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.

Microbiology

Biochemistry

Genetics

Electronics and Computer Sciences

Biotechnology

Food Technology

Biochemical Engineering

Chemical Engineering

Mechanical Engineering

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.

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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. He was Louis Pasteur who in the late 19th 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. Microorganism

Substrate
Specific conditions

Product

Such processes using biomass for making chemicals, constituted the first phase of modern biotechnology. In the early decades of the 20th 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 A shown in the following figure the addition of bisulfite to the generate glycerol.

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

Sugar (glucose) Fructose 1,6 diphosphate Dihydroxyacetone-P 3-Phosphoglyceraldehyde Embeden-Meyerhoff pathway Glycerol NAD NADH Pyruvate

Ethanol Normal route

Acetaledehyde Sodium bisulfite

Acetaldehyde bisulfite addition complex 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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2.Mineral extraction (soil and sea). .Organic acids (citric. gluconic) and mineral extraction. pigments. vaccines. flavors.Medicine Examples .Environment control (air. adhesives. protoplast fusion. 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.Concurrently with the development of antibiotics. Improvement of waste treatment.Feed stocks (agriculture and animal).Single cell protein (SCP). gums. 2. . Recent developments in Biotechnology Category 1.Industry .Energy sources. gene therapy. fibers. replacement of chemical insecticides by biological ones and biodegradation of xenobiotics. introduction of foreign genes into plants and nitrogen fixation. detergents. dyes.Consumer chemicals: …….Agriculture 3. . steroids.Analytical chemical tools 4. 5. monoclonal antibodies.Environment 5. 6.Plant tissue culture.. perfumes.Use of enzymes in detergent industry.Chemicals 4.Production of antibiotics. etc. water and soil) 7. The later was used for production steroid hormones. 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. use of enzymes in food processing and food preservation. recombinant DNA technology drugs and improving diagnosis by enzymes and enzyme sensors. plastics. 4 . gelling and thickening agents.Health care (diagnosis and treatment) 3.Food 6.

Storage on agar slopes at – 4oC and subcultured routinely every few months (depending on the strain) 5.e.etc. 5 . 4. 5. O2 temperature. 2.) by enrichment technique (maintain conditions that favor isolation of the required microorganism).Storage under liquid nitrogen (at – 150oC): cultures are mixed with glycerol. animal and plant) Microbial cultures: Microbial cultures are either obtained from culture collections e.Soil culture: moist sterile soil is inoculated with the microbial culture. Culture maintenance Cultures could be maintained by one or more of the following methods: 1. microorganisms act like chemical factories. 2.Produce the desired product under workable conditions (pH. incubated for few days to allow some growth.). 3.Lyophilization (freeze drying): cultures in small tubes or ampoules are rapidly frozen followed by drying under vacuum. American type culture collection (ATCC) or usually isolated (from soil. 3. transferred to special tubes which withstand low temperature and kept in special tanks containing liquid nitrogen.Introduction to biotechnological resources and techniques Cultures: (microbial.Should be pure culture i.Storage in glycerol at – 70oC in deep freezers.Produce a large amount of the required product. 6.etc. air ….Grow rapidly on inexpensive and readily available media. not contaminated with other species or low producing strains.g. In industry. then sealed under vacuum. Those ones intended to be used in industry should be: 1. then dried at room temperature for few weeks and stored. 4. This is the best and most commonly used one.Be genetically stable (low rate of mutation).Easily cultivated and maintained.….

They are mainly carbohydrates such as cellulose and starch. trace metals and energy source required by the microorganism. 4. food industry and forestry. nitrogen. usually pure defined chemicals are used. 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. 5. Accordingly complex less definable substrates are frequently used to reduce the production costs. production costs and availability of the raw material. Causes minimal problems during the fermentation. product separation and waste disposal. 2. the use of corn steep liquor has the advantage of providing the precursor of benzyl group side chain. nitrogen. whiskey. They must provide the required carbon. while substrates rich in sugars usually obtained from sugar cane and sugar beet. Cheap and available 1ocally throughout the year. In production of benzyl penicillin. 3. If corn steep liquor is not used. 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. Cellulose and lignocellulose is the most abundant and 6 . that is why special types of grapes are used. The media used for cultivation of microorganism must contain the elements needed by the microorganism (carbon. For cultivation of microorganisms in laboratory research.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). but in industrial scale fermentations the use of such pure defined constituents would be too expensive. Easily transported and sterilized. a mixture of natural penicillins is produced. The choice of the raw material for a given process depends on the process. minerals and growth factors) in a form suitable for the synthesis of cell substances and for the production of the products. while in the production of alcoholic beverages (wine. Of definite composition and easily processed.…) flavor is the most important factor. The raw materials should also provide the required precursor for the end product. Most of the substrates used in industrial fermentations are by-products derived from agriculture.

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. and Corn steep liquor or soy meal as a nitrogen source. 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. v) Plant cell and tissue culture. However. peptone. trace elements may be necessary for the growth of the microorganism and maximal yield of the fermentation product. Molasses is a by-product of sugar industry and whey is a by-product of cheese industry. Improvement of product quality and quantity by manipulating the process. maize. iv) Cell fusion for the generation of hybrids with improved productivity. iii) Utilization of recombinant DNA (genetic engineering) to improve an existing process or developing a totally new product. urea. Again starch must be degraded to monosaccharides or oligosaccharides before use in industrial fermentations. wheat. yeast extract. 7 . Starch could be obtained from different types of grains such as rice. other additives such as vitamins. In addition to carbon and nitrogen sources. Much industrial fermentation utilizes ammonium salts.renewable natural resource throughout the world. sweet potatoes and cassava. The success of such programs depends to large extent on the substance to be produced. ii) Utilization of mutation techniques to prepare mutants of better characters. This may include I. In general such programs include i) Selection of the biological entities by a screening program to choose the best biocatalyst of the required product. before use in industrial fermentation it must be degraded to simpler structure.

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.II. iii.Down stream processing: It include 1) Manipulation of the fermentation conditions to specify the product and maximize the yield e.g. 4) Application of the proper methods for separation and purification of the product e. 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. 8 . 3) Improvement of the efficiency of the biocatalyst by immobilization (discussed later). filtration or chromatography. Saccharomyces + Hexoses Saccharomyces + Hexoses Saccharomyces + Hexoses aerobic anaerobic anaerobic &bisulphite Baker’s yeast alcohol glycerol Also citric acid production could be performed by surface culture. Submerged culture is more economic because: i. changing the oxygen potential of growth for Saccharomyces yields different product.g. ii.Les energy is required for sterilizing the containers and the medium.Higher yield. centrifugation.Less space and labor is needed.

9 . larger volume vessels are usually made of stainless steel. aerated fermenters are the ones that are usually used in industry especially in the pharmaceutical industry. Small fermenters are sterilized in autoclave while large fermenters are sterilized by steam generated from distant boiler. 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. however. It is necessary that fermenter and substrate solutions be sterilized (mostly inside the fermenter). agitation. Simple stirred. Different products can be produced in the same apparatus with some modification.g. Bioreactors vary from very simple vessel with few controls to highly complicated ones in which the whole process is under computer control. The defined conditions include the use of the proper substrate (medium) and the proper environmental parameters (e. Small fermenter vessels up to 20 liters are usually made of glass.Fermentation processes Fermentation production products of is an industrial valuable or process utilizing living cells for the commercially either aerobically anaerobically. pH and aeration). In fermentation living cells are allowed to grow under defined conditions in a fermenter (bioreactor). Before construction of a fermenter. temperature. before the addition of the inoculum (microbial strain). All must be optimized to achieve the highest yield and quality at the lowest cost possible.

Compress Outlets Air exhaust Air filter Motor power unit Inlets Monitors Agitator Baffle Cooling or heating coils Impeller … … Design of the fermenter (bioreactor) Air sparger at the end of air 10 .

