environment - Improvement of waste treatment, replacement of chemical

insecticides by biological ones and biodegradation of xenobiotics.

5- Food - Single cell protein (SCP), use of enzymes in food processing and

food preservation.

6- Industry - Use of enzymes in detergent industry, textile and energy production

Future prospects for the development of biotechnology

Biotechnology is expected to make an important contribution to the quality of

life through a wide range of goods and services in many areas for example:

1- Consumer chemicals: adhesives, detergents, dyes, fibers, flavors,

gelling and thickening agents, gums, perfumes, pigments, plastics,

…….. etc.

2- Health care (diagnosis and treatment)

3- Analytical chemical tools

4- Feed stocks (agriculture and animal).

5- Energy sources.

6- Environment control (air, water and soil)

7- Mineral extraction (soil and sea).

4

Introduction to biotechnological resources and techniques Cultures: (microbial, animal and plant) Microbial cultures: Microbial cultures are either obtained from culture collections e.g. American

type culture collection (ATCC) or usually isolated (from soil, air ….etc.) by enrichment

technique (maintain conditions that favor isolation of the required microorganism).

In industry, microorganisms act like chemical factories. Those ones intended to be

used in industry should be:

1- Should be pure culture i.e. not contaminated with other species or low

producing strains.

2- Produce a large amount of the required product.

3- Easily cultivated and maintained.

4- Be genetically stable (low rate of mutation).

5- Grow rapidly on inexpensive and readily available media.

6- Produce the desired product under workable conditions (pH, O2

temperature,….etc.).

Culture maintenance Cultures could be maintained by one or more of the following methods:

1- Lyophilization (freeze drying): cultures in small tubes or ampoules are

rapidly frozen followed by drying under vacuum, then sealed under

vacuum. This is the best and most commonly used one. o2- Storage under liquid nitrogen (at – 150 C): cultures are mixed with

glycerol, transferred to special tubes which withstand low temperature

and kept in special tanks containing liquid nitrogen.

3- Storage in glycerol at – 70oC in deep freezers. o4- Storage on agar slopes at – 4 C and subcultured routinely every few

months (depending on the strain)

5- Soil culture: moist sterile soil is inoculated with the microbial culture,

incubated for few days to allow some growth, then dried at room

temperature for few weeks and stored.

5

Raw materials (substrates) used for the growth and production in biotechnology Raw materials to be used for the cultivation of microorganisms in industry

should be locally available (in nature or by-products from other industries). They

must provide the required carbon, nitrogen, trace metals and energy source required

by the microorganism. The raw materials should also provide the required precursor

for the end product. The media used for cultivation of microorganism must contain

the elements needed by the microorganism (carbon, nitrogen, minerals and growth

factors) in a form suitable for the synthesis of cell substances and for the production

of the products. For cultivation of microorganisms in laboratory research, usually

pure defined chemicals are used, but in industrial scale fermentations the use of such

pure defined constituents would be too expensive. Accordingly complex less

definable substrates are frequently used to reduce the production costs.

The choice of the raw material for a given process depends on the process,

production costs and availability of the raw material. For example in the production

of industrial alcohol the raw material is of an important factor to produce large

quantities of competitive price to chemically synthesized alcohol, while in the

production of alcoholic beverages (wine, whiskey,…) flavor is the most important

factor, that is why special types of grapes are used.

In production of benzyl penicillin, the use of corn steep liquor has the advantage

of providing the precursor of benzyl group side chain. If corn steep liquor is not used,

a mixture of natural penicillins is produced.

In general raw materials used in supporting growth and production should posses the

following characters:

1. Produce maximum yield of the product per gram substrate used.

2. Cheap and available 1ocally throughout the year.

3. Causes minimal problems during the fermentation, product separation and waste

disposal.

4. Of definite composition and easily processed.

5. Easily transported and sterilized.

Most of the substrates used in industrial fermentations are by-products derived

from agriculture, food industry and forestry. They are mainly carbohydrates such as

cellulose and starch, while substrates rich in sugars usually obtained from sugar

cane and sugar beet. Cellulose and lignocellulose is the most abundant and

6

renewable natural resource throughout the world. However, before use in industrial

fermentation it must be degraded to simpler structure. Starch could be obtained from

different types of grains such as rice, wheat, maize, sweet potatoes and cassava.

Again starch must be degraded to monosaccharides or oligosaccharides before use

in industrial fermentations. Molasses is a by-product of sugar industry and whey is a

by-product of cheese industry. Also petrochemicals are now used as substrates. The

biotechnological utilization of these wastes would eliminate much environmental

pollution problems and at the same time convert these wastes into useful products.

Much industrial fermentation utilizes ammonium salts, urea, yeast extract,

peptone, and Corn steep liquor or soy meal as a nitrogen source.

In addition to carbon and nitrogen sources, other additives such as vitamins,

trace elements may be necessary for the growth of the microorganism and maximal

yield of the fermentation product.

Improvement of product quality and quantity by manipulating the process. This may include I- Upstream manipulation (Strain development) The productivity of the wild strains are usually too low for economical processes

and so several years of extensive research programs may be required to develop a

strain that could be used for large-scale production. The success of such programs

depends to large extent on the substance to be produced. In general such programs

include

i) Selection of the biological entities by a screening program to choose

the best biocatalyst of the required product.

ii) Utilization of mutation techniques to prepare mutants of better

characters.

iii) Utilization of recombinant DNA (genetic engineering) to improve an

existing process or developing a totally new product.

iv) Cell fusion for the generation of hybrids with improved productivity.

v) Plant cell and tissue culture.

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II- Down stream processing: It include

1) Manipulation of the fermentation conditions to specify the product and maximize

the yield e.g. changing the oxygen potential of growth for Saccharomyces yields

different product.

Saccharomyces + Hexoses Baker’s yeast aerobic

Saccharomyces + Hexoses alcohol anaerobic

Saccharomyces + Hexoses glycerol anaerobic &bisulphite

Also citric acid production could be performed by surface culture, where spores of

Aspergillus niger are sprayed over the surface of trays containing the medium or

by submerged culture where the fungal spores are mixed and agitated in tanks

containing the medium. Submerged culture is more economic because:

i- Higher yield.

ii- Les energy is required for sterilizing the containers and the

medium.

iii- Less space and labor is needed.

2) Obtaining mathematical models for the fermentation parameters to in the

prediction of the best conditions for maximum yield and cost reduction in scaling

up of the process.

3) Improvement of the efficiency of the biocatalyst by immobilization (discussed

later).

4) Application of the proper methods for separation and purification of the product

e.g. centrifugation, filtration or chromatography.

8

Fermentation processes Fermentation is an industrial

process utilizing living cells for the

production of commercially valuable

products either aerobically or

anaerobically.

In fermentation living cells are

allowed to grow under defined

conditions in a fermenter (bioreactor).

The defined conditions include the use

of the proper substrate (medium) and

the proper environmental parameters

(e.g. temperature, agitation, pH and

aeration). All must be optimized to

achieve the highest yield and quality at the lowest cost possible.

Bioreactors vary from very simple vessel with few controls to highly

complicated ones in which the whole process is under computer control. Before

construction of a fermenter, it must be decided whether the fermenter is to be used

for a special process with certain organism or for a variety of processes with different

microorganisms.

Simple stirred, aerated fermenters are the ones that are usually used in

industry especially in the pharmaceutical industry. Different products can be

produced in the same apparatus with some modification.

Small fermenter vessels up to 20 liters are usually made of glass, however,

larger volume vessels are usually made of stainless steel. It is necessary that

fermenter and substrate solutions be sterilized (mostly inside the fermenter), before

the addition of the inoculum (microbial strain). Small fermenters are sterilized in

autoclave while large fermenters are sterilized by steam generated from distant

boiler.

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Motor power unitCompress Air

filter

Outlets Air

exhaust

Inlets

Monitors

…………

Impeller

Air sparger at the end of air

Agitator

Cooling or heating coils

Baffle

Design of the fermenter (bioreactor)

10

In aerobic fermentations it is important to achieve the optimal oxygen

concentration. It is supplied from compressed, filtered air to the bioreactor and forced

through a sparger to facilitate the dispersion of oxygen to liquid medium. The medium

as well as air bubbles are mixed by impellers arranged on an agitator shaft attached

to bottom or top sealed motor. Careful design is required to achieve optimal oxygen

concentration throughout the fermentation medium since oxygen transfer in large

volume fermenters is difficult. Large fermenters are also supplied with internal baffles

that help in mixing. Aeration and mixing are relatively expensive due to the high-energy costs needed. Fermenters may be supplied with pumps to supply acid or alkali in order to

adjust the pH during the fermentation course. Also pumps to supply nutrients or

antifoams may be also required.

Methods of fermentation:

1- Batch fermentation: the fermenter containing the sterilized culture

medium is inoculated with the microorganism and incubation is allowed to

proceed under optimal conditions for the required period of time. In batch

fermentation, nothing is added to the fermenter during the entire

fermentation process except air, an antifoam agent, acid or alkali to control

the pH. At the end of the fermentation cycle, the fermenter is shut off and

the contents are collected and the product is recovered.

2- Fed-batch process: nutrients are added at intervals as the fermentation

progresses.

3- Continuous Fermentation:, sterile nutrients are added continuously to

the fermenter and equivalent amount of product with microorganism are

simultaneously harvested out of the fermenter. Continuous fermentations

could be achieved through the use of chemostat, or turbidostat. In the

chemostat cell growth is controlled by adjusting the concentration of one

substrate (as a limiting factor). In the turbidostat, cell growth is kept

constant by using turbidity to maintain the biomass concentration and the

rate of nutrient solution addition.

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Scale-up: It is the transfer of small scale laboratory fermentation to an industrial

large scale. Fermentation processes are usually developed in three stages namely

laboratory scale (flasks, laboratory fermenters), pilot plant scale (usually 50-200

liters) and production scale (usually in cubic meter scale depending on the product).

It should be noted that laboratory-scale process may not work or work poorly when

first attempted at large-scale. The aim of the scientists carrying out this development

stages is to find out the optimal fermentation conditions required to maintain the

highest product yield.

Uses of computers in fermentation technology

Data including pH, P02, temperature, pressure, viscosity aeration rate,

exhausted 0 and CO2 2 could be directly fed into computers through sensors and

probes. The whole fermentation process could be done automatically through

special programs, which also alarm when any deviation from the set-up to inform the

production team and sometimes correct it or stop the fermentation process. The data

obtained during the fermentation process can be stored, analyzed and used for

further optimizing the process and can be compared with other batches.

The scheme of fermentation process: 1- preparation of inoculum: The preserved seed let is first either cultivated

on agar slopes or in shaken flasks containing liquid culture medium. A

second series of shake-cultures are made to obtain sufficient inoculum for

small fermenters.

2- Production of the product: Growth obtained in small fermenters is used

as seed for large-scale production fermentations. The inoculum size

usually ranges between 5-10% of the production medium.

3- Recovery of the product: involves the purification of the products. In most

cases, the product represents a very small fraction of the total fermentation

broth and extensive purification procedures must be employed. The

choice of the operations used in recovery depends on the nature of the

desired product, its concentration and stability and the required level of

purity in the end product.

