C hapter 30

1. Chapter 30 Biotechnology

2. Biotechnology is the application of scientific and engineering principles to the production of materials by biological agents. Its origins go back a long way, most were fermentation processes: e.g. • food contamination led to improved flavour and preservation, • beerswas developed from ‘spoilt’ grain while wine from ‘spoilt’ fruit, • contaminated alcohol caused the manufacture of vinegar as food & food preservative, • contaminated milk led to the production of cheese, butter & yoghurt.

3. Modern biotechnology industry began in the First World War: • the Germans used yeast to ferment plant materials in order to produce glycerol for explosives; • the British used bacteria to make acetone & butanol as part of their war effort. • In the Second World War, mass production of penicillin was needed for medical used. • A major expansion of biotechnology advancement occurred when recombinant DNA technologywas employed.

4. 30.1 Growth of Microorganisms 30.1.1 Factors affecting growth – applicable to both plant and animal cell cultures • Nutrients which include 4 elements: carbon, oxygen, hydrogen, nitrogen and significant quantities of phosphorus and sulphur. • Smaller quantities of nutrients include macro-nutrients (Ca, K, Mg & Fe) andmicro-nutrients(Mn, Co, Zn, Cu). • Growth factors like vitamins, amino acids, purines and pyrimidines are also important. • Usually greater concentrations of nutrients will lead to higher rates of growth.

5. 30.1 Growth of Microorganisms Temperature All growth is governed by enzymes which operate within a narrow range of temperatures. Groups are classified according to their preferred temperature ranges: 1 Psychrophiles - optimum growth temperature below 20℃, continuing to grow at temperatures down to 0℃. 2 Mesophiles - optimum growth temperatures in the range 20 – 40℃ 3 Thermophiles - optimum temperature in excess of 45℃, very few can survive as high as 90℃ Microorganisms growing on a nutrient medium

6. pH Microorganisms can tolerate a wider range of pH than plants and animal cells, most prefer acidic conditions. Oxygen Most are obligate aerobes, some are facultative aerobes and obligate anaerobes. Microaerophiles can tolerate oxygen, nevertheless grow better when its concentration is low. Osmotic factors All microorganisms need water for growth. Most cannot grow in environments with high solute concentrations. A few, the halophiles, can survive in conditions of high salt concentration.

7. Pressure Pressure is not a major factor affecting growth of microorganisms. A few species inhabiting the ocean depths, thebarophiles, cannot grow in surface waters where the pressure is too low for their survival. Light Photosynthetic microorganisms require an adequate supply of light to sustain growth. Water Water is needed for a variety of cellular functions. Photosynthetic autotrophs use hydrogen (from water) to reduce carbon dioxide and make food. (H2O) Chemoautotrophs use hydrogen sulphide for making food. (H2S)

8. 30.1.2 Growth Patterns Growth of a population (bacterial growth curve) with the lag phase, exponential phase, stationary phase and death phase:

9. 30.1.3 Biotechnology and Food Production The correct balance of nutrients, an appropriate pH and a suitable medium are essential to microbial growth. Agar is seaweed extract which is metabolically inert and dissolves in hot water but solidifies upon cooling. Minimal medium: a medium composed to satisfy the demands of a single species. Narrow spectrum medium: provides the nutrients for a small group of microorganisms with similar requirements, e.g. acidophiles. Broad spectrum medium: medium for general purposes, designed to grow as wide a range of microorganisms as possible.

10. 30.1.4 Aseptic conditions Pure cultures of single type of microorganism need to be grown free from contamination with others. Sterilizing techniques include heating, very fine filters, UV light illumination or using disinfectants.

11. 30.2 Industrial fermenters and fermentation Fermenters enable specific microorganism to grow inside – a process called fermentation (it might not be anaerobic!) Fermenter for cloned protein

12. Batch cultivation: extract products from time to time with cleaning & sterilization to begin the whole process again Continuous cultivation: used medium and products are continuously removed, raw materials are added throughout the process A stirred tank fermenter

13. Immobilization of cells and enzymes One problem with the fermentation processes so far is that at some point the cell culture is removed and discarded. Any mechanism for immobilizing the microorganism and/or the enzymes they produce, improves the economics of the process. 1 Entrapment – cells or enzyme molecules are trapped in a suitable meshwork of inert material, e.g. agar, cellulose, etc 2 Binding – cells or enzyme become physically attached to the surface of a suitable material, e.g. sand or gravel

14. 3 Cross-linking – cells or enzymes are chemically bonded to a suitable chemical matrix However immobilized, the cells or enzymes are made into small beads which are then either packed into column, or kept in the nutrient medium. The nutrient can be continually added and the product removed without frequent removal of the microorganisms/enzymes. The process cannot be continued indefinitely because impurities may accumulate. Immobilized microbial cell pellets in a packed reactor column used to carry out biotransformations

15. 30.3 Biotechnology and Food Production 1857: Louis Pasteur showed alcoholic fermentation by microorganisms Baking: Use of yeasts in food production (bread) Beer and wine production: also fermentation by yeasts Dairy products: cheese, butter and yoghurt are produced using various bacteria

16. Single Cell Protein (SCP) Single cell protein comprises the cells, or their products, of microorganisms which are grown for animal and human consumption. The product also contains fats, carbohydrates, vitamins and minerals. Raw materials: petroleum chemicals, alcohols, sugars, agricultural & industrial wastes. Microorganisms: bacteria, filamentous fungi, algae, yeast.