3. Large fermenters are also supplied with internal baffles that help in mixing. The medium as well as air bubbles are mixed by impellers arranged on an agitator shaft attached to bottom or top sealed motor. Aeration and mixing are relatively expensive due to the highenergy costs needed. Fermenters may be supplied with pumps to supply acid or alkali in order to adjust the pH during the fermentation course. acid or alkali to control the pH.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.Continuous Fermentation:. Careful design is required to achieve optimal oxygen concentration throughout the fermentation medium since oxygen transfer in large volume fermenters is difficult. cell growth is kept constant by using turbidity to maintain the biomass concentration and the rate of nutrient solution addition. Methods of fermentation: 1. 11 . Also pumps to supply nutrients or antifoams may be also required. 2.Fed-batch process: nutrients are added at intervals as the fermentation progresses. In batch fermentation. In the chemostat cell growth is controlled by adjusting the concentration of one substrate (as a limiting factor). nothing is added to the fermenter during the entire fermentation process except air. At the end of the fermentation cycle. filtered air to the bioreactor and forced through a sparger to facilitate the dispersion of oxygen to liquid medium.In aerobic fermentations it is important to achieve the optimal oxygen concentration. Continuous fermentations could be achieved through the use of chemostat. an antifoam agent. or turbidostat. the fermenter is shut off and the contents are collected and the product is recovered. In the turbidostat. sterile nutrients are added continuously to the fermenter and equivalent amount of product with microorganism are simultaneously harvested out of the fermenter. It is supplied from compressed.

its concentration and stability and the required level of purity in the end product. A second series of shake-cultures are made to obtain sufficient inoculum for small fermenters. pilot plant scale (usually 50-200 liters) and production scale (usually in cubic meter scale depending on the product). the product represents a very small fraction of the total fermentation broth and extensive purification procedures must be employed. P02. The inoculum size usually ranges between 5-10% of the production medium. The whole fermentation process could be done automatically through special programs. In most cases.Recovery of the product: involves the purification of the products. Fermentation processes are usually developed in three stages namely laboratory scale (flasks.Scale-up: It is the transfer of small scale laboratory fermentation to an industrial large scale. The data obtained during the fermentation process can be stored. viscosity aeration rate. exhausted 02 and CO2 could be directly fed into computers through sensors and probes. The scheme of fermentation process: 1.Production of the product: Growth obtained in small fermenters is used as seed for large-scale production fermentations. 2. laboratory fermenters). pressure. It should be noted that laboratory-scale process may not work or work poorly when first attempted at large-scale. analyzed and used for further optimizing the process and can be compared with other batches. 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 aim of the scientists carrying out this development stages is to find out the optimal fermentation conditions required to maintain the highest product yield. 12 . 3. temperature. The choice of the operations used in recovery depends on the nature of the desired product.preparation of inoculum: The preserved seed let is first either cultivated on agar slopes or in shaken flasks containing liquid culture medium. Uses of computers in fermentation technology Data including pH.

g. Precipitation iv. amylase. penicillin acylase 3. iii. Filtration or sedimentation for separation of cells. callus and hairy root. Centrifugation for separation of cells. 8. vii. steroid transformation 5. viii. Extraction with solvents.g. Fractional distillation.Biodegradation product: degradation of xenobiotics (insecticides and petroleum oil) 6. Reverse osmosis and dialysis. baker’s yeast or single cell protein (SCP).Plant tissue culture: cell suspension. Categories of products 1.Genetically engineered therapeutic protein: Insulin.Biomass: The product may be the cells themselves e. 7. which depends on the sensitivity the product and the number of purification stages.Enzyme: e. Chromatography. v.Immunological product: vaccines and monoclonal antibodies (MCA). 2.Energy: alcohol. 10. vi.The following are the general methods for concentration and or purification of the product: i. 4. 13 .Intra or extracellular accumulation of metals. Ultrafiltration. alkaloids or glycosides which are produced during the stationary phase (idiophase).g. ii. It must be noted that in recovery of the product there are always recovery losses. methane (biogas). growth hormone and interferon.Biotransformation product: e. 9.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.

the cells are harvested and used as cakes or dried materials. microbial insecticides and the symbiotic nitrogen-fixing bacteria. 1) Baker’s yeast Baker’s yeast (Saccharomyces cervisiae) is used mainly for baking. Molasses are usually feed gradually to maintain the sugar concentration between 0.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). Certain yeast strains produce characteristic taste and flavor to the bread. which grow rapidly and produce high yield rich in proteins are potential replacement to animal and plant proteins. locally available cheep substrates such as alkanes. methane. SCP) could serve as new sources of proteins and vitamins.I. Microbial protein in the form of SCP compete with soybean meal. Dough amylases convert starch to sugars. Soybean meal is a byproduct of soy oil production.5-1. However. Since the costs of the raw materials are important components for microbial biomass production. 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. 2) Single-cell protein (SCP) Torula utilis or Hansenula anomala (microbial cells) for use in human and animal feed (single cell protein. methanol. there are psychological barriers to the use of microorganisms as food.5%. SCP is now produced for animal feed using waste material from petroleum industry. which are then utilized by the yeast producing CO2. The use of microorganisms.5. 14 . and ethanol that leavens the dough and causes it to rise and increase in volume. the cheapest protein source. Baker’s yeast is produced by growing the organism in molasses mineral salt medium at a 30oC and pH 4. At the end of fermentation. although microorganisms are used as food in case of mushrooms or incorporated in certain foods. cellulose and many other waste materials should be considered.

Among the most attractive alternatives is the use of bioinsecticides. These products are widely applied today for biological control of insects. Rhizobium species are Gram-negative rods that form an association with leguminous plants (symbiosis). Elimination such fertilizers will also reduce the problems associated with nitrification and contamination of ground water. They exhibit high nitrogen-fixing ability (2~3 times higher than the free living nitrogen-fixing soil bacteria).g. Bacillus thuringiensis). 15 . 4) Rhizobium as a fertilizer The most common nutrient limiting the production of the agricultural crops is nitrogen. Bacillus popilliae) or microbes that can produce more safe compounds that can kill harmful insects (e. The commercial product made from Bacillus thuringiensis consists of a mixture of the protein toxin and the bacterial endospores. They are either microbes that can infect and kill harmful insects (e. This symbiotic Rhizobium species are utilized commercially. significant economic savings can be realized if plants could be grown without the need for adding nitrogen fertilizers. The spores of Bacillus popilliae can survive in the soil for long time and can infect more and more larvae. 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. These synthetic chemicals may accumulate in soil and reach the underground water bodies causing adverse effects on the environment and public health. 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. In agriculture.3) Microbes as insecticides There is both public and scientific concern about the widespread use of chemical insecticides.g.

vinegar. oranges. 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. It was simply produced by leaving wine bott1es in open air. The production of vinegar (acetic acid): Production of vinegar from alcohol has been known for thousands of years. which are then hydrolyzed to disaccharide maltose and finally to glucose by beta amylases.II.Microbial enzymes Enzymes could be obtained from different biological sources (animals. Proteases are used primarily in the detergent and laundry industry and in dairy 16 . Amylases are mainly used in production of sweeteners in the food industry. Microbial enzymes are either extracellular or intracellular. corn or vegetables such as potatoes. soy sauce and sauerkraut. cheese). Acid resistant amylase is used as digestive aid. microbial enzymes have the advantages of being large in number and provides an unlimited supply (most economic). 1) Amylase Alpha amylases are enzymes that hydrolyze starch to short chain polymers (dextrins). alcoholic beverages and fermented vegetables such as pickles olives. III. 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. production of fermented dairy products (yogurt. in the removal of starch sizing from cloth and in the removal of spots in drycleaning industry. However. such as baking of bread. fermented fish and meat. Extracellular enzymes are obtained from the culture filtrate and intracellular enzymes are obtained from the cell homogenate after cell lysis. plants and microorganisms).The use of microorganisms in food Microorganisms are used to produce fermented food products. These fermentations usually uses the natural microbiota associated with these vegetables. apples or pears (cider vinegar). The starting materials for vinegar production may he fruits such as grapes (wine-vinegar). Commercially vinegar is produced in two steps.

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. Are enzymes, which hydrolyze pectin to low molecular weight

8) Pectinase:

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

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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.

Substrate for enzyme

Water-insoluble particulate matter

Enzyme molecules bound to particulate matter

Product of enzyme catalyzed

Immobilized-enzyme technology

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. 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 19 These biosensors are designed for potentiometric or amperometric assay of substrates such as glucose, urea and amino

This operon contains an inducer and structural genes for the enzyme luciferase. Bacterial biosensors require a receptor that is activated in the presence of pollutants and a reporter that will make such a change apparent. Another area for which biosensors have been designed is the detection of pollutants and pathogens. 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. The more glucose concentration in the sample. In the presence of a coenzyme called FMNH2. For example glucose oxidase electrodes has been designated for use in determination of glucose concentration.substrate. These electrodes are essentially glucose oxidase layered over a platinum oxygen electrode. Electrolyte Cathode Anode Semi-permeable membrane + + + + + + + + Teflon membrane Product Enzyme Substrate Diagram of simple biosensor For example the lux operon from Photobacterium was studied as a reporter. This has been designed using bacteria that can locate biologically active pollutants. the smaller the amount of oxygen detected by the electrode. the more oxygen consumed by the reaction. 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. 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. Therefore a bacterium containing the lux gene will emit visible light when the receptor is activated. 20 .