12

The following are the general methods for concentration and or purification of the

product:

i. Centrifugation for separation of cells.

ii. Filtration or sedimentation for separation of cells.

iii. Precipitation

iv. Extraction with solvents.

v. Chromatography.

vi. Fractional distillation.

vii. Ultrafiltration.

viii. Reverse osmosis and dialysis.

It must be noted that in recovery of the product there are always recovery

losses, which depends on the sensitivity the product and the number of purification

stages.

Categories of products

1- Biomass: The product may be the cells themselves e.g. baker’s yeast

or single cell protein (SCP).

2- Enzyme: e.g. amylase, penicillin acylase

3- Metabolites: either primary metabolite such as citric acid which are

usually produced during the logarithmic phase of growth (trophophase)

or secondary metabolites such as antibiotics, alkaloids or glycosides

which are produced during the stationary phase (idiophase).

4- Biotransformation product: e.g. steroid transformation

5- Biodegradation product: degradation of xenobiotics (insecticides and

petroleum oil)

6- Immunological product: vaccines and monoclonal antibodies (MCA).

7- Energy: alcohol, methane (biogas).

8- Genetically engineered therapeutic protein: Insulin, growth hormone

and interferon.

9- Intra or extracellular accumulation of metals.

10- Plant tissue culture: cell suspension, callus and hairy root.

13

I- Production of biomass (cell mass) Cells of microorganisms as industrial products

Microorganisms may constitute an industrial product as the case with single

cell protein (SCP), microbial insecticides and the symbiotic nitrogen-fixing bacteria. At

the end of fermentation, the cells are harvested and used as cakes or dried

materials.

1) Baker’s yeast Baker’s yeast (Saccharomyces cervisiae) is used mainly for baking. Dough

amylases convert starch to sugars, which are then utilized by the yeast producing

CO2, and ethanol that leavens the dough and causes it to rise and increase in

volume. Certain yeast strains produce characteristic taste and flavor to the bread.

Baker’s yeast is produced by growing the organism in molasses mineral salt

medium at a 30oC and pH 4.5. Molasses are usually feed gradually to maintain the

sugar concentration between 0.5-1.5%.

2) Single-cell protein (SCP) Torula utilis or Hansenula anomala (microbial cells) for use in human and

animal feed (single cell protein, SCP) could serve as new sources of proteins and

vitamins. The use of microorganisms, which grow rapidly and produce high yield rich

in proteins are potential replacement to animal and plant proteins. However, there

are psychological barriers to the use of microorganisms as food, although

microorganisms are used as food in case of mushrooms or incorporated in certain

foods. It seems that SCP will play a major role in human nutrition only via feeding

animals

SCP for use as human food must be additionally processed before use by

humans to reduce their content of nucleic acid. Microbial protein in the form of SCP

compete with soybean meal, the cheapest protein source. Soybean meal is a by-

product of soy oil production. Since the costs of the raw materials are important

components for microbial biomass production, locally available cheep substrates

such as alkanes, methane, methanol, cellulose and many other waste materials

should be considered. SCP is now produced for animal feed using waste material

from petroleum industry.

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3) Microbes as insecticides There is both public and scientific concern about the widespread use of

chemical insecticides. These synthetic chemicals may accumulate in soil and reach

the underground water bodies causing adverse effects on the environment and public

health. Among the most attractive alternatives is the use of bioinsecticides. They are

either microbes that can infect and kill harmful insects (e.g. Bacillus popilliae) or

microbes that can produce more safe compounds that can kill harmful insects (e.g.

Bacillus thuringiensis).

The spores of Bacillus popilliae can survive in the soil for long time and can

infect more and more larvae. Bacillus thuringiensis is an endospore-forming

bacterium that produces several insecticidal toxins including a water-soluble toxin

called α-exotoxin and a protein crystal called parasporal crystal or δ-endotoxin.

Different strains of Bacillus thuringiensis are used as bioinsecticides for control of

different insect larvae including both insects of agricultural importance and those that

have medical importance.

The commercial product made from Bacillus thuringiensis consists of a mixture

of the protein toxin and the bacterial endospores. These products are widely applied

today for biological control of insects.

4) Rhizobium as a fertilizer The most common nutrient limiting the production of the agricultural crops is

nitrogen. In agriculture, significant economic savings can be realized if plants could

be grown without the need for adding nitrogen fertilizers. Elimination such fertilizers

will also reduce the problems associated with nitrification and contamination of

ground water.

Rhizobium species are Gram-negative rods that form an association with

leguminous plants (symbiosis). They exhibit high nitrogen-fixing ability (2~3 times

higher than the free living nitrogen-fixing soil bacteria). This symbiotic Rhizobium

species are utilized commercially.

15

II- The use of microorganisms in food Microorganisms are used to produce fermented food products, such as baking

of bread, production of fermented dairy products (yogurt, cheese), fermented fish and

meat, vinegar, alcoholic beverages and fermented vegetables such as pickles olives,

soy sauce and sauerkraut. These fermentations usually uses the natural microbiota

associated with these vegetables. The production of vinegar (acetic acid): Production of vinegar from alcohol has been known for thousands of years. It

was simply produced by leaving wine bott1es in open air. Commercially vinegar is

produced in two steps. In the first step carbohydrates are converted to alcohols by

Saccharomyces cervisiae followed by oxidative transformation of alcohol to acetic

acid by Acetobacter or Gluconobacter species. The starting materials for vinegar

production may he fruits such as grapes (wine-vinegar), oranges, apples or pears

(cider vinegar), corn or vegetables such as potatoes.

III- Microbial enzymes Enzymes could be obtained from different biological sources (animals, plants

and microorganisms). However, microbial enzymes have the advantages of being

large in number and provides an unlimited supply (most economic). Microbial

enzymes are either extracellular or intracellular. Extracellular enzymes are obtained

from the culture filtrate and intracellular enzymes are obtained from the cell

homogenate after cell lysis.

1) Amylase

Alpha amylases are enzymes that hydrolyze starch to short chain polymers

(dextrins), which are then hydrolyzed to disaccharide maltose and finally to glucose

by beta amylases. Amylases are mainly used in production of sweeteners in the food

industry, in the removal of starch sizing from cloth and in the removal of spots in dry-

cleaning industry. Acid resistant amylase is used as digestive aid.

Fungal-amylases are commercially produced by Aspergillus species and

bacterial amylases are produced Bacillus species.

2) Proteases: Are a class of enzymes that hydrolyze the peptide bonds of proteins.

Proteases are used primarily in the detergent and laundry industry and in dairy

16

industry. They are also used in leather and pharmaceutical industry and waste

treatment. Commercially they are produced from Bacillus sp. or from Aspergillus

species. Alkaline proteases that act over a wide range of pH and at high

temperatures in the presence of detergents are now produced by genetic

engineering. Before being added to detergents, proteases must be prepared in an

encapsulate form as the dry enzyme powder might result in allergic reactions when

inhaled by production workers or users.

3) Lipases: Are a class of extracellular enzymes, which hydrolyze glycerol esters

(fats) into di- or monoglyceride and fatty acids. They are produced by bacteria,

yeasts and fungi. Lipases are used as digestive enzymes to supplement pancreatic

lipases. They also find use in dairy industry since fatty acids imparts taste of cheese.

4) Glucose isomerase: it causes isomerization of glucose to fructose which is 1.5

times as sweet as glucose and twice as sweet as sucrose. It is an important

sweetening agent in the manufacture of many foods and beverages.

5) L-Asparaginase: It is used as antitumor agents in the treatment or leukemia and

lymp1oma. The enzyme is produced by several bacteria and is usually produced at

low oxygen partial pressure. The enzyme is intracellular and must be purified to be

suitable for injection into human body.

6) Fibrinolysin (streptokinase): Produced from streptococci and used commercially

for treatment of thrombosis

7) Penicillin acylase: It is used for the preparation of 6- aminopenicillanic acid

(6APA), the starting material for the preparation of semisynthetic penicillins. 6-APA is

prepared by hydrolyzing natural penicillin G with penicillin acylase to 6APA and

phenyl acetic acid. 6 APA is then chemically acylated with different acyl groups to

produce semisynthetic penicillins.

8) Pectinase: Are enzymes, which hydrolyze pectin to low molecular weight

dextrins. It is used in food industry to clarify fruit juices.

17

9) Glucose oxidase: It has medical application in the determination of blood glucose

level and in food for the removal of oxygen from various food products to prevent

their deterioration.

Enzyme stabilization During storage or use, physical, chemical and/or biological factors may

inactivate enzymes. There is thus a need to stabilize enzymes. The methods used to

stabilize enzymes include:

a) Substrate stabilization: In this method, the active sites of the enzyme (which are

responsible for its specific activity) are stabilized by adding its substrate. Glucose

isomerase, for instance, can be stabilized against heat damage by adding glucose

and α-amylase can be stabilized by adding starch.

b) Solvent stabilization: Many solvents stabilize the enzyme when used at low

concentration. However, high concentration cause denaturation of enzymes. C) Cation stabilization: For example calcium ions were found to stabilize the

tertiary structure of proteases and α-amylases.

d) Immobilization:

Immobilized-enzyme technology Enzymes can also be stabilized by immobilization. In all the stabilization

methods listed above the enzymes can be stored softly but since they are still

soluble, they can not be recycled after use. On the other hand, immobilized enzymes

have the advantage of being no longer soluble and can be reused or even used

continuously.

Immobilization of enzymes is carried out either by adding an enzyme molecule

to another enzyme by cross-linking, by bonding molecule to a carrier (carrier-bond

enzymes) or encapsulation.

Cross-linked enzymes are prepared by linkage of enzymes with each others

by means of two or more functional groups. Glutraldehyde has been used as a

polymerizing agent for immobilization of several enzymes.

Carrier-bound enzymes are prepared by bonding the enzyme to a carrier

through adsorption, covalent bonding or ionic bonding. Adsorption methods although

causes the least damage are easily accomplished, the bonding is weak and thus the

enzyme may be eluted from the carrier easily during use. Similarly, ionic bonding is

18

weak and can result in enzyme loss from the carrier. On the other hand, covalent

bonding is strong bonding force and is the most widely used commercially. However,

the preparation of a covalently bonded enzyme is expensive and difficult to carry out.

Encapsulated enzymes are prepared by enclosing the enzyme physically in,

microcapsules, gels or fibrous polymers. There must be pores small enough to trap

the enzyme molecules so that they can not be washed out, which are still large

enough to permit the diffusion of the substrate and production.

Similarly whole cells with enzymatic activities can be immobilized. The use of

enzymes immobilized within cells have several advantages including shorter recovery

and purification times (i.e. 1ow costs), the cofactors that may be required for enzyme

activity may be already present within the cells and multi-enzyme reactions can be

carried out. However, undesirable side reactions by other enzymes ma occur.

Biosensors Immobilized enzymes had also found wide application in manufacture of the

so-called enzyme electrodes or biosensors. These biosensors are designed for

potentiometric or amperometric assay of substrates such as glucose, urea and amino

acids.

The electrode is composed of an electrochemical sensor in close contact with

a thin permeable membrane, in which the enzyme is embedded, capable of reacting

specifically with a specific substrate. Depending on the enzymatic reaction a small

molecule is produced (e.g. 02, H+, C02) which, can be readily detected by the specific

sensor. The magnitude of the response determines the concentration of the

Immobilized-enzyme technology

Substrate for enzyme

Water-insoluble

Product of enzyme catalyzed

particulate matterEnzyme molecules bound to particulate matter

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substrate. For example glucose oxidase electrodes has been designated for use in

determination of glucose concentration. These electrodes are essentially glucose

oxidase layered over a platinum oxygen electrode. The more glucose concentration

in the sample, the more oxygen consumed by the reaction, the smaller the amount of

oxygen detected by the electrode.

