CChapter 30hapter 30

Biotechnology

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,

beers was 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.

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 technology was employed.

30.1 Growth of Microorganisms30.1.1 Factors affecting growth – applicable to

both plant and animal cell culturesNutrients 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) and micro-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.

30.1 Growth of Microorganisms

Microorganisms growing on a nutrient medium

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℃

pHMicroorganisms can tolerate a wider range of pH than

plants and animal cells, most prefer acidic conditions.

OxygenMost are obligate aerobes, some are facultative aerobes

and obligate anaerobes.Microaerophiles can tolerate oxygen, nevertheless grow

better when its concentration is low.Osmotic factorsAll 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.

PressurePressure is not a major factor affecting growth of

microorganisms. A few species inhabiting the ocean depths, the

barophiles, cannot grow in surface waters where the pressure is too low for their survival.

 LightPhotosynthetic microorganisms require an

adequate supply of light to sustain growth.WaterWater 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)

30.1.2 Growth PatternsGrowth of a population (bacterial growth curve) with

the lag phase, exponential phase, stationary phase and death phase:

30.1.3 Biotechnology and Food ProductionThe 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.

30.1.4 Aseptic conditionsPure 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.

30.2 Industrial fermenters and fermentationFermenters enable specific microorganism to grow inside – a process called fermentation (it might not be anaerobic!)

Fermenter for cloned protein

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

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

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

30.3 Biotechnology and Food Production1857: Louis Pasteur showed alcoholic fermentation

by microorganismsBaking: Use of yeasts in food production (bread)Beer and wine production: also fermentation by

yeastsDairy products: cheese, butter and yoghurt are

produced using various bacteria

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.

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).

30.4 Biotechnology and PharmaceuticalsAntibiotics 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 antibioticChloremphenicol – a broad spectrum antibiotic

Penicilin fermentation

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.

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

30.5 Biotechnology and Fuel ProductionGasohol production:Use yeasts to ferment sugar cane into alcohol and

produce a relatively cheap, renewable fuel.Processes include:1 Growing & cropping sugar cane2 Extract the sugars from plants3 Crystallizing out of the sucrose for sale and leaving a

syrup of glucose & fructose as molasses4 Fermentation of the molasses by yeast to dilute alcohol5 Distillate to pure alcohol, using the waste (bagasse) as

a power source

Biogas productionA 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

30.6 Biotechnology and Waste Disposal Sewage disposalAnaerobic 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

Biodegradable plasticsPolythene 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

Disposal of oilC. 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.

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.

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.

30.7 Other products of the biotechnology industryExamples: 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

30.8 Cell, tissue and organ culturePlant and animal cells can also be grown in vitro to

make a variety of products.

Anther callus on agar

In vitro: by artificial means outside the body

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.

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.

Plant organ culture from 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

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.

2 Manufacture of useful chemicals by plant culture – atropine (dilation of pupil),

codeine (pain killer),digoxin (treatment of cardiovascular problems),jasmine (perfume),menthol (flavouring).

Animal cell cultureOnly 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 cultures but 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.

Application:1 Production of viral vaccines, e.g. polio,

measles, German measles, rabies2 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