Such water-soluble biopolymers are rapidly emerging as source of gums. used as plasma substitute and as complex with iron for iron deficiency anemia. 21 . dextran or cellulose and heteropolysaccharides e. produced commercially by the Gram-negative bacterium Leuconostic mesenteroid. organic nitrogen and phosphates. Commercially the process could be carried by the direct or indirect methods: exopolysaccharides are classified into two types namely.Extracellular polysaccharides Microbial cell surface (cell wall and glycocalyx) is rich in polysaccharides. the microorganism produces an extracellular inducible dextransucrase enzyme. Traditional gums are produced from plants and algae. The type of semiconductors used for this purpose are all field effect transistors (FET). which hydrolyze sucrose to glucose and fructose and at the same time utilizes glucose for dextran synthesis (polymerization). In the production of dextran. the substrate does not enter the cell.g.g. but if is disorganized and attached loosely to the cell wall. so that the medium containing the bacteria becomes highly viscous. Microbial xanthan. The slime layer tends to be soluble in water. it is called capsule. The growth and production medium contain sucrose. When the glycocalyx is organized and attached firmly to the cell wall. homopolysaccharides e. They have wide application in food. pharmaceutical and other industries due to their unique physical properties.Biochips It is possible to combine enzymes (or biological systems) with semiconductors. 1. emulsifying agents. When an FET is combined with an enzyme it is called ENFET. Instead. Microbial gums also are not subjected to crop failure due climatic conditions and are supplied in constant quality. binders and lubricants. thickening agents. stabilizers. suspending agents. it is described as a slime layer. The polysaccharides that covers the microbial cell surface are also termed exopolysaccharides or biopolymers. Microbial gums are used as gelling agents. 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.Dextran: (polymer of α-D-glucose).

pH is controlled to 7. which depends on precipitation with ethanol. ammonium nitrate) and salts. 2. 22 . then the cells are separated and the medium containing the enzyme is then used to transform sucrose into dextran. The problem with this method is that during recovery of the product. sucrose. nitrogen source (yeast extract. The enzymatic process is carried out at 25oC-30oC and pH 5. Xanthan is used in paints.i. Xanthan is recovered by precipitation with methanol. 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. The process is carried out under anaerobic conditions at 25oC-30oC and pH 5.Direct method: The medium is supplied with enough sucrose (10 %) to support growth and dextran synthesis. and textile industries as well as in oil industry as drilling fluid additive and for enhanced oil recovery. printing. Viscosity increases during the course of fermentation. which supports growth only. peptone. or corn starch hydrolysate). The precipitated xanthan is then dried and ground.Xanthan: (heteropolysaccharide) produced commercially by Xanthomonas campestris. bacteria may be trapped with the product. Production of xanthan is carried out in medium containing carbohydrate (glucose. ii. which takes about 30 hrs.Indirect method (Enzymatic): The bacteria are allowed to grow in presence of 3% sucrose.

Primary metabolites of biotechnological many others. Moreover the regulation of differ significantly from that of the primary Trophophase Log b Idiophase Screening microorganisms: In screening for new metabolites. Primary metabolic pathways are similar in all microorganisms whereas secondary metabolites are formed only by few organisms and their formation is secondary metabolites biosynthesis metabolites. A useful antibiotic- 23 . relatively few possess the criteria for an industrially valuable microorganism. Of the many species. Samples from various sources are collected and examined as a potential source of antibiotic-producing microorganism. Examples of secondary metabolites produced by microorganisms are antibiotics and glycosides by plants. (trophophase). They are produced during the stationary phase of growth (idiophase). butanol. They are synthesized during the lag and logarithmic phases of growth include hand. importance On the organic acids. Time extremely dependent on environmental conditions. The search for antibiotics in the pharmaceutical industry presents a good example of how screening programs are employed to select microorganism for industrial applications.V. Primary metabolites are essential for life and reproduction of cell.Production of metabolites Metabolites are either primary or secondary. researchers try to isolate strains from the environment with the hope that they are potentially valuable in producing a commercially useful product. acetone and other secondary metabolites seem to be non essential for growth and reproduction.

Starch can also be used if amylases are formed by the producing fungus (or should be added to the fermentation broth). In pharmaceutical and cosmetic industry.Organic acids 1. The use of methanol during fermentation render the mycelium not sensitive to the presence of iron in the medium (i. which have been prohibited in many localities worldwide. A positive result in such a primary screening procedure does not ensure the discovery of an industrially useful strain. 24 . Beside the use of optimum carbon and nitrogen source concentrations. The success of a screening program depends on both the kind of organism used and the method for detection of activity.O. V. poison the M. It simply identifies strains of microorganisms that have the potential for further development. Citric acid is a primary metabolite in the tricarboxylic acid cycle. the carbon source must be re-treated with either precipitation or exchanger to remove cations. For this reason.). Citric acid is also used in the detergent industry to replace the use of polyphosphates. The main carbon source used for citric acid production is sugar cane syrup.producing strain must produce metabolites that inhibit the growth or reproduction of pathogen. The choice of strain is reported to have a 30-40% influence on the outcome and the test procedure has a 60-70% influence.e. 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. It is used widely in food industry especially in the production of soft drinks. citric acid is used as preservative for different products and as iron citrate for treatment of iron-deficiency anemia.Primary metabolites A. Citric acid: Citric acid has long been isolated from unripe fruits but today its production is entirely produced microbiologically by fermentation. Citric acid is produced commercially using mutants of Aspergillus niger and Aspergillus wentii. Metals are removed from molasses with calcium hexacyanoferrate or with cation exchanger. metal salts and phosphate concentration in the medium were found to influence the yield. sugar cane molasses or sugar beet molasses.

After sterilization of the medium. The low pH. The precipitate is then filtered and dried or subjected to further purification if needed. Optimum production is achieved at low pH (3. it must be precipitated first as calcium oxalate at low pH and the precipitated calcium oxalate is removed with the mycelium. Gluconic acid is produced commercially by Aspergillus niger using the 25 .Gluconic acid: Gluconic acid finds wide application in pharmaceutical industry.5). filamentous mycelium with limited branches produces little citric acid. but it is sensitive to oxygen deficiency. For recovery of citric acid. If oxalic acid is present as byproduct. Submerged cultures are more widely used today for citric acid production. During citric acid production the aeration rate must be kept at optimum levels as the fungus require little oxygen. On the other hand. the mycelia are removed by centrifugation. metal industry and leather industry. however. Citric acid is then precipitated as calcium citrate at pH 7. Although the process have the advantage of being simple and needs low investment.The fermentation should be carried out as two phase fermentation. In this process. The structure of the mycelium formed in submerged culture during the trophophase is very important for high citric acid yield. loose. which usually takes 5-8 days. during the first phase the conditions should be adjusted for maximum growth to obtain enough mycelium. ferrous gluconate is used to supply iron for the treatment of anemia. the produced citric acid is extracted with hot water and then purified. Very small solid pellet is the best for optimum citric acid production. Surface processes use solid or liquid media. the Aspergillus niger strains are not as sensitive to trace elements as in the submerged processes. followed by restriction of cations to enhance the production of citric acid. the labor costs high. the medium is inoculated with Spores and spread in shallow layers (about 5 cm thick) on trays and incubated at 28oC. The fermenter used must be constructed from resistant material such as stainless steel. Those employing solid substrates may use either wheat bran or pulp from sweet potato starch production as culture medium. 2.2 and 70-90oC. At the end of the process. Calcium gluconate is used to supply calcium to the body. Citric acid is produced both by surface and submerged fermentation. have the advantage of lowering the risk of contamination.

is concentrated by using a vacuum then subjected to further purification. Lactobacillus bulgaricus is used with whey.1% boron is added to the fermentation medium to prevent precipitation of calcium gluconate (35% of calcium gluconate remains in solution). When glucose is used as carbon source Lactobacillus delbruecki is used for production. 3. the pH is controlled to 5.5-6. Calcium lactate is used in the treatment of calcium deficiency. further incubation results in utilization of gluconic acid. potato starch. The fermenter is agitated for mixing but not aerated. The free acid is liberated from the crystalline calcium gluconate by the addition of acid. 0.5vvm). Borate is added to the medium to stabilize calcium gluconate (which have low solubility) and prevents its precipitation. Ammonium phosphate and trace amounts of other nitrogen sources are usually used. iron lactate is used in treatment of anemia and sodium lactate is used as placticizer and moistening agent. Lactic acid fermentation is carried out under anaerobic or microaerophilic conditions at temperature range of 40-500C and pH range of 5.submerged culture. Fermentation usually takes about 2-3 days. Calcium hydroxide is added to the fermentation broth to control the pH.5 using calcium carbonate and the temperature is kept at 30oC.5. molasses and whey can be used for the production of lactic acid. Gluconic acid is recovered by addition of calcium hydroxide to the fermentation broth after removal of the mycelium. The fermentation is conducted under highly aerobic condition (aeration rate between 1-1. and Lactobacillus pentosus is used with sulfite waste liquor. 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. Lactic acid: Lactic acid is a valuable industrial product because its derivatives have a variety of uses. The filtrate. 26 . which contains the soluble calcium lactate. Substrates containing starch must be hydrolyzed first to glucose either by acid treatment or enzymatically. The process takes about 36-48 hr when all glucose is utilized. Glucose is usually used as carbon source. Several carbon sources such as corn-starch.

