Microorganisms and Their Applications in Biotechnology

MICROORGANISMS AND THEIR APPLICATIONS IN BIOTECHNOLOGY

Biology Concepts

Microorganisms and biotechnology Content Microorganisms Food biotechnology Industrial biotechnology biotechnology • • •

Learning outcomes Candidates Candidates should be able to: (a) List the main characteristics characteristics of the following groups: viruses, bacteria and fungi; (b) Outline the role of microorganisms in decomposition; (c) Explain the role of yeast in the production of bread and alcohol; (d) Outline the role of bacteria in yoghurt and cheese production; (e) Describe the use of fermenters for large-scale production of antibiotics and single cell protein; (f) Describe the role of the fungus Penicillium in the production of penicillin.

Prepared by: Ahmed Kaleem Khan Niazi

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Biology Concepts

EXPERIMENTS TO SHOW THE GROWTH OF BACTERIA - basic techniques (similar techniques are used to grow fungi such as moulds and yeasts) Microbiological media Bacteria will grow on practically any source of organic food which provides carbon compounds to be respired for energy for energy,, and nitrogen compounds to be incorporated into proteins for growth for growth.. These substances are normally provided dissolved in water. However, in nature, bacteria can break down solid and insoluble substances by releasing enzymes into the substrate in which they are growing. These substances are thus broken down or digested to simpler substances and the process is called extracellular digestion because it takes place outside the bacterial cells. The two normal media used in bacteriology are a clear soup-like liquid nutrient broth, broth, usually in tubes, and nutrient agar , which is set into a jelly by the addition of a seaweed extract called agar, and when melted poured into glass or plastic Petri dishes - also known as "plates". "plates".


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A standard carbon source is glucose, glucose, and nitrogen is often provided by peptones (partially digested proteins), proteins), or inorganic or inorganic salts. salts. Minerals and vitamins may also be provided, according to the growth requirements of the bacteria. Combinations of chemicals (buffers) may be used to keep the pH stable. Measured amounts of the concentrates are added t o water, and dissolved to reconstitute the media. Sometimes, substances are mixed into media, in order to suppress growth of other types of bacteria. There are many such selective media.

Microbiological techniques Sterilisation, aseptic techniques, inoculation, incubation

These media must then be sterilised by heating in an autoclave (like a pressure cooker) at 121°C (pressure 1 bar or 15 lb/sq. in.) for 15 minutes, which kills all living organisms, including spores. All apparatus used from this point onwards must be sterilised by heat (glassware - 160 °C for 2 hrs) or exposure to radiation. Aseptic techniques must be used to reduce the likelihood of bacterial contamination. This usually involves disinfection of working areas, minimising possible access by bacteria from the air to exposed media, and use of flames of flames to kill bacteria which might enter vessels as they are opened.


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Bacteria may be introduced to the media (inoculated ( inoculated)) by various means. Usually the bacteria e.g. from a drop in a heat-sterilised loop are spread on the surface of (ready set) agar. A similar technique is used with broth cultures.


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Sometimes bacteria in a liquid are introduced using a sterile pipette to the Petri dish before the (fairly cool) agar medium is poured on top ("pour plates"). Then the Petri dishes containing containing agar or tubes containing broth are incubated, incubated, i.e. put in a special apparatus at a fixed temperature (usually 37°C - human body temperature, for possible pathogens - or 25°C for bacteria from the t he environment). In schools, lower incubation temperatures are used in order to discourage the growth of potential pathogens. When growing bacteria, bacteria, it is usual to invert the Petri dishes, so as t o prevent condensation droplets droplets from falling onto the surface of the agar. Petri dishes are often "sealed" at this stage to prevent people who handle them from contamination by bacteria, which will multiply multiply greatly. It is normal to use 2 strips of adhesive tape from base to lid rather than attempt seal the circular edge of t he Petri dish. This is to guard against the possibility of anaerobic organisms organisms growing due to lack of air. However, it must be borne in mind that any drips from a partially sealed Petri dish are potential sources of infection.

Results Cultures are usually examined after 24 hrs incubation. incubation. Liquid media such as broth become cloudy if bacteria are present. This could be the result of only one bacterial cell originally entering the medium, then dividing repeatedly to produce millions!

Bacteria on agar "plates" become visible as distinct circular colonies circular colonies;; each colony should represent an individual individual bacterial cell (or group) which has divided repeatedly but, being kept in one place, the resulting cells have accumulated to form a visible patch.