Another area for which biosensors have been designed is the detection of pollutants

and pathogens. This has been designed using bacteria that can locate biologically

active pollutants. Bacterial biosensors require a receptor that is activated in the

presence of pollutants and a reporter that will make such a change apparent.

Electrolyte Anode

Cathode

Teflon membrane

For example the lux operon from Photobacterium was studied as a reporter.

This operon contains an inducer and structural genes for the enzyme luciferase. In

the presence of a coenzyme called FMNH2, luciferase react with the molecule in such

a way that the enzyme-substrate complex emits blue-green light, which then oxidizes

the FMNH2 to FMN. Therefore a bacterium containing the lux gene will emit visible

light when the receptor is activated.

The concept have been used for preparation of Lactobacillus bacteria

containing the lux operon for use in the detection of the presence of antibiotics in milk

to be used for cheese production. In another application, the marine bacterium

Photobacterium is used directly to detect pollutants. Other biosensors use

recombinant bacteriophages that contain the lux genes for detection of Listeria and

Escherichia coli in foods and to detect drug-resistant mycobacteria.

+

+ + +

+

+++

Product Enzyme Substrate

Semi-permeable membrane

Diagram of simple biosensor

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Biochips It is possible to combine enzymes (or biological systems) with semiconductors.

The type of semiconductors used for this purpose are all field effect transistors (FET).

When an FET is combined with an enzyme it is called ENFET. The active area of the

semiconductor used in ENFET is very small and it is possible to implant them in

human body for different medically useful applications.

IV- Extracellular polysaccharides Microbial cell surface (cell wall and glycocalyx) is rich in polysaccharides.

When the glycocalyx is organized and attached firmly to the cell wall, it is called

capsule, but if is disorganized and attached loosely to the cell wall, it is described as

a slime layer. The slime layer tends to be soluble in water, so that the medium

containing the bacteria becomes highly viscous. The polysaccharides that covers the

microbial cell surface are also termed exopolysaccharides or biopolymers. Such

water-soluble biopolymers are rapidly emerging as source of gums. Traditional gums

are produced from plants and algae. Microbial gums are used as gelling agents,

thickening agents, emulsifying agents, stabilizers, suspending agents, binders and

lubricants. They have wide application in food, pharmaceutical and other industries

due to their unique physical properties. Microbial gums also are not subjected to crop

failure due climatic conditions and are supplied in constant quality. Microbial exopolysaccharides are classified into two types namely,

homopolysaccharides e.g. dextran or cellulose and heteropolysaccharides e.g.

xanthan.

1- Dextran: (polymer of α-D-glucose), produced commercially by the Gram-negative

bacterium Leuconostic mesenteroid, used as plasma substitute and as complex with

iron for iron deficiency anemia.

The growth and production medium contain sucrose, organic nitrogen and

phosphates. In the production of dextran, the substrate does not enter the cell.

Instead, the microorganism produces an extracellular inducible dextransucrase

enzyme, which hydrolyze sucrose to glucose and fructose and at the same time

utilizes glucose for dextran synthesis (polymerization). Commercially the process

could be carried by the direct or indirect methods:

21

i- Direct method: The medium is supplied with enough sucrose (10 %) to

support growth and dextran synthesis. The process is carried out under

anaerobic conditions at 25o oC-30 C and pH 5. Viscosity increases during

the course of fermentation, which takes about 30 hrs. The problem with

this method is that during recovery of the product, which depends on

precipitation with ethanol, bacteria may be trapped with the product.

ii- Indirect method (Enzymatic): The bacteria are allowed to grow in

presence of 3% sucrose, which supports growth only, then the cells are

separated and the medium containing the enzyme is then used to

transform sucrose into dextran. The enzymatic process is carried out at

25o oC-30 C and pH 5. Dextran is recovered by fractional precipitation

with increasing amounts of ethanol to obtain the proper molecular

weight and higher molecular weight dextran is again hydrolyzed with

acid and further fractionated.

2- Xanthan: (heteropolysaccharide) produced commercially by Xanthomonas

campestris. Production of xanthan is carried out in medium containing carbohydrate

(glucose, sucrose, or corn starch hydrolysate), nitrogen source (yeast extract,

peptone, ammonium nitrate) and salts, pH is controlled to 7. Xanthan is recovered

by precipitation with methanol. The precipitated xanthan is then dried and ground.

Xanthan is used in paints, printing, and textile industries as well as in oil

industry as drilling fluid additive and for enhanced oil recovery.

22

V- Production of metabolites

Trophophase Idiophase Log b Time

Metabolites are either primary or

secondary. Primary metabolites are

essential for life and reproduction of cell.

They are synthesized during the lag and

logarithmic phases of growth

(trophophase). Primary metabolites of

biotechnological importance include

organic acids, butanol, acetone and

many others. On the other hand,

secondary metabolites seem to be non

essential for growth and reproduction. They

are produced during the stationary phase of

growth (idiophase). Examples of secondary

metabolites produced by microorganisms

are antibiotics and glycosides by plants.

Primary metabolic pathways are

similar in all microorganisms whereas

secondary metabolites are formed only by

few organisms and their formation is

extremely dependent on environmental conditions. Moreover the regulation of

secondary metabolites biosynthesis differ significantly from that of the primary

metabolites.

Screening microorganisms:

In screening for new metabolites, researchers try to isolate strains from the

environment with the hope that they are potentially valuable in producing a

commercially useful product. Of the many species, relatively few possess the criteria

for an industrially valuable microorganism.

The search for antibiotics in the pharmaceutical industry presents a good

example of how screening programs are employed to select microorganism for

industrial applications. Samples from various sources are collected and examined as

a potential source of antibiotic-producing microorganism. A useful antibiotic-

23

producing strain must produce metabolites that inhibit the growth or reproduction of

pathogen.

The success of a screening program depends on both the kind of organism

used and the method for detection of activity. The choice of strain is reported to have

a 30-40% influence on the outcome and the test procedure has a 60-70% influence.

A positive result in such a primary screening procedure does not ensure the

discovery of an industrially useful strain. It simply identifies strains of microorganisms

that have the potential for further development. The success of a screening

procedure is quite dependent on the development of intelligent tests with which

known or undesirable antibiotics can be eliminated and compounds with the required

properties can be recognized.

V- Primary metabolites

A- Organic acids

1. Citric acid: Citric acid has long been isolated from unripe fruits but today its production is

entirely produced microbiologically by fermentation. It is used widely in food industry

especially in the production of soft drinks. In pharmaceutical and cosmetic industry,

citric acid is used as preservative for different products and as iron citrate for

treatment of iron-deficiency anemia. Citric acid is also used in the detergent industry

to replace the use of polyphosphates, which have been prohibited in many localities

worldwide.

Citric acid is produced commercially using mutants of Aspergillus niger and

Aspergillus wentii.

Citric acid is a primary metabolite in the tricarboxylic acid cycle. The main

carbon source used for citric acid production is sugar cane syrup, sugar cane

molasses or sugar beet molasses. Starch can also be used if amylases are formed

by the producing fungus (or should be added to the fermentation broth). Beside the

use of optimum carbon and nitrogen source concentrations, metal salts and

phosphate concentration in the medium were found to influence the yield. For this

reason, the carbon source must be re-treated with either precipitation or exchanger

to remove cations. Metals are removed from molasses with calcium hexacyanoferrate

or with cation exchanger. The use of methanol during fermentation render the

mycelium not sensitive to the presence of iron in the medium (i.e. poison the M.O.).

24

The fermentation should be carried out as two phase fermentation, during the first

phase the conditions should be adjusted for maximum growth to obtain enough

mycelium, followed by restriction of cations to enhance the production of citric acid.

Optimum production is achieved at low pH (3.5). The low pH, however, have

the advantage of lowering the risk of contamination. Citric acid is produced both by

surface and submerged fermentation.

Surface processes use solid or liquid media. Those employing solid

substrates may use either wheat bran or pulp from sweet potato starch production as

culture medium. After sterilization of the medium, the medium is inoculated with

Spores and spread in shallow layers (about 5 cm thick) on trays and incubated at

28oC. At the end of the process, which usually takes 5-8 days, the produced citric

acid is extracted with hot water and then purified. Although the process have the

advantage of being simple and needs low investment, the labor costs high. In this

process, the Aspergillus niger strains are not as sensitive to trace elements as in the

submerged processes.

Submerged cultures are more widely used today for citric acid production. The

fermenter used must be constructed from resistant material such as stainless steel.

During citric acid production the aeration rate must be kept at optimum levels as the

fungus require little oxygen, but it is sensitive to oxygen deficiency. The structure of

the mycelium formed in submerged culture during the trophophase is very important

for high citric acid yield. Very small solid pellet is the best for optimum citric acid

production. On the other hand, loose, filamentous mycelium with limited branches

produces little citric acid.

For recovery of citric acid, the mycelia are removed by centrifugation. If oxalic

acid is present as byproduct, it must be precipitated first as calcium oxalate at low pH

and the precipitated calcium oxalate is removed with the mycelium. Citric acid is then

precipitated as calcium citrate at pH 7.2 and 70-90oC. The precipitate is then filtered

and dried or subjected to further purification if needed.

2. Gluconic acid: Gluconic acid finds wide application in pharmaceutical industry, metal industry

and leather industry. Calcium gluconate is used to supply calcium to the body,

ferrous gluconate is used to supply iron for the treatment of anemia.

Gluconic acid is produced commercially by Aspergillus niger using the

25

submerged culture. The process takes about 36-48 hr when all glucose is utilized,

further incubation results in utilization of gluconic acid. The fermentation is conducted

under highly aerobic condition (aeration rate between 1-1.5vvm), the pH is controlled

to 5.5 using calcium carbonate and the temperature is kept at 30oC. 0.1% boron is

added to the fermentation medium to prevent precipitation of calcium gluconate (35%

of calcium gluconate remains in solution). Glucose is usually used as carbon source.

Borate is added to the medium to stabilize calcium gluconate (which have low

solubility) and prevents its precipitation.

Gluconic acid is recovered by addition of calcium hydroxide to the

fermentation broth after removal of the mycelium. The free acid is liberated from the

crystalline calcium gluconate by the addition of acid.

3. Lactic acid:

Lactic acid is a valuable industrial product because its derivatives have a

variety of uses. Calcium lactate is used in the treatment of calcium deficiency, iron

lactate is used in treatment of anemia and sodium lactate is used as placticizer and

moistening agent.

Several carbon sources such as corn-starch, potato starch, molasses and

whey can be used for the production of lactic acid. Substrates containing starch must

be hydrolyzed first to glucose either by acid treatment or enzymatically. When

glucose is used as carbon source Lactobacillus delbruecki is used for production,

Lactobacillus bulgaricus is used with whey, and Lactobacillus pentosus is used with

sulfite waste liquor. Ammonium phosphate and trace amounts of other nitrogen

sources are usually used.

Lactic acid fermentation is carried out under anaerobic or microaerophilic

conditions at temperature range of 40-500C and pH range of 5.5-6.5. Calcium

hydroxide is added to the fermentation broth to control the pH. The fermenter is

agitated for mixing but not aerated. Fermentation usually takes about 2-3 days.