1.3 vvm at 28oC. The fermentation takes 7 days with an aeration rate of 0. The fermentation is carried out in two stages process. The culture filtrate may be dried and used as animal feed additive. which lasts between 2-4 days. The culture medium consists of soybean meal or fish or meat extract as nitrogen source and cobalt. peptone and soybean oil. 27 . Cobalamine is then converted to cyanocobalamine with KCN. In the second aerobic phase. Riboflavin is formed both extracellular and bound to mycelium. chromatography. The bound vitamin is released from the cell heat treatment at 120oC for 60 minutes and the mycelium is separated and discarded. commercial production for economical purposes is restricted for the production of vitamin B12 and riboflavin. In the first anaerobic phase. which lasts between 3-4 days cobalamine (coenzyme B12) is produced. Vitamin B12 could be also produced using Bacillus megatherium. Vitamin B12 (cyanocobalamine): Vitamin B12 can be commercially produced by Propionobacterium shermanii.B) Vitamins Although microorganisms produces many vitamins.5. for pharmaceutical use the vitamin is extracted with butanol. 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.5-8. Vitamin B2 (Riboflavin): Riboflavin can be produced by Ashbya gossypii or Eremathecium ashbyii. B The vitamin is then purified by When radioactive Vitamin B12 (used in schilling test for pernicious anemia) is required. The medium used for production contains corn steep liquor. radioactive cobalt is added to the medium. Cobalamine is almost completely intracellular and is released into solution by heat treatment at 80-120oC for 10-30 minutes at pH 6. Both are cotton pathogens and so are not suitable for production of riboflavin in Egypt. cobinamide is produced. Both stages can also be operated continuously in two fermenters operated in cascade fashion. 2. B The vitamin is used for treatment of pernicious anemia and as animal feed supplement.

1. which is formed in the tricarboxylic acid cycle. amino acids are used as starting materials for the manufacture of polymers (e. Glutamic acid. α-ketoglutarate is then converted into L-glutamic acid through reductive amination with free ammonium ions. lysine and tryptophan for example are produced industrially by microbial strains that produce an excess of these amino acids. In some instances the amount of amino acid synthesized can exceed the cells need for that amino acid. sodium glutamate). Ammonia feeding permits pH control of the process. 3 . as antoxidants (e.tryptophan). Lysine is produced commercially by microbial fermentation using an auxotroph of Corynebacterium glutamicum.g.g. The excess amino acid may be excreted into the culture medium. 2. 28 . In food industry. Lysine: Lysine is an amino acid essential for human and animal nutrition. L-cysteine and L. Cane molasses is usually used as carbon source and ammonia or urea as nitrogen source. The yield is so high to the extent that part of the tryptophan precipitated out (nearly 78 g/l). In medicine. The key precursor of glutamic acid biosynthesis is αketoglutaric. Ammonia feeding permits pH control of the process (near neutrality). Amino acids find wide applications in the food and chemical industry as starting materials. Addition of indole and serine to an extract of this enzyme results in conversion of indole into L. L-Tryptophan Studies using recombinant DNA technology led to a 230-fold increase in tryptophan synthetase enzyme of Escherichia coli. amino acids are used to enhance flavors (e. L-Glutamic acid: Glutamic acid is produced commercially by a mutant strain of Corynebacterium glutamicum. polyalanin fibers). Ammonium salts or ammonia can be used as nitrogen source. In chemical industry. High PO4 causes growth and production inhibition. amino acids are used as ingredients in infusion solutions.tryptophan. Sucrose and molasses are widely used as carbon sources.g.C) Amino acids Many microorganisms can synthesize amino acids from inorganic nitrogen compounds such as ammonium sulfate.

1. Later on penicillium notatum was replaced by Penicillium chrysogenum. during World War II. If the fermentation of penicillin is conducted without the addition of side chain precursors. They are effective against microorganisms in low concentration. in USA it was suggested that through the use of corn steep liquor. Penicillin: Penicillin is an acylated 6-aminopenicillanic acid (6-APA). In 1940. but they were unable to develop an industrial. 6APA consists of athiazolidine ring fused to a β-lactam ring.V) Secondary metabolites Antibiotics Antibiotics are compounds produced by living microorganisms and are used as therapeutic antimicrobial agents. Only benzyl penicillin or penicillin G of this mixture is 29 . an intensive screening programs for antibiotics started and streptomycin then chloramphenicol and several hundreds of antibiotics were discovered. However. Later. which contain phenyl alanine. and a submerged fermentation process with high yield of penicillin was developed. it was possible to develop a fermentation process. which is then replaced by various side chains during the fermentation. Penicillin was the first discovered antibiotic by Fleming in 1929 as a product from penicillium notatum contaminating Staphylococcal culture. Acylase β-lactam ring Thiazolidine ring β-lactamase Biosynthesis of penicillin depends on the condensation of valine and cysteine amino acids to yield 6APA and L-α-aminoadipate as side chain. a mixture of natural penicillins are produced. the British scientists continued the research.

so that only the desired penicillin G is produced.5% total vegetable oil and the process was carried out by continuous feed. The yield of penicillin has been increased about 50. which extends for further 50 h. followed by the penicillin production phase.8% total phenylacetic acid and 0. By fed batch the production phase could be extended to up to 160 h.5-7. During the first 40 h of fermentation (growth phase) cell mass is built up. Rapid Rapid Easily utilizable sugar (glucose) Slowly utilizable sugar (lactose) Slow Acids Acids Rapid Gas Gas Growth Glucose utilization pH change Lactose utilization Penicillin production The pH of the fermentation during the production of penicillin should be kept constant at 6.0v/v depending on the strain used.5-0. The medium used today contained 10% total glucose (or molasses).0 and the optimal temperature range between 25-270C. submerged fermentation and proper recovery. if phenyl-alanine or phenyl acetic acid are added as a side-chain precursor. 0.5-1. However. Penicillin was originally produced by a strain of Penicillium notatum . During 30 .000 times through the use of the non pigmented Penicillium chrysogenum mutants. The aeration rate is between 0. The course of the fermentation takes about seven days (longer times when very large fermenters are used).therapeutically effective and all the other produced penicillins are by-products that create problems during the down-stream processing. which produced pigments and only about 2 international units per milliliter (lU/ml) of penicillin by surface culture fermentation . 4-5%corn steep liquor. proper medium.

glucose as the carbon source and sodium chloride. the acids are first utilized before the M. As in penicillin fermentation.the growth phase the M. For recovery of penicillin. utilizes glucose (easily utilizable) sugar with the production of acids leading to drop in pH.0vvm and the temperature is kept between 28-30oC with pH in the neutral range. with production of mycelial biomass.O. During the first phase of streptomycin fermentation. there is rapid growth of Streptomyces griseus. 2. The mycelium inoculum is used to initiate the fermentation process in the production fermenter. starts to utilize lactose (slowly utilizable) and the ph starts to rise again. 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. Oxygen supply is adjusted between 0.O. During the production the M.O. however.5-1. Penicillin should be recovered before the third phase starts. dried and used as animal feed supplement. Streptomycin: Aminoglycosides are oligosaccharides antibiotics primarily effective against Gram-negative bacteria. Streptomycin is produced by strains of Streptomycin griseus. The pH of the medium rises due to proteolytic enzymatic activity and release of ammonia from soybean 31 . The filtrate is cooled to 2-30C. 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. but mainly used in the treatment of tuberculosis. 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%. The length of the fermentation ranges between 4-7 days depending on the strain. The medium used for the production contains soy-bean meal as the nitrogen source. the liquid medium containing the penicillin is separated from the fungal cell using rotating vacuum filter. utilized lactose with no acid production and so the pH remains constant around neutrality. The last (third) phase starts when the mycelium starts to lyse and so the pH starts to increase. The fungal biomass is scraped from the surface of the filter drum. spores of Streptomyces griseus are inoculated into a medium to establish a culture with a high mycelial biomass for use as inoculum.