17. Others: Vinegar, Sauerkraut (salted cabbage), Olive and cucumber preservation, Coffee and cocoa beans, Soy sauce, Enzymes: lipase (flavour development in cheese), protease (meat tenderizers), -amylase (improve flour, breakdown starch in beer production).

18. 30.4 Biotechnology and Pharmaceuticals Antibiotics are chemical substances produced by microorganisms which are effective in dilute solution in preventing the spread of other microorganisms. Most inhibit growth rather than kill microorganisms. Penicillin – a narrow spectrum antibiotic Chloremphenicol – a broad spectrum antibiotic Penicilin fermentation

19. Comparison of primary & secondary metabolite production Antibiotics are made when growth of the producer organism is slowing down rather than it is at its maximum. They are secondary metabolites and their production takes longer than primary metabolites. As a result, only batch fermentation can be employed.

20. Hormones are produced utilizing recombinant DNA technology, e.g. insulin, growth hormone, testosterone, oestrogen, etc.

21. 30.5 Biotechnology and Fuel Production Gasohol production: Use yeasts to ferment sugar cane into alcohol and produce a relatively cheap, renewable fuel. Processes include: 1 Growing & cropping sugar cane 2 Extract the sugars from plants 3 Crystallizing out of the sucrose for sale and leaving a syrup of glucose & fructose as molasses 4 Fermentation of the molasses by yeast to dilute alcohol 5 Distillate to pure alcohol, using the waste (bagasse) as a power source

22. Biogas production A simple process of utilizing a digester (container) to hold the wastes with anaerobic bacteria to produce methane, a gas which is ready for cooking, lighting and heating. Biogas generator

23. 30.6 Biotechnology and Waste Disposal Sewage disposal Anaerobic digestion: microorganisms break down organic sewage into acetic acid, carbon dioxide and hydrogen. Aerobic digestion: aerobic microbes oxidize organic compounds in sludge by pumping air through Sewage farm showing filter beds

24. Biodegradable plastics Polythene and polyester polyurethanes of low molecule mass are developed and can be degraded by microorganisms, e.g. C.resinae (a fungus). In general, more flexible plastics are broken down more easily than rigid ones. Biodegradable plastic bottles

25. Disposal of oil C.resinae can also degrade oil. Microorganisms, developed from recombinant DNA technology, have been useful in cleaning oil spills. Emulsifier, produced commercially from bacteria, is used to cause oil to mix with water and so both disperse it and speed up microbial breakdown.

26. Disposal of industrial wastes An increased awareness of ecological issues and tighter legislation have prompted safer waste disposal. Examples: Biogas production utilizing many forms of wastes (paper, cotton); Brewery waste could be converted to citric acid by a fungus; Potato processing plant wastes could be converted to animal feeds. Genetically engineered microorganisms are making the task increasing easy.

27. Recovery of valuable material from low-level sources including wastes Bacteria can be used to extract copper and uranium from waste ores, and remove sulphur from high sulphur coal. A alga is used to absorb metal ions against a concentration gradient. Enhanced oil recovery, utilizes microbes to extract oil out of the rocks.

28. 30.7 Other products of the biotechnology industry Examples: Interferon for treatment of viral diseases, Vitamin B12 as food supplement, Protease as detergent additive, Indigo as a textile dye, Artificial snow at winter holiday resorts. Interferon for treatment of viral diseases

29. The plant meristems retain the growing ability of plant cells. If a tissue containing meristematic cells, e.g. a bud, root tip, etc., is removed from the plant and grown aseptically on a nutrient medium, an undifferentiated mass (callus) develops in the presence of hormones and growth regulators. 30.8 Cell, tissue and organ culture Plant and animal cells can also be grown in vitro to make a variety of products. In vitro: by artificial means outside the body Anther callus on agar

30. Plant tissue culture is the production of undifferentiated callus. If the callus is suspended in a liquid medium and broken into individual cells it forms a plant cell culture. These can be maintained indefinitely if sub-cultured giving rise to a cell-line. With the help of pectinases & cellulases to dissolve the cell walls, the protoplasts of different cells can fuse to form hybrids to grow into new plant varieties.

31. Plant organ culturefrom apical shoot tips growing with cytokinin can develop a cluster of shoots, each of which may be grown into new clusters of genetically identical individuals – a form of micropropagation. Micropropagation of plants growing in medium

32. Applications: 1 Generation of plants for agricultural or horticultural use – Vast quantities of plants can be grown in sterile controlled conditions ensuring a much higher survival rate. A uniform crop with desired characters can be maintained. Plants are pathogen free and can develop defence mechanisms against many diseases.

33. 2 Manufacture of useful chemicals by plant culture – atropine (dilation of pupil), codeine (pain killer), digoxin (treatment of cardiovascular problems), jasmine (perfume), menthol (flavouring).

34. Animal cell culture Only in recent years has it proved possible to culture vertebrate cells on any scale. With the help of proteolytic enzymes to separate the cells, a monolayer of cells attach themselves to the bottom of the container are capable of cell division – primary culture. Cells from these can be used to establish secondary culturesbut their life span is limited, division often ceasing after 50-100 divisions. It is possible to make these cultures continue to divide indefinitely by adding chemicals or viruses to transform them into cancer cells – neoplastic, which can induce cancers if transplanted into a related species.

35. Application: 1 Production of viral vaccines, e.g. polio, measles, German measles, rabies 2 Pharmaceutical products from cell lines, e.g. interferon, human growth factor and clotting factor 3 Monoclonal antibodies production through recombinant DNA technology to clone viral vectors