At the end of fermentation the fermentation mesh is boiled to coagulate the

microbial proteins which is trapped on a filter and dried for use as animal feed

supplement. The filtrate, which contains the soluble calcium lactate, is concentrated

by using a vacuum then subjected to further purification.

26

B) Vitamins Although microorganisms produces many vitamins, commercial production for

economical purposes is restricted for the production of vitamin B and riboflavin. 12

1. Vitamin B (cyanocobalamine): 12

Vitamin BB12 can be commercially produced by Propionobacterium shermanii.

The vitamin is used for treatment of pernicious anemia and as animal feed

supplement.

The culture medium consists of soybean meal or fish or meat extract as

nitrogen source and cobalt. The carbon source is either oil or when easily utilizable

carbon is to be used it is preferable to add it continuously during the fermentation

process.

The fermentation is carried out in two stages process. In the first anaerobic

phase, which lasts between 2-4 days, cobinamide is produced. In the second aerobic

phase, which lasts between 3-4 days cobalamine (coenzyme B12) is produced. Both

stages can also be operated continuously in two fermenters operated in cascade

fashion.

Cobalamine is almost completely intracellular and is released into solution by

heat treatment at 80-120oC for 10-30 minutes at pH 6.5-8.5. Cobalamine is then

converted to cyanocobalamine with KCN. The vitamin is then purified by

chromatography.

Vitamin BB12 could be also produced using Bacillus megatherium. When

radioactive Vitamin B12 (used in schilling test for pernicious anemia) is required,

radioactive cobalt is added to the medium.

(Riboflavin): 2. Vitamin B2

Riboflavin can be produced by Ashbya gossypii or Eremathecium ashbyii.

Both are cotton pathogens and so are not suitable for production of riboflavin in

Egypt. The medium used for production contains corn steep liquor, peptone and

soybean oil. The fermentation takes 7 days with an aeration rate of 0.3 vvm at 28oC.

Riboflavin is formed both extracellular and bound to mycelium. The bound

vitamin is released from the cell heat treatment at 120oC for 60 minutes and the

mycelium is separated and discarded. The culture filtrate may be dried and used as

animal feed additive; for pharmaceutical use the vitamin is extracted with butanol.

27

C) Amino acids Many microorganisms can synthesize amino acids from inorganic nitrogen

compounds such as ammonium sulfate. In some instances the amount of amino acid

synthesized can exceed the cells need for that amino acid. The excess amino acid

may be excreted into the culture medium. Glutamic acid, lysine and tryptophan for

example are produced industrially by microbial strains that produce an excess of

these amino acids.

Amino acids find wide applications in the food and chemical industry as

starting materials. In food industry, amino acids are used to enhance flavors (e.g.

sodium glutamate), as antoxidants (e.g. L-cysteine and L- tryptophan). In medicine,

amino acids are used as ingredients in infusion solutions. In chemical industry, amino

acids are used as starting materials for the manufacture of polymers (e.g. polyalanin

fibers).

1. L-Glutamic acid: Glutamic acid is produced commercially by a mutant strain of

Corynebacterium glutamicum. The key precursor of glutamic acid biosynthesis is α-

ketoglutaric, which is formed in the tricarboxylic acid cycle. α-ketoglutarate is then

converted into L-glutamic acid through reductive amination with free ammonium ions.

Sucrose and molasses are widely used as carbon sources. Ammonium salts or

ammonia can be used as nitrogen source. Ammonia feeding permits pH control of

the process. High PO causes growth and production inhibition. 4

2. Lysine:

Lysine is an amino acid essential for human and animal nutrition. Lysine is

produced commercially by microbial fermentation using an auxotroph of

Corynebacterium glutamicum. Cane molasses is usually used as carbon source and

ammonia or urea as nitrogen source. Ammonia feeding permits pH control of the

process (near neutrality).

3 . L-Tryptophan

Studies using recombinant DNA technology led to a 230-fold increase in

tryptophan synthetase enzyme of Escherichia coli. Addition of indole and serine to an

extract of this enzyme results in conversion of indole into L- tryptophan. The yield is

so high to the extent that part of the tryptophan precipitated out (nearly 78 g/l).

28

V) Secondary metabolites

Antibiotics Antibiotics are compounds produced by living microorganisms and are used

as therapeutic antimicrobial agents. They are effective against microorganisms in

low concentration.

Penicillin was the first discovered antibiotic by Fleming in 1929 as a product

from penicillium notatum contaminating Staphylococcal culture. In 1940, during World

War II, the British scientists continued the research, but they were unable to develop

an industrial. However, in USA it was suggested that through the use of corn steep

liquor, which contain phenyl alanine, it was possible to develop a fermentation

process. Later on penicillium notatum was replaced by Penicillium chrysogenum,

and a submerged fermentation process with high yield of penicillin was developed.

Later, an intensive screening programs for antibiotics started and streptomycin

then chloramphenicol and several hundreds of antibiotics were discovered.

1. Penicillin: Penicillin is an acylated 6-aminopenicillanic acid (6-APA). 6APA consists of a-

thiazolidine ring fused to a β-lactam ring.

Acylase

β-lactamase

Thiazolidine ring β-lactam

ring

Biosynthesis of penicillin depends on the condensation of valine and cysteine

amino acids to yield 6APA and L-α-aminoadipate as side chain, which is then

replaced by various side chains during the fermentation. If the fermentation of

penicillin is conducted without the addition of side chain precursors, a mixture of

natural penicillins are produced. Only benzyl penicillin or penicillin G of this mixture is

29

therapeutically effective and all the other produced penicillins are by-products that

create problems during the down-stream processing. However, if phenyl-alanine or

phenyl acetic acid are added as a side-chain precursor, so that only the desired

penicillin G is produced.

Penicillin was originally produced by a strain of Penicillium notatum , which

produced pigments and only about 2 international units per milliliter (lU/ml) of

penicillin by surface culture fermentation . The yield of penicillin has been increased

about 50,000 times through the use of the non pigmented Penicillium chrysogenum

mutants, proper medium, submerged fermentation and proper recovery.

The medium used today contained 10% total glucose (or molasses), 4-5%corn

steep liquor, 0.5-0.8% total phenylacetic acid and 0.5% total vegetable oil and the

process was carried out by continuous feed.

Easily utilizable sugar (glucose) Acids Gas

Slowly utilizable sugar (lactose) Acids Gas

The pH of the fermentation during the production of penicillin should be kept

constant at 6.5-7.0 and the optimal temperature range between 25-270C. The

aeration rate is between 0.5-1.0v/v depending on the strain used.

The course of the fermentation takes about seven days (longer times when very

large fermenters are used). During the first 40 h of fermentation (growth phase) cell

mass is built up, followed by the penicillin production phase, which extends for further

50 h. By fed batch the production phase could be extended to up to 160 h. During

Growth

Glucose utilization

pH change

Lactose utilization

Penicillin production

Rapid Rapid

Slow Rapid

30

the growth phase the M.O. utilizes glucose (easily utilizable) sugar with the

production of acids leading to drop in pH, however, the acids are first utilized before

the M.O. starts to utilize lactose (slowly utilizable) and the ph starts to rise again.

During the production the M.O. utilized lactose with no acid production and so the pH

remains constant around neutrality. Also at the end of the growth phase phenyl acetic

acid (precursor of benzyl penicillin) is released from phenyl alanine found in corn

steep liquor. The last (third) phase starts when the mycelium starts to lyse and so the

pH starts to increase. Penicillin should be recovered before the third phase starts.

For recovery of penicillin, the liquid medium containing the penicillin is

separated from the fungal cell using rotating vacuum filter. The fungal biomass is

scraped from the surface of the filter drum, dried and used as animal feed

supplement. The filtrate is cooled to 2-30C, the pH is adjusted to liberate the free

acid. Penicillin is extracted from the filtrate using organic solvent and then extracted

back into the aqueous solution after adjusting the pH. Potassium ions are added and

the result in crystalline potassium salt of penicillin G is removed by filtration (or

centrifugation) and then dried to yield a penicillin salt with purity over 99%.

2. Streptomycin: Aminoglycosides are oligosaccharides antibiotics primarily effective against

Gram-negative bacteria, but mainly used in the treatment of tuberculosis.

Streptomycin is produced by strains of Streptomycin griseus. As in penicillin

fermentation, spores of Streptomyces griseus are inoculated into a medium to

establish a culture with a high mycelial biomass for use as inoculum. The mycelium

inoculum is used to initiate the fermentation process in the production fermenter.

The medium used for the production contains soy-bean meal as the nitrogen

source, glucose as the carbon source and sodium chloride.

Oxygen supply is adjusted between 0.5-1.0vvm and the temperature is kept

between 28-30oC with pH in the neutral range. The length of the fermentation ranges

between 4-7 days depending on the strain.

During the first phase of streptomycin fermentation, there is rapid growth of

Streptomyces griseus, with production of mycelial biomass. The pH of the medium

rises due to proteolytic enzymatic activity and release of ammonia from soybean

31

meal. During the second phase, streptomyin accumulates in the medium. The

remaining glucose in the medium and the ammonia released from the soybean meal

are consumed during this phase. After depletion of the carbohydrate from the

medium, the production of streptomycin ceases and the bacterial cells begin to lyse.

For recovery of streptomycin the mycelium is separated by filtration.

Streptomycin is adsorbed on charcoal or ion exchange resin and then eluted from the

charcoal with acid alcohol. The antibiotic is then precipitated with acetone and further

purified by chromatographic methods. Infection with bacteriophages may cause

problems in streptomycin production. To avoid such problem, it is better to isolate

and use of phage-resistant mutants for production.

Other commercially produced antibiotics Antibiotic Producing microorganism

Bacitracin Bacillus lichenformis

Polymyxin Bacillus polymyxa

Spectinomycin Streptomyes spectabilis

Kanamycins A, B & C Streptomyces kanamyceticus

Tobramycin Streptomyces tenebrarius

Erythromycin Streptomyces halstedii

Amphotericin B Streptomyces nooses

Nystatin Streptomyces noursei

Neomycins B & C Streptomyces fradiae

VI- Bioremediation

Bioremediation denotes ability of microorganisms to detoxify or degrade toxic

pollutants (mainly xenobiotics) from the environment. In other words, the use of the

metabolic activities of microorganisms to change an undesirable chemicals into ones

that has less objectionable properties.

During the last few decades man has used fossil fuel resources and produced

many novel synthetic compounds (xenobiotics). The disposal or accidental spillage of

these compounds creates serious environmental pollution problems. One of the

approaches to save the environment from the adverse effects of these pollutants is

the use of bioremediation.

The capacity of different microorganisms to degrade different organic

compounds and transform various substances forms the basis for bioremediation.

32

Bioremediation of petroleum hydrocarbons. chlorinated solvents and chemical

insecticides residues are among the most important bioremediation processes.

Oil-spills from wrecked tankers represent some of the most dramatic examples

of chemical pollution. The economic losses from contaminated fisheries and beaches

can be enormous. Natural bioremediation occurs and can be enhanced by providing

the resident bacteria with nitrogen and phosphorous through the addition of a

fertilizer since petroleum hydrocarbons are deficient in essential elements such as

nitrogen and phosphorous. Another approach to clean up the oil from a spill is to inoculate the spill area

with a microorganism that can degrade the oil. For example a genetically engineered

strain of Pseudomonas putida has been constructed for this purpose. The strain has

the ability to metabolize hydrocarbons present in crude oil (octane, xylene, and

naphthalene).