Bioremediation Bioremediation denotes ability of microorganisms to detoxify or degrade toxic pollutants (mainly xenobiotics) from the environment. In other words. The antibiotic is then precipitated with acetone and further purified by chromatographic methods. For recovery of streptomycin the mycelium is separated by filtration.meal. During the second phase. After depletion of the carbohydrate from the medium. B & C Tobramycin Erythromycin Amphotericin B Nystatin Neomycins B & C Producing microorganism Bacillus lichenformis Bacillus polymyxa Streptomyes spectabilis Streptomyces kanamyceticus Streptomyces tenebrarius Streptomyces halstedii Streptomyces nooses Streptomyces noursei Streptomyces fradiae VI. One of the approaches to save the environment from the adverse effects of these pollutants is the use of bioremediation. To avoid such problem. During the last few decades man has used fossil fuel resources and produced many novel synthetic compounds (xenobiotics). streptomyin accumulates in the medium. Streptomycin is adsorbed on charcoal or ion exchange resin and then eluted from the charcoal with acid alcohol. the use of the metabolic activities of microorganisms to change an undesirable chemicals into ones that has less objectionable properties. it is better to isolate and use of phage-resistant mutants for production. The disposal or accidental spillage of these compounds creates serious environmental pollution problems. Infection with bacteriophages may cause problems in streptomycin production. The remaining glucose in the medium and the ammonia released from the soybean meal are consumed during this phase. the production of streptomycin ceases and the bacterial cells begin to lyse. 32 . Other commercially produced antibiotics Antibiotic Bacitracin Polymyxin Spectinomycin Kanamycins A. The capacity of different microorganisms to degrade different organic compounds and transform various substances forms the basis for bioremediation.

Another approach to clean up the oil from a spill is to inoculate the spill area with a microorganism that can degrade the oil. chlorinated solvents and chemical insecticides residues are among the most important bioremediation processes. VII. The design of novel synthetic compounds. For example a genetically engineered strain of Pseudomonas putida has been constructed for this purpose. Accordingly such strains can be used to protect the pollution of irrigation water with selenium which can kill wildlife. This process is termed microbial transformation or biotransformation. The results showed 96% degradation of glyphosate after 21 days.Bioremediation of petroleum hydrocarbons. changing the design of this synthetic compound to a linear chain renders the product easily degradable. For example. and naphthalene). This mechanism involves converting selenium to less toxic form. Pilot field trials were carried out in aerated reactors maintained at pH 7-8 and 250C and supplemented with ammonium nitrate as nitrogen source. which is detergents is resistant to biodegradation. xylene. transformation is a very helpful tool for: 33 Microbial . 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. due to branching of the alkyl chain. 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. 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. which could be degraded. The biodegradation of the world s most widely used herbicide glyphosate by Flavobacterium species. alkyl sulfonates. The strain has the ability to metabolize hydrocarbons present in crude oil (octane. is now studied. It is now used in industrial waste streams by immobilized bacteria technology. Agrobacterium and Achromobacter species is extensively studied.Microbial transformation Microorganisms are able to modify a wide variety of organic compounds. However.

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. The price of 1g cortisone was 200$. epoxidation.The steroid substrate is added in suitable solvent. enzymes or immobilized enzymes are currently applied.the production of more useful and expensive products. Steroids such estrogen. immobilized cells. reduction.1. spores. Derivatives of progesterone and estrogens are used as contraceptives and derivatives of cortisone are used as anti-inflammatory in rheumatoid arthritis. Different biotransformation processes using growing cultures. When growing cells are used.The microorganism is allowed to grow for about 17-48 h under controlled conditions (pH. Biotransformation reactions include various types of hydroxylation. microorganism using induced enzymes. 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 method applies for steroids. Steroids can be produced by chemical synthesis. However. Steroid biotransformation: Steroids are group of lipid compounds having the same basic cyclopentano phenanthrene nucleus.) 2. hydrocarbons. temperature. 2. 34 The transformation reaction is considered to be a detoxification reaction by the . but the process is laborious and expensive. Description of fermentation: 1. resting cells. oxidation. epoxide cleavage. progesterone and androgens are used as therapeutics. For instance. 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. terpines and antibiotics. the price was reduced to one $ or less when the process was conducted by Rhizopus nigricans. oxygen. …etc. hydrolytic or condensation reactions.Elucidation of the chemical structure of complex compounds. the chemical process of conversion of deoxycholic acid (bile acids) to cortisone required 37 steps and yielded 1g cortisone from 615g deoxycholic acid.

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. 5. 1. The technology is already well established and is particularly 35 . sometimes the growth medium is different from that used for transformation. 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.Simple medium. 2.Extraction of the steroid product with organic solvent and its crystallization.Ease of recovery of the product.No fear of contamination.After transformation spores are separated and recycled. purified and freed from any vegetative debris. 6. 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. diminished reserves and unpredictable supplies are forcing many industrialized nations to seek alternative fuel resources. 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.Removal of the mycelium by filtration or centrifugation.Cost is much reduced. Steroid transformation by fungal spores Fungal spores are often active as vegetative cells in transformation of steroids. VIII. The microorganism is allowed to grow in presence of the steroid as inducer for long period to produce enough spores.Energy and biotechnology The increasing fuel prices. Steroid transformation by fungal spores provides the following advantages: 1.The conditions are readjusted for steroid transformation. 4. Spores are then collected in buffer. Ethanol: Ethanol production as an alternative liquid fuel is one of few viable options that are currently available. 4.3. 3.Recovery of the product 5.

Sweden and many other countries. Among the organisms that have been extensively studied for alcohol production are Zymomonas mobilis and Thermoanaerobacter ethanolicus. Non-metabolic accumulation may result from biosorpition and precipitation of metals extracellularly at or within the cell-wail matrix and intracellularly. 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. The use of strains that can grow at temperatures above the boiling point of ethanol. The methanogenic bacteria are able to utilize only a restricted group of substrates for the production of methane. Metal accumulation by microbes may occur through metabolic or nonmetabolic processes. Accumulation of metals by microorganisms: Certain microorganisms have been found to accumulate large quantities of metals. nutrient-rich residue used as fertilizer and improves sanitation. and/or by reducing the recovery costs through. The largest ethanol program is in Brazil.Microorganisms and the recovery of metals 1. It means that a biogas production unit produces clean fuel. Brazil produces and uses large amounts of ethanol as fuel for cars (20% ethanol + 80% gasoline). 2. Accumulation of metals at the cell surface is strongly affected by environmental factors such as temperature and pH. 36 .valuable in countries that have abundant supplies of plant residues. 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. Metabolic accumulation is usually intracellular. or natural gas or biogas is a microbial product of the anaerobic digestion of organic matter. Philippines. Methane is produced by methanogenic bacteria. Gasohol (gasoline containing 10% ethanol) is also sold in the USA. Methane: Methane. Methanogenesis is a widely used process in organic waste disposal. Indonesia. Other programs for ethanol production are also reported in Australia. IX. Cars were also built or converted to run on pure hydrated ethanol (96% ethanol + 4% water).

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. Copper bioleaching. 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. Another problem with the traditional methods of processing ores is air pollution. This process is called bioleaching. it is possible to leach the ore in situ. 2. but most commercial bioleaching processes are carried out after mining the ore. It is usually present in low-grade ore in the 37 . 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. Biotechnology and mining: The extraction of various metals from ores has become a problem for the mining industry because easily accessible.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. The leaching liquor containing Thiobacillus ferrooxidans. Microorganisms can play an important role in recovering minerals from lowgrade ores. Fe2+ and SO42+. This has made it necessary to process lower-grade ores and to find techniques for more efficient extraction of metal in the ores. The process is currently applied on commercial scale for copper and uranium recovery from low-grade ores. is carried out by sprinkling the leaching liquor over the ore heap. 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. If the ore is porous and overlays a water-impermeable stratum. Uranium is used as fuel by the nuclear power generation industry. nutrients for the bacterium. for example. high-grade mineral deposits of ores are becoming depleted. The solution percolates through the rock pile to the lower level where the copper rich liquor is collected.

2.Separation of the bacterial cells is often carried out by centrifugation while ammonium sulphate is used to precipitate diphtheria and tetanus toxoids. during this step preservative and adjuvants are added.Vaccines: a) Production of bacterial vaccines 1.At the end of the growth period the cultures (from different fermenters) are pooled to form a bulk harvest. 5. Most modern viral vaccines are live attenuated virus strains and so no inactivation step is required. The skin of calves is used for smallpox vaccine. The bulk harvest is often a dangerous mixture of bacterial cells and metabolic products (toxins). The required virus is inoculated and the living cells are incubated until the virus growth is maximal.The incubation conditions are adjusted. X. poliomyelitis and rabies. There are three important exceptions: influenza.Inactivation of the cultures either by heating or the addition of a bactericide. to ferric iron. Formalin at a concentration of 0. It is also used to detoxify the toxins of diphtheria and tetanus. Subsequently ferric iron. converting them into harmless toxoids. 38 .form of the insoluble uranium oxide. 3. Blending: various components of the vaccine are mixed to form a final bulk. Thiobacillus ferrooxidans can oxidize the pyrite. which is usually present in uranium ore. 4.Immunological products 1. can oxidize uranium oxide to the readily leachable form UO2SO4. The culture fluids containing the virus are then harvested and pooled.5% is used to kill the cells of whooping cough. b) Production of viral vaccines The growth of viruses requires living cells.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. Phenol is also effective in killing bacteria in cholera and typhoid vaccines. the fertile eggs (chick embryo) for influenza and yellow fever and cell cultures prepared from monkey kidney are used for rabies vaccine.