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By an extension of this method using serial dilutions in sterilised liquids, the number of bacteria in a given amount of sample, e.g. food, can be calculated. calculated. After use, bacterial cultures, etc. must be sterilised by the use of heat, before disposal


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Production of yoghurt This process relies on the growth of bacteria - usually mixed cultures including Lactobacillus species - on milk, and the conversion is due to anaerobic respiration by the bacteria. The milk is usually skimmed milk "low fat"- often concentrated with extra "milk solids"


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1. Give 2 reason reasons s for the first heat treatmen treatment: t: …………………………………………………………………………………………………………………… …………………………………………………………………………………………………………………… …………………………………………………………………………………………………………………… 2. Why is is the incuba incubation tion phase phase so short short compar compared ed to brewin brewing? g? …………………………………………………………………………………………………………………… …………………………………………………………………………………………………………………… …………………………………………………………………………………………………………………… 3. Give 2 advanta advantages ges for for the the second second heat treatmen treatment: t: …………………………………………………………………………………………………………………… …………………………………………………………………………………………………………………… As the bacteria respire anaerobically, they produce lactic acid: lactose > lactic acid + energy (milk sugar) In fact, it is said that Lactobacillus Lactobacillus bulgaricus breaks down milk protein to chains of amino acids, which are then used by Streptococcus thermophil t hermophilus us to make methanoic acid (formic acid) which Lactobacillus bulgaricus uses to convert lactose into lactic acid. The acidic conditions (pH 3.7-4.3) cause milk proteins to be coagulated, which provides the t hickened texture of yoghurt. Other chemical products from the bacterial growth, e.g. ethanal (acetaldehyde) (acetaldehyde) contribute to the f lavour. Keeping qualities As the yoghurt is cooled, the bacterial growth rate is reduced and the product can keep for about 10 days at 5 °C. The normal numbers of bacteria are about 10 8 (100,000,000) per gram. After some time, acid slowly released by the bacteria gradually kills them and causes the proteins to separate into curds and whey. Yeasts and moulds can cause problems if they contaminate yoghurt - causing pots to become "blown", "blown", due to the production of carbon dioxide


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MANIPULATING GENES Gene transfer or genetic engineering involves the transfer of ……………….from one species of organism to another species, i.e.from a donor into a recipient organism. It is seen by some as a simple extension of other biotechnological biotechnological processes, whereas to others it is considered as a development development with much more sinister implications. implications. In fact there has been pressure to use the term biotechnology, which has gained some public acceptance, to cover both A gene is a unit of hereditary information (i.e. it normally passes on characteristics characteristics from one generation to another), and is composed of ……………. Gene manipulation may be advantageous because it makes the resulting ……………………… or …….. …… organism easier to grow or manage, or to transfer a characteristic to a different crop, etc. It differs from f rom selective breeding which only involves members of the same species, in that usually only single genes are moved, often in addition to that organism's normal complement of genes ("…………..…."). Because selective breeding involves the normal methods of sexual reproduction reproduction (gamete transfer, fertilisation and development, development, etc.), it only results in large combinations of genes being transferred (the ……………. number of chromosomes contained contained in a gamete is in effect half a genome), and the effect of these genes may be masked or diluted due to ……………………. by other genes.


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Production of antibiotics 1. Antibiotics are waste products excreted by microbes mainly soil bacteria and fungi. 2. Antibiotics are very important in the control of bacterial diseases because they kill bacterial cells but seldom affect human or animals cells. 3. They are complex in nature and are uneconomical to synthesis on a large scale in the laboratory. They are more economically and easily produced by a relevant biotechnological biotechnological process. 4. Commercial production of penicillin: penicillin: •

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Penicillin is a metabolic waste product of the blue-green filamentous fungus, Penicillium . The antibiotic only begins to accumulate in the surrounding medium medium after the mould’s growth slows down. Fermenter size used is between 100, 000 to 1500, 000 liters.

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The fermenter is first sterilized and filled with a fixed volume of culture medium. Conditions Conditions are adjusted (e.g. temperature at 26 0C; aeration and pH 5-6) to support the optimum growth of Penicillium of Penicillium . Nutrients supplied include lactose (the main carbohydrate sucrose), and organic nitrogen source (e.g. corn-steep liquor-an organic waste) and mineral salts. Oxygen is supplied as Penicillium grows under aerobic conditions.

Once conditions for optimal growth are achieved, a selected Penicillium Penicillium strain is introduced into the culture medium in the fermenter. No or very little penicillium is produced when the fungus is growing rapidly. As growth slows down, the fungus starts to produce and excrete penicillin. Conditions in the f ermenter are adjusted to suit penicillium production. When the desired amount of penicillin is reached, the culture medium is filtered to remove the fungus. A solvent is used to extract penicillin from the mixture and pure penicillin penicillin is obtained by crystallization. crystallization.

system. (batch process). 5. Penicillin is produced by the batch operating system. •

A fixed volume of culture medium is used and the microbe is freely dispersed throughout the culture. The conditions in the culture are altered by the microbe activity. i.e. nutrient content decreases and product content increases. (This is closed system.)

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Microorganisms and Their Applications in Biotechnology

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