Selenium at high concentration is toxic to humans and animals but some

bacteria have a protective mechanism that prevents selenium from being toxic to

them. This mechanism involves converting selenium to less toxic form. Accordingly

such strains can be used to protect the pollution of irrigation water with selenium

which can kill wildlife.

The biodegradation of the world s most widely used herbicide glyphosate by

Flavobacterium species, Agrobacterium and Achromobacter species is extensively

studied. It is now used in industrial waste streams by immobilized bacteria

technology. Pilot field trials were carried out in aerated reactors maintained at pH 7-8

and 250C and supplemented with ammonium nitrate as nitrogen source. The results

showed 96% degradation of glyphosate after 21 days.

The design of novel synthetic compounds, which could be degraded, is now

studied. For example, alkyl sulfonates, which is detergents is resistant to

biodegradation, due to branching of the alkyl chain. However, changing the design of

this synthetic compound to a linear chain renders the product easily degradable.

VII- Microbial transformation Microorganisms are able to modify a wide variety of organic compounds. This

process is termed microbial transformation or biotransformation. Microbial

transformation is a very helpful tool for:

33

1- the production of more useful and expensive products.

2- Elucidation of the chemical structure of complex compounds.

Biotransformation reactions include various types of hydroxylation, oxidation,

epoxidation, reduction, epoxide cleavage, hydrolytic or condensation reactions. The

method applies for steroids, hydrocarbons, terpines and antibiotics. The

transformation reaction is considered to be a detoxification reaction by the

microorganism using induced enzymes.

Different biotransformation processes using growing cultures, resting cells,

spores, immobilized cells, enzymes or immobilized enzymes are currently applied.

When growing cells are used, the microorganism is cultured in a suitable medium

until a suitable growth of the culture is reached. The substrate is then added in a

concentrated form and the biotransformation is monitored until maximum product is

reached. The use of spores or resting cells has many advantages over growing

cultures processes including reduction of cost and the lowering the risk of

contamination and reduction of byproducts.

Steroid biotransformation: Steroids are group of lipid compounds having the same basic cyclopentano

phenanthrene nucleus. Steroids such estrogen, progesterone and androgens are

used as therapeutics. Derivatives of progesterone and estrogens are used as

contraceptives and derivatives of cortisone are used as anti-inflammatory in

rheumatoid arthritis.

Steroids can be produced by chemical synthesis, but the process is laborious

and expensive. For instance, the chemical process of conversion of deoxycholic acid

(bile acids) to cortisone required 37 steps and yielded 1g cortisone from 615g

deoxycholic acid. The price of 1g cortisone was 200$. However, the price was

reduced to one $ or less when the process was conducted by Rhizopus nigricans.

Low-cost sterols of animal origin such as cholesterol and of plant origin such

as diosgenin are converted chemically to progesterone which in turn is used as

substrates for biotransformation.

Description of fermentation:

1- The microorganism is allowed to grow for about 17-48 h under controlled

conditions (pH, oxygen, temperature, …etc.)

2- The steroid substrate is added in suitable solvent.

34

3- The conditions are readjusted for steroid transformation; sometimes the

growth medium is different from that used for transformation.

4- Recovery of the product

5- Removal of the mycelium by filtration or centrifugation.

6- Extraction of the steroid product with organic solvent and its crystallization.

Steroid transformation by fungal spores

Fungal spores are often active as vegetative cells in transformation of steroids.

The microorganism is allowed to grow in presence of the steroid as inducer for long

period to produce enough spores. Spores are then collected in buffer, purified and

freed from any vegetative debris. Spores are then mixed with the steroid substrate in

sugar medium (no nitrogen source to avoid contamination) and transformation is

carries out as under vegetative cells.

Steroid transformation by fungal spores provides the following advantages:

1- Simple medium.

2- No fear of contamination.

3- After transformation spores are separated and recycled.

4- Ease of recovery of the product.

5- Cost is much reduced.

VIII- Energy and biotechnology

The increasing fuel prices, diminished reserves and unpredictable supplies are

forcing many industrialized nations to seek alternative fuel resources. Among the

various alternatives is the production of energy using biomass including the ethanol

production by fermentation and methane gas (biogas) production by anaerobic

digestion.

The success of the commercial production of fuel using biomass depends on

finding the right microorganisms that are able to efficiently produce the desired fuel

using an inexpensive available substrates. The process can be more attractive

economically and environmentally when waste materials such as municipal garbage,

sewage and industrial wastes can be used as substrates.

1. Ethanol: Ethanol production as an alternative liquid fuel is one of few viable options that

are currently available. The technology is already well established and is particularly

35

valuable in countries that have abundant supplies of plant residues.

The largest ethanol program is in Brazil. Brazil produces and uses large

amounts of ethanol as fuel for cars (20% ethanol + 80% gasoline). Cars were also

built or converted to run on pure hydrated ethanol (96% ethanol + 4% water).

Gasohol (gasoline containing 10% ethanol) is also sold in the USA. Other

programs for ethanol production are also reported in Australia, Indonesia,

Philippines, Sweden and many other countries.

Cost reduction for successful production can be achieved by finding strains

that can readily utilize waste materials that can tolerate high concentration of ethanol

resulted from its accumulation during the fermentation process, and/or by reducing

the recovery costs through. The use of strains that can grow at temperatures above

the boiling point of ethanol. Among the organisms that have been extensively studied

for alcohol production are Zymomonas mobilis and Thermoanaerobacter ethanolicus.

2. Methane: Methane, or natural gas or biogas is a microbial product of the anaerobic

digestion of organic matter. Methanogenesis is a widely used process in organic

waste disposal. Methane is produced by methanogenic bacteria. The methanogenic

bacteria are able to utilize only a restricted group of substrates for the production of

methane.

The main advantage of anaerobic digestion is that it allows the extraction of

the energy value of the organic feed stocks without destroying the nutrients that are

contained. It means that a biogas production unit produces clean fuel, nutrient-rich

residue used as fertilizer and improves sanitation.

IX- Microorganisms and the recovery of metals

1. Accumulation of metals by microorganisms: Certain microorganisms have been found to accumulate large quantities of

metals. Metal accumulation by microbes may occur through metabolic or non-

metabolic processes. Non-metabolic accumulation may result from biosorpition and

precipitation of metals extracellularly at or within the cell-wail matrix and

intracellularly. Metabolic accumulation is usually intracellular. Accumulation of metals

at the cell surface is strongly affected by environmental factors such as temperature

and pH.

36

This phenomenon can be utilized in the recovery of metals of high commercial

value such as silver from industrial solutions and can also be used in removal of

metals from waste to avoid their accumulation and the consequences of their toxicity

to humans.

2. Biotechnology and mining: The extraction of various metals from ores has become a problem for the

mining industry because easily accessible, high-grade mineral deposits of ores are

becoming depleted. This has made it necessary to process lower-grade ores and to

find techniques for more efficient extraction of metal in the ores. Another problem

with the traditional methods of processing ores is air pollution.

Microorganisms can play an important role in recovering minerals from low-

grade ores. This process is called bioleaching. The process uses microbial metabolic

activities to gain access to the desired product by physically or chemically altering the

properties of ores so that metals can be extracted. Thiobacillus thiooxidans and

Thiobacillus ferrooxidans are the most commonly used microorganisms in

bioleaching industry.

Thiobacillus ferrooxidans can oxidize the metal sulfide into the more readily

leachable metal sulfate or may exert its action in an indirect manner by oxidizing

ferrous iron of the ore into ferric iron and subsequently the ferric iron can chemically

oxidizes the metal to more readily leachable form.

The process is currently applied on commercial scale for copper and uranium

recovery from low-grade ores. If the ore is porous and overlays a water-impermeable

stratum, it is possible to leach the ore in situ, but most commercial bioleaching

processes are carried out after mining the ore.

Copper bioleaching, for example, is carried out by sprinkling the leaching

liquor over the ore heap. The leaching liquor containing Thiobacillus ferrooxidans,

nutrients for the bacterium, Fe2+ 2+ and SO4 . The solution percolates through the rock

pile to the lower level where the copper rich liquor is collected. Thiobacllus

ferrooxidans carry out direct oxidation of covellite to the readily leachable cupper

sulfate and chalcopyrite is oxidized in presence of sulfuric acid to cupper sulfate. The

copper is then recovered by solvent extraction or by using scrap iron as following:

CuSO4 + Feo Cuo + FeSO4

Bioleaching of uranium is another important example. Uranium is used as fuel by

the nuclear power generation industry. It is usually present in low-grade ore in the

37

form of the insoluble uranium oxide. Thiobacillus ferrooxidans can oxidize the pyrite,

which is usually present in uranium ore, to ferric iron. Subsequently ferric iron, can

oxidize uranium oxide to the readily leachable form UO SO . 2 4

X- Immunological products

1- Vaccines: a) Production of bacterial vaccines

1- The required bacterial either isolated from a clinical specimen or obtained as

lyophilized culture is cultivated in liquid suitable medium (preferred to solid

medium) as it can be used in large fermenter vessels.

2- The incubation conditions are adjusted.

3- At the end of the growth period the cultures (from different fermenters) are

pooled to form a bulk harvest. The bulk harvest is often a dangerous mixture of

bacterial cells and metabolic products (toxins).

4- Inactivation of the cultures either by heating or the addition of a bactericide.

Formalin at a concentration of 0.5% is used to kill the cells of whooping cough.

It is also used to detoxify the toxins of diphtheria and tetanus, converting them

into harmless toxoids. Phenol is also effective in killing bacteria in cholera and

typhoid vaccines.

5- Separation of the bacterial cells is often carried out by centrifugation while

ammonium sulphate is used to precipitate diphtheria and tetanus toxoids.

b) Production of viral vaccines

The growth of viruses requires living cells. The skin of calves is used for

smallpox vaccine, the fertile eggs (chick embryo) for influenza and yellow fever and

cell cultures prepared from monkey kidney are used for rabies vaccine.

The required virus is inoculated and the living cells are incubated until the

virus growth is maximal. The culture fluids containing the virus are then harvested

and pooled.

Most modern viral vaccines are live attenuated virus strains and so no inactivation

step is required. There are three important exceptions: influenza, poliomyelitis and

rabies.

Blending: various components of the vaccine are mixed to form a final bulk, during

this step preservative and adjuvants are added.

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Quality control of vaccines To provide assurance that both efficacy and safety of every batch of every

product is achieved.

1) In process control

Is the control carried out over the starting materials and intermediates. The

quality control for diphtheria and tetanus toxoids is tested for absence of free toxin,

due to inadequate detoxification with formalin. An example from virus vaccine

manufacture is the titration prior to inactivation of the infectivity of live poliovirus used

to make inactivated poliomyelitis vaccine.

2) Final product control

The final product should be tested for identity, potency and safety.