1) In process control Is the control carried out over the starting materials and intermediates. 2) Final product control The final product should be tested for identity.Quality control of vaccines To provide assurance that both efficacy and safety of every batch of every product is achieved. a. The potency of live viral vaccines is estimated in the same way except that living cells are used. An example from virus vaccine manufacture is the titration prior to inactivation of the infectivity of live poliovirus used to make inactivated poliomyelitis vaccine. b. Vaccines containing killed microorganisms or their products. Preparations containing combined agents are required the pass the tests prescribed for each separate component. such as bacterial toxoids are tested for potency by the ability to stimulate the production of antibodies in a group of laboratory animals.Identity tests: the identity of bacterial vaccines by in vivo agglutination and precipitation methods. The quality control for diphtheria and tetanus toxoids is tested for absence of free toxin. c. Antibodies are then assayed by vaccination of group of animals and titration of blood samples for antibodies and comparison with the corresponding standard vaccine. 39 . Vaccines containing live microorganisms are generally tested for potency by counting their viable particles. Inactivated viral vaccines are tested by observation of the specific antibody responses that they produce after neutralization with specific antisera. Usually laboratory animals are used to detect any harmful or disease effect.Potency assay: each immunological product is intended to exert a unique prophylactic or therapeutic effect.Safety tests: killed bacteria or bacterial products must be completely free from the living microbes and toxoids require testing for inadequate detoxification. due to inadequate detoxification with formalin. potency and safety.

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. but myeloma cells have a metabolic defect and so can not use this bypass pathway. HAT is a mixture of hypoxanthin. Aminopterin is a powerful toxin which block certain metabolic pathway. spleen cells and fused cells. Monoclonal antibody production Consequently. HAT is non toxic to spleen cells medium. This pathway can be bypassed if the cell is provided with intermediate metabolites hypoxanthin and thymidine. they die in HAT medium.2. they have metabolic defect. Once the animals exhibited a good antibody response.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. It is usually impossible to separate the different types of antibodies.e. Animals (usually mice or rats) are immunized with antigen. When the culture is set up in HAT medium. indefinitely in cell culture condition. it contains myeloma cells. The myeloma cells are selected because of their inability to grow in 8-azaguanine as they lack certain enzyme i. the spleens are removed and a cell suspensions is prepared (lymph node cells may be also used). polyethylene glycol is the agent used to fuse the two types of cells. The fusion mixture is then set-up in culture medium containing "HAT". 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. Aminopterin and thymidine. 40 The spleen cells dies . 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.

the situation was changed and scientists have developed techniques making it possible to move genes from one cell type to another e. 41 . For instance a gene that codes production of limited quantities of a valuable compound from normal plant or animal tissue. consisted of separately incorporating synthetic genes of the A and B chains of human insulin into the βgalactosidase regions of the pBR322 plasmid. producing only one type of antibody.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. The culture is then distributed in wells. so that the patient needs a larger dose. the myeloma cells are killed by HAT. from plants and mammals to bacteria. The patients injected with these types of insulin developed antiinsulin antibodies which led to minor problems. can be isolated from a plant or animal cell and cloned into a bacterial cell.naturally in culture after 1-2 weeks..g. These two plasmids were cloned separately in Escherichia coli. Any well containing growing cells are tested for the production of the desired antibody. The bacterial cell may then synthesize unlimited quantities of the gene product. Since the discovery of recombinant DNA technology in 1973. 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. 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 process of insulin production using recombinant DNA technology. The future of genetic engineering is considered almost unlimited in its commercial applications. which wasdeveloped by Eli Lilly in collaboration with Genentech Inc. XI. The transformed bacteria synthesized the A and B chains separately. 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.

α-interferon: Interferons are produced by eukaryotic cells in response to viral infection or foreign double stranded RNAs (viral or synthetic). the two chains were mixed and connected together to prepare an active insulin preparation. mRNA was copied into cDNA and a methionine codon (ATG) was chemically synthesized and attached to the 5’end of the proinsulin cDNA. In another approach. the interferon causes the cells to produce molecules that prevent replication of the infecting virus. the A and B chains were released from the βgalactosidase. There are several kinds of interferons each made by a different cell type. Interferon is extracted and used by the cells. Several modifications were then developed. This was cloned in a plasmid vector and transferred in Escherichia coil. coli β.Synthesize A-chain gene and insert into a plasmid Synthesize B-chain gene and insert into a plasmid Insert into E. α-Interferon is produced by leukocytes. The C peptide (connecting chain) was cleaved with enzymatic reaction yielding pure human insulin. After purification. The proinsulin was then released from the β-galactosidase enzyme by cyanogen bromide which destroys the methionine linker residue.Gal B chain Lyse cells and cleave with CNBr Oxidize A chain B chain Insulin By means of cyanogen bromide. while β- 42 . When the cells become infected with virus. The infected cell produces interferon for a few hours.Gal A chain β.

Interferons are now available commercially for clinical use. coli. 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. it was possible to clone the gene for hepatitis B surface antigen (HBs antigen) into cells of the yeast Saccharomyces cerevisiae. it was possible to clone naphthalene oxidase gene from Pseudomonas sp. most of the genes for human interferons have now been cloned in bacteria.interferon is produced by fibroblasts and γ-interferon is produced by sensitized T cells. Using DNA from HBV.Chemicals Indigo dye The dye indigo is a plant product but was manufactured chemically to reduce the cost. 43 . 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. coli produced indigo. However. Human-growth hormone: Human growth hormone is another pharmaceutical product made more efficiently by a genetically engineered bacterium. Interferon α was found to reduce the duration of viral infection. coli. This was the first vaccine against a human disease produced with genetic engineering methods. The yeast expressed the gene and made HBs antigen particles that could be extracted after the cells were broken. effective against herpes virus infection of the eye and reduces the incidence of attacks of multiple sclerosis. it was possible to obtainunlimited quantities of HBs antigen particles. Previously the hormone was obtained only in extremely small quantities by extracting it from the pituitary glands of the animals. However. as the naphthalene oxidase enzyme oxidized indole of E. Hepatitis B vaccine: Production of certain vaccines such as hepatitis B. into E. Since yeast cells are easy to propagate. coli to 2-3 dihydrodiol which spontaneously oxidize and dimerize to indigo resulting in blue E. yeast or mammalian cells. 2. The modified E. The genetically engineered product is being used to treat children pituitary dwarfism and other conditions related to growth hormone deficiency.

primer annealing and polyrmerase extension. A genetically engineered ice-minus strain with the surface protein deleted is sprayed to replace the indigenous population and protect the crop. the primers 44 . The starting material for PCR is a segment of double stranded DNA containing the target sequence. double stranded template DNA is denatured to single stranded DNA by heating. The technique involves three temperature-dependent steps namely template denaturation. However. the release of genetically engineered raised environment questions. primers complementary to the 3' ends of the target DNA are added. 5.3.Polymerase chain reaction and hybridization techniques: Both are used as diagnostic techniques. This technique was applied to tyrosyl RNA synthetase.Improvement of performance: The key control gene for lysine biosynthesis was identified. The manipulated gene is cloned and reintroduced at a high copy number. These bacteria are conditional plant.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. In the second step. Similar principles have been applied to antibiotic-producing organisms. causing death due to frost damage only at temperatures that can initiate the freezing process. In the first step. 4. manipulated to be insensitive to repression. 6. which results in frost damage to the plant. Substitution of certain amino acid at a specific position can be achieved by site-directed mutation in the cloned gene. Polymerase chain reaction (PCR) is a technique that allows a specific DNA sequence to be amplified. When the mixture of primers and DNA is cooled. 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. Using this technique.pathogens.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.

Each cycle takes no more than a few minutes. By the twenty-fifth cycle. become a powerful tool for genetic diseases. At the end of this cycle two copies of the gene are formed from each initial copy. which can be obtained without harming the fetus. there are millions of copies of the target sequence. forensic medicine and basic research. For instance. 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. In subsequent cycles the number of copies of the target gene increases exponentially. 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. The technique enabled clinicians to detect infection by the AIDS virus when other methods used for PCR diagnosis have failed. 45 . can be amplified by PCR and analyzed to see whether the DNA is identical to that of a person suspected of committing a crime. trace amounts of DNA. enough to he easily analyzed by routine laboratory procedures. DNA amplification has many applications in clinical diagnosis.bind to the gene. Hybridization techniques probes using a DNA wide finds application in the detection of bacteria and diagnosis of hereditable diseases. in fluids such as blood or semen or in tissue such as hair. of also various Today the technique is diagnosis has of several other diseases.

The compound is excreted in their milk. Unless certain precautions are taken. 8. There is a requirement for the removal of the cell wall when fusion of bacteria. cells tend to delete sections of the plasmid or even the whole plasmid. 1.7. where two whole genomes are mixed. re-assorted for selection of the required characteristics. 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. It is usual for cloned genes to be carried on a plasmid. 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. However. poultry and fish. The use genetically engineered strains generated new problems. have been produced in transgenic sheep.Cell fusion: Gross genetic engineering is possible by cell fusion. On a laboratory scale. Problems and solution The problems which faced the biotechnology companies in both regulatory and safety issues were new to the industry. Over-expression of growth hormone has also been tried in order to increase the rate of growth of livestock. a protein used as replacement therapy for genetically-deficient individuals at risk from emphysema. Production of foreign proteins in transgenic farm animals find a more significant progress. Subsequent regeneration of cell wall and outgrowth of the fused cell is necessary before selection of the desired phenotype. For example α1-antitrypsin. yeast.Modification of macroscopic animals: Transgenic animals Transgenesis is the use of gene manipulation to permanently modifying germ cells of animals. strict control of culture storage and fermentation conditions has maintained productivity.Strain stability Reversion of mutants to low productivity is a common problem in fermentation industry. fungi or plants cells before fusion. 46 . For example the production of super mice was a result of the overproduction of human growth hormone. by regulation of the temperature of the process.

Inactivation of cells before processing. DNA can be constructed by incorporating the gene coding for the desired product together with DNA coding for an additional amino acid sequence.Environmental monitoring.Engineering design a) Scale: fermentaions with genetically engineered organisms are often conducted at small fermenters such as growth hormone. 6. The expressed protein will be composed of the target protein plus a tail.Validation of the above system.Use of detailed operating instructions.Incineration or filtration of exit gas. which need special considerations such as: 1. 8. enzyme activity and specific binding. 5. Macro-modification of proteins for facilitated processing utilizes the recombinant DNA technology to facilitate the product recovery and hence reduce the production costs.The fermenter is kept at low temperature during growth and high temperature during the production phases. 2. 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. compared to the traditional fermentations of alcoholic beverages. 2.Emergency planes for large spillages or leaks.Training of personnel. b) Containment: to prevent accidental release of genetically engineered organisms into the environment. c) Separation of the product: Downstream processing accounts for about 80% of the production costs of proteins. high level expression results in the formation 47 .Use of leak proof agitator seals. 3. 7. For example in many prokaryotes. The additional properties may include differences in solubility and/or hydrophobicity. antibiotics and organic acids which use vessels with several thousands of liters. 9. 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.Inoculation and sampling through non-aerosol producing system. 4.

Plants that have gained new genetic information from foreign sources are called transgenic plants. For example glyphosate. The downstream processing of such products may affect their activity. is known to inhibit an enzyme called EPSP synthetase. The transgenic tomato plants expressing this gene were found to be glyphosate tolerant. The transgenic plants prepared were found to express higher levels of the enzyme and were found to be more tolerant to glyphosate. the most commonly used herbicide. In this method the gene sequence coding for the desired protein is extended by insertion of the codons for arginine. a mutant gene encoding EPSP synthetase was introduced into tomato plants. In another approach. The polyarginine tail is then cleaved with the enzyme carboxypeptidase B.of inclusion bodies (insoluble aggregates of the product). particularly in E. engineered and introduced into the plant. 48 . Another example of such purification fusions is polyarginine tailing. The detailed protein is rechromatographed on an identical ion exchange resin to separate it from any more basic contaminating protein.Plant biotechnology Altering the genotypes of plants is an important application of recombinant DNA technology. This will have the advantage of avoiding loss of activity as well as no cell disruption is required. Signal peptides can direct the product into the culture medium. which are more acidic using ion exchange chromatography. coli. XII. The produced enzyme was found to retain its specific activity but has decreased affinity for the herbicide. a key enzyme in the biosynthesis of aromatic amino acids in plants. The resultant protein will have a polyarginine tail which makes it more basic. 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. The product is then separated from other proteins. The gene encoding the EPSP synthetase was isolated. 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 enzyme is involved in softening and over ripening of tomatoes. 49 . 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. 3. They were engineered so that it has reduced levels of polygalacturonase enzyme.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.

Suspended biomass (for both environmental and industrial applications) ii. Biopolymers 50 . Classification according to the mode of bioreaction within the bioreactors: i.1. Higher yields 2. Batch ii. Classification according to the configuration: i. Classification according to the flow within the bioreactors: i. Closed (well controlled) for industrial application ii.Scope and state of art: Bioreactors can be classified according to different aspects: 1. containers or systems in which the bioreaction takes place. All bioreaction conditions can be properly controlled through the bioreactor and its design. Well mixed (for homogenous distribution of the nutrients and substrates) ii. Types of bioreactors i. Bioreactors vary from very simple vessel with few controls to highly complicated ones in which the whole process is under computer control ii.Background: Bioreactors are the vessels. Fed batch iii.Recent development and approaches: 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 Different steps or stages Inlet Outlet Membrane bioreactors: Immobilized biological systems on membranes with different pore size or permeability characters to facilitate the product recovery (DSP) The use of living higher organisms as bioreactors: • • Plastics from the plant (biopolymer assignment) Transgenic animals for production of α1-antitrypsin protein (main course) 2. Opened most commonly used for environmental applications 2. Easier down stream processing 3. Continuous iii. Immobilized biomass for both applications with advantages of: 1. Classification according to the biomass retention mode: i. Plug flow (Directional flow for controlled flow of the nutrients and substrates) 4.

DNA. Plants create the plastic through the conversion of sunlight into energy. In oil industry as drilling fluid additive and for enhanced oil recovery (Xanthans) iii. ii. those which need to be polymerized but come from renewable resources. 5. and textile industries (Xanthans and polyhydroxylbutyrate (PHB)) 4. The plant is harvested and the plastic is extracted from it using a solvent. and RNA are all examples of biopolymers. The plant contains the enzymes used by bacteria to create plastics. proteins and peptides. Biocatalysis 51 . respectively. 2. Non toxic Biodegradable (easy to be removed from the environment) Biopolymers can be used as: 1. and nucleic acids. Scope and state of art: Biopolymers have several advantages such as: 1.i. 3. as a result the plant produces plastic through its cellular processes. 3. living organisms. 4. Background: All polymers. amino acids. are sugars. Biopolymers are polymers which are present in. These biopolymers are used for production of biodegradable plastics called green plastics. Starch. Sustainable and renewable Well defined structure and specific upon synthesis = monodispersity Biocompatible and hence is suitable for medical applications. printing. Therefore it has advantage of CO2 fixation and conversion. are made of repetitive units called monomers. There are two main types of biopolymers: those that come from living organisms. These are currently replacing the petrochemical based plastics in all fields Plastics from the plant: Plants are becoming factories for the production of plastics. including biopolymers. Biopolymers inherently have a well defined structure. Used in paints. Researchers created a Arabidopis thaliana plant through genetic engineering. or created by. a versatile family of biobased polymers. Recent development and approaches: Green plastics: through the production of polyhydroxyalkanoates (PHAs). Plasma substitute (Dextrans) 2. in which the monomer units. Complex with iron for iron deficiency anemia (Dextrans) 3. The researchers have transferred the gene that codes for this enzyme into the plant. and.

etc. Ultimate and sustainable resources 2. Bioseparation 52 . Scope and state of art: The use of biocatalysts especially enzymes revealed many advantages such as: 1. Environmentally acceptable 4. Wide diversity 3. Background: Biocatalysis is the use of natural living catalysts. produce the product with maximum purity (up to the enatioselectivity level) Type Amylase Applications and use Production of sweeteners in the food industry 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 dairy industry leather and pharmaceutical industry and waste treatment Digestive enzymes to supplement pancreatic lipases. Proteases Lipases Glucose isomerase L-Asparaginase Fibrinolysin (streptokinase) Glucose oxidase iii. such as protein enzymes or even the whole living cell to perform chemical transformations. production of alcoholic beverages.i. These have been used in many industries such as detergents production. 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 Renantiomer will remain in a pure form) Further more the converted S-enantiomer can be stereoinverted to the pure R-enantiomer monoamine oxidase 4. Highly selective and specific and therefore. In dairy industry since fatty acids imparts taste of cheese Manufacture of many foods and beverages In the treatment or leukemia and lymphoma Treatment of thrombosis Determination of blood glucose level (Biosensor) Removal of O2 from various food products to prevent their deterioration. food industries. ii.

• The additional properties may include differences in solubility and/or hydrophobicity. Biomonitoring 53 .i. added to the desired protein product. 2. Affinity legends such as the use of specific antibody to separate the Adsorption desired protein product as shown in the Desorption Washing following scheme iii. enzyme activity and specific binding. ii. This is of a great importance since the separation (down stream processing) accounts for more than 50 . 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 2. 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). 6. Macro-modification of the desired product: Concept = incorporating the gene coding for the desired product + DNA coding for an additional amino acid sequence 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. 3. Molecular imprinting techniques: Protein + 5. Recent development and approaches: Immobilized Metal Affinity Chromatography (IMAC): based on high affinity between certain metal and polypeptide chain called his tag.70 % of the total cost. Precipitation Centrifugation Microfilteration or ultrafiltration Membrane chromatography Ion exchange chromatography Electrophoresis Bioseparation using biomaterials as separating agents: 1. 4. Scope and state of art: Bioseparation of bioproducts such as enzymes and other active protein is commonly carried out through: 1. 5.