Preparations containing combined agents are required the pass the tests prescribed

for each separate component.

a- Identity tests: the identity of bacterial vaccines by in vivo agglutination and

precipitation methods. Inactivated viral vaccines are tested by observation

of the specific antibody responses that they produce after neutralization

with specific antisera.

b- Potency assay: each immunological product is intended to exert a unique

prophylactic or therapeutic effect. Vaccines containing killed

microorganisms or their products, such as bacterial toxoids are tested for

potency by the ability to stimulate the production of antibodies in a group of

laboratory animals. Antibodies are then assayed by vaccination of group of

animals and titration of blood samples for antibodies and comparison with

the corresponding standard vaccine. Vaccines containing live

microorganisms are generally tested for potency by counting their viable

particles. The potency of live viral vaccines is estimated in the same way

except that living cells are used.

c- Safety tests: killed bacteria or bacterial products must be completely free

from the living microbes and toxoids require testing for inadequate

detoxification. Usually laboratory animals are used to detect any harmful or

disease effect.

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2- Monoclonal antibodies A very important advance in the rapidly expanding field of biotechnology

comes from the cell lines capable of producing antibody of any required specificity,

indefinitely in cell culture condition.

In normal immunization procedure injection of purified antigen leads to the

production of an antiserum containing antibodies with a wide rang of specificities for

different antigenic determinants on the antigen molecule. It is usually impossible to

separate the different types of antibodies.

The principle behind the method is the fusion of cells (lymphocytes) obtained

from an immunized animal with cells from cultured myeloma cell line to produce

hyberidoma. polyethylene glycol is the agent used to fuse the two types of cells. The

myeloma cells are selected because of their inability to grow in 8-azaguanine as they

lack certain enzyme i.e. they have metabolic defect.

Monoclonal antibody production

Animals (usually mice or rats) are immunized with antigen. Once the animals

exhibited a good antibody response, the spleens are removed and a cell suspensions

is prepared (lymph node cells may be also

used). These cells are fused with a

myeloma cell line by the addition of

polyethylene glycol (PEG) which promote

membrane fusion. Small proportion of the

cells fuses successfully. The fusion

mixture is then set-up in culture medium

containing "HAT". HAT is a mixture of

hypoxanthin, Aminopterin and thymidine.

Aminopterin is a powerful toxin which

block certain metabolic pathway. This

pathway can be bypassed if the cell is

provided with intermediate metabolites

hypoxanthin and thymidine. HAT is non

toxic to spleen cells medium, but myeloma

cells have a metabolic defect and so can

not use this bypass pathway.

Consequently, they die in HAT medium. When the culture is set up in HAT medium,

it contains myeloma cells, spleen cells and fused cells. The spleen cells dies

40

naturally in culture after 1-2 weeks; the myeloma cells are killed by HAT, but fused

cells (Hybridoma) survive as they have the immortality of myeloma cells and the

bypass of the spleen cells and are in the same time antibody producing cells. The

culture is then distributed in wells. Any well containing growing cells are tested for

the production of the desired antibody. Positive cultures are plated out serially so that

only one cell is placed in each well. This produces a clone of cells derived from a

single progenitor, producing only one type of antibody.

XI- Genetic engineering and biotechnology

Impact of genetics on fermentation technology The main impact of classic genetics on fermentation was the improvement of

the industrial strains which resulted in increased yield and reduction in costs. Since

the discovery of recombinant DNA technology in 1973, the situation was changed

and scientists have developed techniques making it possible to move genes from

one cell type to another e.g. from plants and mammals to bacteria.

The future of genetic engineering is considered almost unlimited in its

commercial applications. For instance a gene that codes production of limited

quantities of a valuable compound from normal plant or animal tissue, can be

isolated from a plant or animal cell and cloned into a bacterial cell. The bacterial cell

may then synthesize unlimited quantities of the gene product.

Potential of genetic engineering 1- Therapeutic proteins and peptides Human insulin: Traditionally insulin has been extracted and purified from pancreases of beef

and pork which are different from human insulin for treatment of insulin-dependent

diabetes mellitus. The patients injected with these types of insulin developed anti-

insulin antibodies which led to minor problems, so that the patient needs a larger

dose.

The process of insulin production using recombinant DNA technology, which was-

developed by Eli Lilly in collaboration with Genentech Inc., consisted of separately

incorporating synthetic genes of the A and B chains of human insulin into the β-

galactosidase regions of the pBR322 plasmid. These two plasmids were cloned

separately in Escherichia coli. The transformed bacteria synthesized the A and B

chains separately.

41

Synthesize A-chain gene and insert into a plasmid

Synthesize B-chain gene and insert into a plasmid

Insert into E. coli

Lyse cells and cleave with CNBr

β- Gal A chain β- Gal B chain

A chain B chain Oxidize

Insulin

By means of cyanogen bromide, the A and B chains were released from the β-

galactosidase. After purification, the two chains were mixed and connected together

to prepare an active insulin preparation. In another approach, mRNA was copied into cDNA and a methionine codon

(ATG) was chemically synthesized and attached to the 5’end of the proinsulin cDNA.

This was cloned in a plasmid vector and transferred in Escherichia coil. The

proinsulin was then released from the β-galactosidase enzyme by cyanogen bromide

which destroys the methionine linker residue. The C peptide (connecting chain) was

cleaved with enzymatic reaction yielding pure human insulin. Several modifications

were then developed.

α-interferon:

Interferons are produced by eukaryotic cells in response to viral infection or

foreign double stranded RNAs (viral or synthetic). The infected cell produces

interferon for a few hours. Interferon is extracted and used by the cells. When the

cells become infected with virus, the interferon causes the cells to produce molecules

that prevent replication of the infecting virus. There are several kinds of interferons

each made by a different cell type. α-Interferon is produced by leukocytes, while β-

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interferon is produced by fibroblasts and γ-interferon is produced by sensitized T

cells.

Interferons have been previously available in extremely minute quantities

extracted from cultured human cells that made it not possible even to test the

production for potential clinical value. However, most of the genes for human

interferons have now been cloned in bacteria, yeast or mammalian cells. Interferons

are now available commercially for clinical use. Interferon α was found to reduce the

duration of viral infection, effective against herpes virus infection of the eye and

reduces the incidence of attacks of multiple sclerosis.

Human-growth hormone: Human growth hormone is another pharmaceutical product made more

efficiently by a genetically engineered bacterium. Previously the hormone was

obtained only in extremely small quantities by extracting it from the pituitary glands of

the animals. The genetically engineered product is being used to treat children

pituitary dwarfism and other conditions related to growth hormone deficiency.

Hepatitis B vaccine: Production of certain vaccines such as hepatitis B, has been difficult because

the virus was unable to grow in cell cultures and the extreme hazards of working

with large quantities of the virus which can be obtained from the blood of humans

and experimentally infected chimpanzees but.

Using DNA from HBV, it was possible to clone the gene for hepatitis B surface

antigen (HBs antigen) into cells of the yeast Saccharomyces cerevisiae. The yeast

expressed the gene and made HBs antigen particles that could be extracted after the

cells were broken. Since yeast cells are easy to propagate, it was possible to obtain-

unlimited quantities of HBs antigen particles. This was the first vaccine against a

human disease produced with genetic engineering methods.

2- Chemicals

Indigo dye

The dye indigo is a plant product but was manufactured chemically to reduce

the cost. However, it was possible to clone naphthalene oxidase gene from

Pseudomonas sp. into E. coli. The modified E. coli produced indigo, as the

naphthalene oxidase enzyme oxidized indole of E. coli to 2-3 dihydrodiol which

spontaneously oxidize and dimerize to indigo resulting in blue E. coli.

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3- Construction of new microbes

Ice-minus Pseudomonas syringae:

An interesting ecological relationship between bacteria and plants involves the

role of Pseudomonas syringae which produce a surface protein initiating ice crystals

formation, which results in frost damage to the plant. These bacteria are conditional

plant- pathogens, causing death due to frost damage only at temperatures that can

initiate the freezing process. A genetically engineered ice-minus strain with the

surface protein deleted is sprayed to replace the indigenous population and protect

the crop. However, the release of genetically engineered raised environment

questions.

4- Improvement of performance: The key control gene for lysine biosynthesis was identified, manipulated to be

insensitive to repression. The manipulated gene is cloned and reintroduced at a high

copy number. Similar principles have been applied to antibiotic-producing organisms.

5- Protein engineering: Knowledge of the tertiary structure of an enzyme with knowledge of its DNA

sequence can enable the rational modification of the molecule to produce the desired

change such as substrate specificity and temperature stability. Substitution of certain

amino acid at a specific position can be achieved by site-directed mutation in the

cloned gene. This technique was applied to tyrosyl RNA synthetase.

6- Polymerase chain reaction and hybridization techniques:

Both are used as diagnostic techniques. Polymerase chain reaction (PCR) is a

technique that allows a specific DNA sequence to be amplified. Using this technique,

enormous number of copies of one or more genes can be obtained from very tiny

initial quantities of DNA that are impossible to detect by routine methods.

The starting material for PCR is a segment of double stranded DNA containing

the target sequence. The technique involves three temperature-dependent steps

namely template denaturation, primer annealing and polyrmerase extension.

In the first step, double stranded template DNA is denatured to single stranded

DNA by heating. In the second step, primers complementary to the 3' ends of the

target DNA are added. When the mixture of primers and DNA is cooled, the primers

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bind to the gene. In the third step the enzyme DNA polymerase adds energized

nucleotides one at a time to one end of each primer and synthesize a long strand of

complementary DNA. At the end of this cycle two copies of the gene are formed from

each initial copy. In subsequent cycles the number of copies of the target gene

increases exponentially. Each cycle takes no more than a few minutes. By the

twenty-fifth cycle, there are millions of copies of the target sequence, enough to he

easily analyzed by routine laboratory procedures.

DNA amplification has many applications in clinical diagnosis, forensic

medicine and basic research. For instance, trace amounts of DNA, in fluids such as

blood or semen or in tissue such as hair, can be amplified by PCR and analyzed to

see whether the DNA is identical to that of a person suspected of committing a crime.

The technique enabled clinicians to detect infection by the AIDS virus when other

methods have failed.

Today the technique is

used for diagnosis of

several other diseases.

PCR has also

become a powerful tool for

diagnosis of various

genetic diseases, such as

sickle cell anemia in fetus

still in its mother’s uterus,

by amplifying the genetic

information provided by

just a few fetal cells, which

can be obtained without

harming the fetus.

Hybridization

techniques using DNA

probes finds a wide

application in the detection

of bacteria and diagnosis

of hereditable diseases.

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7- Cell fusion: Gross genetic engineering is possible by cell fusion, where two whole

genomes are mixed, re-assorted for selection of the required characteristics. There is

a requirement for the removal of the cell wall when fusion of bacteria, yeast, fungi or

plants cells before fusion. Subsequent regeneration of cell wall and outgrowth of the

fused cell is necessary before selection of the desired phenotype.

8- Modification of macroscopic animals: Transgenic animals Transgenesis is the use of gene manipulation to permanently modifying germ

cells of animals. For example the production of super mice was a result of the over-

production of human growth hormone. Over-expression of growth hormone has also

been tried in order to increase the rate of growth of livestock, poultry and fish. Production of foreign proteins in transgenic farm animals find a more significant

progress. For example α1-antitrypsin, a protein used as replacement therapy for

genetically-deficient individuals at risk from emphysema, have been produced in

transgenic sheep. The compound is excreted in their milk.

Problems and solution The problems which faced the biotechnology companies in both regulatory

and safety issues were new to the industry.