Electrophoresis d. b. 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.C iii. Microscopical examination using different stains and biochemical reactions b. Scope and state of art: Biomonitoring of the flora (biological systems) can be in: a. Recent development and approaches: Biomonitoring of the flora using LC/MS via soft Ionization of proteins: Electron spray. Serological reactions c.i.g. Bioassays Biomonitoring using biological system have many applications such as: a. Medicine to detect the infective agent (the present microbe(s) b. MALDI Biomonitoring using biological system in High through put techniques: • ELISA techniques • Microarrays for detection of biomarkers • Quartz crystal balance 6. Medicinal applications such as the sensitivity testing of antimicrobial agents. Industrial applications as a tool for final product Q. Biosensors (see biosensors) b. Green chemistry 54 . 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. Industrial biotechnology as a quality control on the in use bioelement purity c. Molecular identification e. 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. determination of the pollution degree with certain group of xenobotics (LC 50) c. Environmental applications e.g. antibiotic assays and biosensors for blood sugar measurement. ii.C or in process Q.

not stoichiometric reagents Avoid chemical derivatives 7 8 9 10 11 12 Maximize atom economy Use safer solvents and reaction conditions Increase energy efficiency Design chemicals and products to degrade after use Analyze in real time to prevent pollution Minimize the potential for accidents Many green products are currently or very soon would be available such as: 1. Organic acids 3. Much attention is currently focused on bio-ethanol and bio-diesel. Biopolymers (through the use of polymerizing biological systems) 6. the former from carbohydrate rich feedstock and the latter from oilseed crops. Bioscrubber 55 . Scope and state of art: Application of green chemistry resulted in reduced waste. This is through the application of the 12 principles: 1 2 3 4 5 6 Prevent waste Design safer chemicals and products Design less hazardous chemical syntheses Use renewable feedstocks Use catalysts. • 7. Oxo alcohols and plasticizers 4. the development and application of clean processes based on biocatalysis for production of chemical products from renewable raw materials) ii. Environmentally friendly coat reagent such as epoxides and acrylates from the vegetable oils 5. • 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.i. Background: Green chemistry is also known as sustainable chemistry. o Screening for extremophilic biocatalysts such as thermostable enzymes The use of renewable biomass to produce energy primarily for transportation purposes. refers to environmentally friendly chemicals and processes (i. eliminating costly end-of-thepipe treatments. Formalin technology (production plants and catalysts for formalin production) 2. and reduced use of energy and resources—all improving the competitiveness of chemical manufacturers and their customers. safer products. Biosurfactants such as sugar based surfactants synthesized enzymatically iii.e.

where a continuous flow of liquid containing microalgae is scrubbing the CO2 (the more soluble gas than CH4). they will be biodegraded and neutralized inside the liquid by the used biological system (mainly bacteria) Bioscrubber for removal of H2S in Mining Continuous flow of scrubbing liquid containing Fe+3 and bacteria (Thiobacillus ferrooxidans). 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. The biological system tends to continuously biodegrade the pollutant or scrubs the desired product creating a continuous state of sink conditions ii. Biosensors 56 . The H2S is converted to readily soluble SO4-2 via the oxidation with the Fe+3. 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 CO2 from Methane) The produced biogas is forced through the bioscrubber.i. Therefore the out put will contain only the methane gas with very minor concentrations of O2. Scope and state of art: Bioscrubbers for removal of organic emissions The scrubbing liquid containing the bacteria will solubilise the pollutants. 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 traces of O2 are removed by applying mild pressure 8.

Glucose conc. Medicinal applications e. Lux opeons based biosensors: Lux operon from Photobacterium = receptor (inducer) + structural genes for luciferase enzyme. amperometric or capacitive assay of substrates such as glucose. inversely proportion to the O2 detected Electrolyt e Cathode Anode Semi-permeable membrane + + + + + + + Teflon membrane Product Enzyme Substrate General diagram of biosensors 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.coli in foods and to detect drug-resistant mycobacteria 9. Scope and state of art: Principal: Enzymatic reaction → a small molecule is produced (e.g. ii. The more the interaction between the antitoxin and the toxins the more change in the capacitance. 02. designed for potentiometric. Background: Biosensor = Immobilized Bioelement electrodes. etc The electrode = an electrochemical sensor in close contact with a thin permeable membrane. in which the bioelement is embedded. Glucose oxidase electrodes = glucose oxidase layered over a platinum O2 electrode.g. Extremophiles in biotechnology i.i. H+. Recent development and approaches: Capacitive biosensors for detection of toxins: using the immobilized specific antibody (antitoxin) on the capacitive platinum electrode. urea and amino acids. C02) → detected by the specific sensor The magnitude of the response determines the concentration of the substrate. • Biosensors using recombinant bacteriophages that contain the lux genes for detection of Listeria and E. xenobiotics. Pollutant (substrate) + receptor → activation → production of luciferase enzyme Luciferase + substrate • FMNH2 Enzyme-substrate complex (blue-green light) 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. Background: 57 .

Organisms having growth temperature optimum of 15°C or lower. The screening extremophiles for natural products especially novel antimicrobials. 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. ice cream Applications Detergents and digestive aids Recovery of metals Organic acids and solvents iii. Some extremophiles can tolerate extremely high levels of radiation or toxic compounds. food coloring and feedstock Applications Food additives. These extremcondition could as shown in table 1. Thermophile Halophile Psychrophile Alkaliphile Acidophile Piezophile Organisms having growth temperature optimum of 50°C or higher. dietary supplements Artificial snow. Spirulina and Dunaliella Psychrophiles Polyunsaturated fatty acids Ice nucleating proteins Alkaliphiles and Acidophiles Proteases. pH 2. Oganisms with a pH optimum for growth at.e. Fortunately.Extremophiles are organisms that live in conditions that would kill other creatures (i. b-carotene and cell extracts. lipases and amylases Sulphur oxidizing acidophiles Acidophiles Applications DNA amplification by PCR Detergents Baking and brewing Applications Health foods. The screening of relevant and novel extremophilic organisms in untapped extreme environments. (some can survive at – 10°C).e. extremozymes can be produced through recombinant DNA technology (genetic engineering) without the need to massive culturing of the source extremophiles. and are unable to grow above 20°C. cellulases. dietary supplements. mainly antiviral and anticancer agents 10.g. Examples of common applications of extremophiles Thermophiles and Hyperthermophiles DNA polymerases Lipases. food industry e. thrive in extreme environments where no other organisms are found). Organisms requiring at least 0. ii. Gene Therapy 58 . pullulanases and proteases Amylases Halophiles g-Linoleic acid. or below. Table 2. Recent development and approaches: • • • Improvement of the cultivation techniques for extremophiles as a base for screening these extremophiles. (previously termed barophile) = Organisms that lives optimally at high hydrostatic pressure. Organisms with optimal growth at pH values above 10.2 M (3–30%) salt for growth.g.

This can be done: In-vivo: through. 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. Recent development and approaches: is the introduction of genes (naked DNA). cancer. DNA Vaccines = 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. with the objective of triggering the immune system to produce antibodies for certain infectious diseases. Background: Gene therapy = the replacement of a defective gene in an organism suffering from a genetic disease. 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. The corrected cells were re-injected into her. 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. White blood cells were taken from her. or some autoimmune diseases.i. a genetic disease which leaves her defenseless against infections. Genomics 59 . 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. She had adenosine deaminase (ADA) deficiency. and the normal genes for making adenosine deaminase were inserted into them. His can also done using stem cells Gene therapy is being studied for treatment of many diseases such as: ADA deficiency AIDS Asthma Brain tumor Breast cancer Colon cancer Diabetes Heart diseases Hemophilia Liver cancer Lung cancer Melanoma Muscular dystrophy Neurodegenerative conditions Ovarian cancer Prostate cancer iii. ii.

3. how it is regulated and how it interacts with other genes. 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. directing growth and development. 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 60 . in determining structure. individually and collectively.i. These two main genomics cover all other fields such as: 1. 2. 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. It consists of two branches: • • Structural genomics Functional genomics. to identify useful small molecules and alter their structure to improve their efficacy. 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. Detect microbial contaminants in cell cultures Functional genomics = a field of research that aims to understand what each gene does. Identify the genes involved in complex traits that are controlled by many genes. Background: Genomics is the scientific study of the genome and the role genes play. especially proteins. ii. or no response at all. a bad response to a drug. Fungal genomics Plant genomics Microbial genomics Any thing genomics iii. and controlling biological functions. 4.

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