1- Strain stability

Reversion of mutants to low productivity is a common problem in fermentation

industry. However, strict control of culture storage and fermentation conditions has

maintained productivity. The use genetically engineered strains generated new

problems. It is usual for cloned genes to be carried on a plasmid. Unless certain

precautions are taken, cells tend to delete sections of the plasmid or even the whole

plasmid. On a laboratory scale, it is feasible to apply selective pressure such as

incorporation of antibiotics to insure maintenance of the recombinant plasmid as it

contain resistance factor to such an antibiotic. On large scale it is possible to control

expression, through:

a) Incorporation of a heat sensitive gene on the plasmid which allows maintaining

low copy number during growth of the microorganism and high copy number

during the production stage, by regulation of the temperature of the process.

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The fermenter is kept at low temperature during growth and high temperature

during the production phases.

b) Incorporation of partition (par) gene which helps to regulate the inheritance of

plasmid within the population during growth and production phases of the

fermentation.

2- Engineering design

a) Scale: fermentaions with genetically engineered organisms are often conducted

at small fermenters such as growth hormone, compared to the traditional

fermentations of alcoholic beverages, antibiotics and organic acids which use

vessels with several thousands of liters.

b) Containment: to prevent accidental release of genetically engineered organisms

into the environment, which need special considerations such as:

1- Incineration or filtration of exit gas.

2- Use of leak proof agitator seals.

3- Inoculation and sampling through non-aerosol producing system.

4- Inactivation of cells before processing.

5- Emergency planes for large spillages or leaks.

6- Environmental monitoring.

7- Use of detailed operating instructions.

8- Training of personnel.

9- Validation of the above system.

c) Separation of the product: Downstream processing accounts for about 80% of

the production costs of proteins. Macro-modification of proteins for facilitated

processing utilizes the recombinant DNA technology to facilitate the product

recovery and hence reduce the production costs. DNA can be constructed by

incorporating the gene coding for the desired product together with DNA coding

for an additional amino acid sequence. The expressed protein will be composed

of the target protein plus a tail. The tail can be designed so that the hybrid

protein can have a wide range of additional properties which can be used to

facilitate recovery and purification. The additional properties may include

differences in solubility and/or hydrophobicity, enzyme activity and specific

binding.

For example in many prokaryotes, high level expression results in the formation

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of inclusion bodies (insoluble aggregates of the product), particularly in E. coli.

The downstream processing of such products may affect their activity. Signal

peptides can direct the product into the culture medium. This will have the

advantage of avoiding loss of activity as well as no cell disruption is required. Another example of such purification fusions is polyarginine tailing. In this

method the gene sequence coding for the desired protein is extended by

insertion of the codons for arginine. The resultant protein will have a

polyarginine tail which makes it more basic. The product is then separated from

other proteins, which are more acidic using ion exchange chromatography. The

polyarginine tail is then cleaved with the enzyme carboxypeptidase B. The de-

tailed protein is rechromatographed on an identical ion exchange resin to

separate it from any more basic contaminating protein.

XII- Plant biotechnology

Altering the genotypes of plants is an important application of recombinant DNA

technology. Plants that have gained new genetic information from foreign sources

are called transgenic plants. Efforts in plant biotechnology are focused on three main

directions that are discussed below:

1) Improving agronomic traits:

Crop plants were modified so that they become resistant to herbicides. This

can be achieved either by introduction of genes into plant that enables the plant to

degrade or detoxify the herbicide or by engineering the plant so that the target

molecule in the plant cell is rendered insensitive or is over-produced.

For example glyphosate, the most commonly used herbicide, is known to

inhibit an enzyme called EPSP synthetase, a key enzyme in the biosynthesis of

aromatic amino acids in plants. The gene encoding the EPSP synthetase was

isolated, engineered and introduced into the plant. The transgenic plants prepared

were found to express higher levels of the enzyme and were found to be more

tolerant to glyphosate.

In another approach, a mutant gene encoding EPSP synthetase was

introduced into tomato plants. The produced enzyme was found to retain its specific

activity but has decreased affinity for the herbicide. The transgenic tomato plants

expressing this gene were found to be glyphosate tolerant.

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2) Development of plants with improved food processing characteristics:

For example transgenic tomatoes with an increased shelf-life and increased

resistance to bruising were produced. They were engineered so that it has reduced

levels of polygalacturonase enzyme. This enzyme is involved in softening and over

ripening of tomatoes.

3. Use of plants as factors for production of biological and chemical products:

For example plants have been also used to produce mammalian proteins such

as interferons and human serum albumen.

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1. Types of bioreactors

i- Background:

Bioreactors are the vessels, containers or systems in which the bioreaction takes place. All bioreaction conditions can be properly controlled through the bioreactor and its design. Bioreactors vary from very simple vessel with few controls to highly complicated ones in which the whole process is under computer control

ii- Scope and state of art:

Bioreactors can be classified according to different aspects: 1. Classification according to the configuration:

i. Closed (well controlled) for industrial application ii. Opened most commonly used for environmental applications

2. Classification according to the biomass retention mode: i. Suspended biomass (for both environmental and industrial applications)

Immobilized biomass for both applications with advantages of: ii. Higher yields 1. Easier down stream processing 2.

3. Classification according to the flow within the bioreactors: i. Well mixed (for homogenous distribution of the nutrients and substrates)

Plug flow (Directional flow for controlled flow of the nutrients and substrates) ii. 4. Classification according to the mode of bioreaction within the bioreactors:

i. Batch Fed batch ii. Continuous iii.

iii- Recent development and approaches:

Inlet

Different steps or stages

Outlet

Multiple steps bioreactors: The same bioreactor contain multiple stages of immobilized biological systems each to carry out single step of a series of reactions in a consequent way Membrane bioreactors: Immobilized biological systems on membranes with different

ore size or permeability characters to facilitate the product recovery (DSP) p The use of living higher organisms as bioreactors:

• Plastics from the plant (biopolymer assignment) • Transgenic animals for production of α -antitrypsin protein (main course) 1

2. Biopolymers

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i. Background:

All polymers, including biopolymers, are made of repetitive units called monomers. Biopolymers are polymers which are present in, or created by, living organisms. There are two main types of biopolymers: those that come from living organisms; and, those which need to be polymerized but come from renewable resources. Biopolymers inherently have a well defined structure. Starch, proteins and peptides, DNA, and RNA are all examples of biopolymers, in which the monomer units, respectively, are sugars, amino acids, and nucleic acids.

ii. Scope and state of art:

Biopolymers have several advantages such as:

1. Sustainable and renewable 2. Well defined structure and specific upon synthesis = monodispersity 3. Biocompatible and hence is suitable for medical applications. 4. Non toxic 5. Biodegradable (easy to be removed from the environment)

Biopolymers can be used as:

1. Plasma substitute (Dextrans) 2. Complex with iron for iron deficiency anemia (Dextrans) 3. Used in paints, printing, and textile industries (Xanthans and polyhydroxylbutyrate

(PHB)) 4. In oil industry as drilling fluid additive and for enhanced oil recovery (Xanthans)

iii. Recent development and approaches:

Green plastics: through the production of polyhydroxyalkanoates (PHAs), a versatile family of biobased polymers. These biopolymers are used for production of biodegradable plastics called green plastics. These are currently replacing the petrochemical based plastics in all fields

Plastics from the plant: Plants are becoming factories for the production of plastics. Researchers created a Arabidopis thaliana plant through genetic engineering. The plant contains the enzymes used by bacteria to create plastics. Plants create the plastic through the conversion of sunlight into energy. Therefore it has advantage of CO2 fixation and conversion. The researchers have transferred the gene that codes for this enzyme into the plant; as a result the plant produces plastic through its cellular processes. The plant is harvested and the plastic is extracted from it using a solvent.

Biocatalysis3.

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i. Background:

Biocatalysis is the use of natural living catalysts, such as protein enzymes or even the whole living cell to perform chemical transformations. These have been used in many industries such as detergents production, food industries, production of alcoholic beverages, etc.

ii. Scope and state of art:

The use of biocatalysts especially enzymes revealed many advantages such as: 1. Ultimate and sustainable resources 2. Wide diversity 3. Environmentally acceptable 4. Highly selective and specific and therefore, produce the product with

maximum purity (up to the enatioselectivity level)

Type Applications and use Production of sweeteners in the food industry

Amylase Removal of starch sizing from cloth spots dry-cleaning industry Acid resistant amylase is used as digestive aid

Primarily in the detergent and laundry industry Proteases dairy industry

leather and pharmaceutical industry and waste treatment Digestive enzymes to supplement pancreatic lipases. Lipases In dairy industry since fatty acids imparts taste of cheese

Glucose isomerase Manufacture of many foods and beverages

L-Asparaginase In the treatment or leukemia and lymphoma

Fibrinolysin (streptokinase) Treatment of thrombosis

Determination of blood glucose level (Biosensor) Glucose oxidase Removal of O from various food products to prevent their deterioration. 2

iii. Recent development and approaches:

Discovering Novel Biocatalysts: The main concern in this regard is the screening of new biocatalysts from extremophiles since life in unusual habitats makes for unique biocatalysts and the great majority of those biochemical potential remains untapped Improving Existing Biocatalysts: Through the use of genetic engineering The use of chiral biocatalysts: Racemic nicotine Oxidation of the S-enantiomer only (The R-enantiomer will remain in a pure form)

monoamine oxidase

Further more the converted S-enantiomer can be stereoinverted to the pure R-enantiomer

4. Bioseparation

52

i. Background:

Bioseparation is the separation of biological materials (bioproducts) or the use of biological element as separating agent and sometime can be both (using bioelements to separate bioproducts). This is of a great importance since the separation (down stream processing) accounts for more than 50 - 70 % of the total cost.

ii. Scope and state of art:

Bioseparation of bioproducts such as enzymes and other active protein is commonly carried out through:

1. Precipitation 2. Centrifugation 3. Microfilteration or ultrafiltration 4. Membrane chromatography 5. Ion exchange chromatography 6. Electrophoresis

Bioseparation using biomaterials as separating agents:

1. The use of corn based adsorbents or polysaccharides adsorbents to remove the water content from ethanol and also algae can be used for purification of gases from impurities

Adsorption Washing Desorption

2. Affinity legends such as the use of specific antibody to separate the desired protein product as shown in the following scheme

iii. Recent development and approaches:

Immobilized Metal Affinity Chromatography (IMAC): based on high affinity between certain metal and polypeptide chain called his tag, added to the desired protein product. Macro-modification of the desired product: Concept = incorporating the gene coding for

the desired product + DNA coding for an additional amino acid sequence Protein +

tail

The tail• can be designed so that the hybrid protein can have a wide range of

additional properties which can be used to facilitate recovery and purification.

• The additional properties may include differences in solubility and/or hydrophobicity,

enzyme activity and specific binding.

Molecular imprinting techniques:

5. Biomonitoring

53

i. Background:

Biomonitoring is the testing (monitoring) of the present flora in a system or the use of biological system to measure qualitatively or quantitatively the presence of certain chemical or group of natural or synthetic chemicals or asses the exposure of human to natural or synthetic chemicals.

ii. Scope and state of art:

Biomonitoring of the flora (biological systems) can be in: a. Medicine to detect the infective agent (the present microbe(s) b. Industrial biotechnology as a quality control on the in use bioelement purity c. Environmental biotechnology as in bioremediation to investigate the most

predominant strains to properly control the conditions towards the desired function

Biomonitoring of the flora (biological systems) can be done by: a. Microscopical examination using different stains and biochemical reactions b. Serological reactions c. Electrophoresis d. Molecular identification e.g. FISH (Flourscence In Situ Hybridization) and PCR

reactions

Biomonitoring using biological system to measure or asses a specific chemical or group of chemicals can be done either by: a. Biosensors (see biosensors) b. Bioassays

Biomonitoring using biological system have many applications such as: a. Medicinal applications such as the sensitivity testing of antimicrobial agents, antibiotic

assays and biosensors for blood sugar measurement. b. Environmental applications e.g. determination of the pollution degree with certain

group of xenobotics (LC 50) c. Industrial applications as a tool for final product Q.C or in process Q.C

iii. Recent development and approaches:

Biomonitoring of the flora using LC/MS via soft Ionization of proteins: Electron spray, MALDI Biomonitoring using biological system in High through put techniques:

• ELISA techniques

• Microarrays for detection of biomarkers

• Quartz crystal balance

6. Green chemistry

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i. Background:

Green chemistry is also known as sustainable chemistry, refers to environmentally friendly chemicals and processes (i.e. the development and application of clean processes based on biocatalysis for production of chemical products from renewable raw materials)

ii. Scope and state of art:

Application of green chemistry resulted in reduced waste, eliminating costly end-of-the-pipe treatments; safer products; and reduced use of energy and resources—all improving the competitiveness of chemical manufacturers and their customers. This is through the application of the 12 principles:

1 7 Prevent waste Maximize atom economy 2 8 Design safer chemicals and products Use safer solvents and reaction conditions 3 9 Design less hazardous chemical syntheses Increase energy efficiency 4 10Use renewable feedstocks Design chemicals and products to degrade after use 5 11Use catalysts, not stoichiometric reagents Analyze in real time to prevent pollution 6 12 Minimize the potential for accidents Avoid chemical derivatives

Many green products are currently or very soon would be available such as:

1. Formalin technology (production plants and catalysts for formalin production) 2. Organic acids

3. Oxo alcohols and plasticizers

4. Environmentally friendly coat reagent such as epoxides and acrylates from the vegetable oils

5. Biopolymers (through the use of polymerizing biological systems)

6. Biosurfactants such as sugar based surfactants synthesized enzymatically

iii. Recent development and approaches:

• The screening of relevant and novel biocatalysts: o Improvement of the screening methods for novel biocatalysts o Adaptation of the currently available biocatalysts to extreme conditions like high

temp. o Screening for extremophilic biocatalysts such as thermostable enzymes

• The use of renewable biomass to produce energy primarily for transportation purposes. Much attention is currently focused on bio-ethanol and bio-diesel, the former from carbohydrate rich feedstock and the latter from oilseed crops.

7. Bioscrubber

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i. Background:

Bioscrubber is a scrubber with a continuous recirculation of the liquid containing the biological system specifically placed during the start up of the installation. The biological system tends to continuously biodegrade the pollutancreating a continuous state of sink conditions

t or scrubs the desired product

ii. Scope and state of art:

Bioscrubbers for removal of organic emissions ll

ioscrubber for removal of H

The scrubbing liquid containing the bacteria wisolubilise the pollutants; they will be biodegraded and neutralized inside the liquid by the used biological system (mainly bacteria) B 2S in Mining

ing Fe+3 Continuous flow of scrubbing liquid containand bacteria (Thiobacillus ferrooxidans). The H2S is converted to readily soluble SO4

-2 via the oxidation with the Fe+3. Then the resulted Fe+2 will regenerated by the bacteria to Fe+3 again Two phase bioscrubbers for continuous separation of the products: Continuous flow of liquid containing biological system as legend to specifically bind the desired product and therefore will increase the partitioning and consequently the efficiency and capacity of the product recovery

iii. Recent development and approaches:

Submerged membrane bioreactors acting as bioscrubbers

The main medication to immobilize the biological system on membrane filters instead of being continuously circulating with the liquid to improve the efficiency and the stability Purification of the biogas (removal of the CO from Methane) 2

The produced biogas is forced through the bioscrubber, where a continuous flow of liquid containing microalgae is scrubbing the CO2 (the more soluble gas than CH4). Therefore the out put will contain only the methane gas with very minor concentrations of O2. The traces of O2 are removed by applying mild pressure

Biosensors8.

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i. Background:

Biosensor = Immobilized Bioelement electrodes, designed for potentiometric, amperometric or capacitive assay of substrates such as glucose, urea and amino acids, xenobiotics, etc The electrode = an electrochemical sensor in close contact with a thin permeable membrane, in which the bioelement is embedded.

General diagram of biosensors

Anode Electrolyte Cathode

Teflon membraneProduct Enzyme Substrate

++ + +

+++

Semi-permeablemembrane

ii. Scope and state of art:

Principal: Enzymatic reaction → a small molecule is produced (e.g. 02, H+, C02) → detected by the specific sensor The magnitude of the response determines the concentration of the substrate.

Medicinal applications e.g. Glucose oxidase electrodes = glucose

oxidase layered over a platinum O2 electrode.

Glucose conc. inversely proportion to the O2

detected

Similar Biosensors have been developed to be used in wide verities of assays: • Alcohol (using alcohol oxidase enzyme) • Pollution (using highly sensitive biological system such as yeast)

iii. Recent development and approaches:

Capacitive biosensors for detection of toxins: using the immobilized specific antibody (antitoxin) on the capacitive platinum electrode. The more the interaction between the antitoxin and the toxins the more change in the capacitance.

Lux opeons based biosensors: Lux operon from Photobacterium = receptor (inducer) + structural genes for luciferase

enzyme.

Pollutant (substrate) + receptor → activation → production of luciferase enzyme

Luciferase + substrate Enzyme-substrate complex (blue-green light) FMNH2

Similar biosensors have been developed for verities of applications :

• Lactobacillus bacteria containing the lux operon for detection of antibiotics in milk to

be used for cheese production.

• Biosensors using recombinant bacteriophages that contain the lux genes for

detection of Listeria and E.coli in foods and to detect drug-resistant mycobacteria

9. Extremophiles in biotechnology

i. Background:

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Extremophiles are organisms that live in conditions that would kill other creatures (i.e.

thrive in extreme environments where no other organisms are found). These extremcondition could as shown in table 1. Some extremophiles can tolerate extremely high levels of radiation or toxic compounds.

Thermophile Organisms having growth temperature optimum of 50°C or higher. Halophile Organisms requiring at least 0.2 M (3–30%) salt for growth.

Organisms having growth temperature optimum of 15°C or lower, (some can survive at –10°C), and are unable to grow above 20°C.

Psychrophile

Alkaliphile Organisms with optimal growth at pH values above 10. Acidophile Oganisms with a pH optimum for growth at, or below, pH 2. Piezophile (previously termed barophile) = Organisms that lives optimally at high hydrostatic pressure.

ii. Scope and state of art:

Applications of the extremophiles based mainly on the corresponding isolated extremozymes or proteins and to less extent on whole cells. Fortunately, extremozymes can be produced through recombinant DNA technology (genetic engineering) without the need to massive culturing of the source extremophiles.

Table 2. Examples of common applications of extremophiles Thermophiles and Hyperthermophiles Applications DNA polymerases DNA amplification by PCR Lipases, pullulanases and proteases Detergents Amylases Baking and brewing Halophiles Applications g-Linoleic acid, b-carotene and cell extracts,e.g. Spirulina and Dunaliella

Health foods, dietary supplements, food coloring and feedstock

Psychrophiles Applications Polyunsaturated fatty acids Food additives, dietary supplements Ice nucleating proteins Artificial snow, food industry e.g. ice cream Alkaliphiles and Acidophiles Applications Proteases, cellulases, lipases and amylases Detergents and digestive aids Sulphur oxidizing acidophiles Recovery of metals Acidophiles Organic acids and solvents

iii. Recent development and approaches:

• Improvement of the cultivation techniques for extremophiles as a base for screening these extremophiles.

• The screening of relevant and novel extremophilic organisms in untapped extreme environments.

• The screening extremophiles for natural products especially novel antimicrobials, mainly antiviral and anticancer agents

10. Gene Therapy

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i. Background:

Gene therapy = the replacement of a defective gene in an organism suffering from a genetic disease. Recombinant DNA techniques are used to isolate the functioning gene and insert it into cells to fix any defects The first patient treated with gene therapy was a four-year old girl in 1990. She had adenosine deaminase (ADA) deficiency, a genetic disease which leaves her defenseless against infections. White blood cells were taken from her, and the normal genes for making adenosine deaminase were inserted into them. The corrected cells were re-injected into her.

ii. Scope and state of art:

There are 2 main lines of gene therapy either:

• Germ-line gene therapy • Somatic cell gene therapy

Germ-line cell therapy involves the introduction of corrective genes into reproductive cells (sperm and eggs) or zygotes, with the objective of creating a beneficial genetic change that is transmitted to the new generation

Somatic cell gene therapy is the introduction of genes in an organ or tissue to enable it to function normally. This can be done: In-vivo: through, systemic infusion or tissue injection Ex-vivo: the defected cell type is being removed to outside the body and genetically treated to avoid such defect then the treated cells re-injected to the body. His can also done using stem cells Gene therapy is being studied for treatment of many diseases such as:

ADA deficiency Hemophilia AIDS Liver cancer Asthma Lung cancer Brain tumor Melanoma Breast cancer Muscular dystrophy Colon cancer Neurodegenerative conditions Diabetes Ovarian cancer Heart diseases Prostate cancer

iii. Recent development and approaches: DNA Vaccines = is the introduction of genes (naked DNA), with the objective of triggering the immune system to produce antibodies for certain infectious diseases, cancer, or some autoimmune diseases.

Stem Cell Therapy: in the future it might be used in conjunction with gene therapy for regeneration of tissue and organs after they have been treated with corrective genes

11. Genomics

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i. Background:

Genomics is the scientific study of the genome and the role genes play, individually and collectively, in determining structure, directing growth and development, and controlling biological functions. It consists of two branches:

• Structural genomics • Functional genomics.

ii. Scope and state of art:

Structural Genomics = the field of structural genomics includes the construction and comparison of various types of genome maps and large-scale DNA sequencing

• Used to isolate specific recombinant molecules or microbes with unique biochemistry. • Identify the genes involved in complex traits that are controlled by many genes. • Detect microbial contaminants in cell cultures

Functional genomics = a field of research that aims to understand what each gene does, how it is regulated and how it interacts with other genes. These two main genomics cover all other fields such as:

1. Fungal genomics 2. Plant genomics 3. Microbial genomics 4. Any thing genomics

iii. Recent development and approaches: Metagenomics = Environmental Genomics = Ecogenomics = Community Genomics) = the study of genetic material recovered directly from environmental samples Chemical genomics = using structural and functional genomic information about biological molecules, especially proteins, to identify useful small molecules and alter their structure to improve their efficacy. Pharmacogenomics = personalized medicine = the science that examines the inherited variations in genes that dictate drug response and explores the ways these variations can be used to predict whether a patient will have a good response to a drug, a bad response to a drug, or no response at all. This will enable the development of drugs tailored to specific subpopulations based on genes Pharmacogenomics has the potential to:

• Decrease side effects of drugs • Increase drug effectiveness • Make drug development faster and less costly

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