-- = aped curves, He then boiled the broth in J a ch of the flasks to kill any microorganisms in em, The steam produced by boiling the broth co forced any air out of the flask. When the Basks were left to cool, the air slowly returned to flask. But Pasteur reasoned that any icroorganisms that were present floating in the ir, being heavier, would settle in the moisture hat condensed on the sides of the long S-shaped .ecks of the flasks. In this way, the air entering ithe flasks would be washed free of dust and smicroorganisms. The boiled broth should remain clear and ster broth in Pasteur’s special flasks? When Pasteur broke the flasks open after several months, he found that the broth was clear and smelt sweetly. What does this tell you about the theory of spontaneous generation? If Pasteur cut the neck off one of the flasks or tipped some of the broth into the S- shaped neck and then poured it back into the flask, the broth would become cloudy and smell bad in 24 to 48 hours, How does this observation support Pasteur’s theory? Why did Pasteur have to leave his flasks open to the air and show that microorganisms could grow in the boiled broth in order to disprove the theory of spontaneous generation? a ‘A mleroblologist ploking. up some bacteria growing on ‘an agar plate to examine under a microscope Ber cus Wention of the microscope opened up the world of very minute organisms called microorganisms. Known commonly a8 germs robes, they are found almost everywhere in nature: roth in Snecked flask remains clear for along time. . Pasteur's flasks | Ifthe theory of spontaneous generation was correct, what should happen to thea. BIOLOGY: A Course for ‘0 Level @ in air, water and soil; E> RK jects and living organisms; and hee @ on the surface of objects ant < cy ; , \> within living organisms, where they are able to invade 4 vind cause harm, or live in harmony with them, alr a inne ; ‘Microorganisms from the We shall study three types of microorganism: viruses, bacteria ang certain fungi. / Viruses: Biochemical Parasites Viruses cause diseases in living cells and are the smallest disease. causing particles known. They are not visible under the ordinary light microscope. To examine their detailed structure, a special instrument, This plate of nutrient agar was the electron microscope, is required. This instrument can magnify exposed briefly to the air. After a objects to a maximum of x 250 000. few hours, the microorganisms in the air that landed on the agar ‘Multiplied to form these patches ll Viruses are not considered as cells because they do not have an} protoplasm. A virus is made up of one nucleic acid (either DNA or RNA) surrounded by a protein coat. They may be spherical in shape (eg. influenza virus), rod shaped (e.g. tobacco mosaic virus which attacks tobacco plants) or tadpole shaped (e.g. bacteriophages which attack bacteriall¢ells). The living cell, which the virus attacks, is called the host cell. Structure and mode of life H\ | Outside the host cell, the virus do not carry out any characteristics of living things. They do not feed, respire, excrete, Stow or reproduce. But inside the host cell, the virus can Viral diseases and our defence against them Viral diseases include the common cold, influenza, chi dengue haemorrhagic fever, herpes and AIDS Qa a Immune Deficiency Syndrome; refer to Chapter 21). Figure 17.2 Structure of viruses Figure 17.2 Electron micrographs of some viruses Influenza virusMicroorganisms and Their Applications in Biotechnology ised to destroy bacteria, are not effective against viruses, only be destroyed by antibodies produced by our white living cells, barely visible under the ordinary light ‘They are larger than viruses. nd types . like any other living cell, has a cell surface membrane im. Surrounding the cell surface membrane is the cell wall. | ria are enveloped in a layer of slime (slime capsule). A S Joes not possess a nuclear envelope, so it does not have a js. But it has a single, circular loop of DNA. This is the romosome. It contains the genes that give the bacterium haracteristics and properties. There are also smaller rings jown as plasmids, in the cytoplasm, Plasmids are of great snetic engineers because, like viruses, they can be used as ransfer genes between cells of two different species ) ; ). ria are non-motile. They cannot move by themselves or lever, some are motile, They possess long, hair-like threads to their shapes, bacteria may be classified into three general (singular: coccus)-spherical shaped bacteria ingular: bacillus)—rod shaped bacteria, (vibrio~ shaped bacteria which are a variation of bacilli) | (singular: spirillum)-spiral shaped bacteria | Structure of a bacterium ‘Figure 47.4 Some forms of bacteria >,@ Useful viruses We tend to think of viruses as being simply disease-causing agents. However, they have proved to be very useful to biologists in three ways: © Firstly, viruses reproduce very ly which means they are logists called 10 wish to study ‘the processes by which information is passed from ‘one generation to another. The small size of the virus ‘and its rapid life cycle mean it is possible to study these processes in small dishes in the laboratory. Soe, oder oe saan eee eee scree one one a type of tomato plant which eo =eSP...» Antibiotic resistance Antibiotics are potentially the most powerful drugs we have to fight disease-causing bacteria and fungi. However, many types of bacteria are becoming resistant to antibiotics so that drugs like Penicilin are becoming less Useful. This has been caused by the misuse of antibiotics through ‘over prescription and their use as growth promoters in animals. You might like to find out what uidelines are used in Singapore in the prescription of antibiotics by doctors. Figure 17.5 Yeast cells chitinous cel wall —__ Figure 47.6 Bread mould BIOLOGY: A Course for ‘0’ Level Mode of life Bacteria occur in almost every environment, They in soil, air, water and in the bodies of animals ( intestines). Bacteria may be: are fang in saprophytic, feeding on decaying organic matter, parasitic, causing diseases in plants and animals; ang © autotrophic, able to manufacture food using energy from | of inorganic compounds. e Some bacteria are aerobic, requiring oxygen for respi anaerobic, surviving in the absence of oxygen, and live in the presence or absence of oxygen. ration, some are some others can Bacteria are known for their ability to reproduce rapidly. They reproduce by a process called binary fission, in which the bacterial cell copies the DNA it contains and then splits in half form two new cells. In adverse conditions, some bacteria form spores, each protected by a thick wall. Such spores are very resistant to cold or heat, and very drastic conditions are n {o kill them, Ifthe spores settle on a suitable medium, their walls break and they begin to grow and multip ly. Fungi may be unicellular, (Rhizopus), mushro antibiotic. 8; yeasts, or multicellular, eg, bread mould oms and Penicillium which Produces the penicillin Structure ‘Spore prod structires.Charter Microorganisms icroorganisms and Thelr Applications in Biotechnology mui may reproduce by budding, F ; uungi of spores. The spores are minute and ynmental conditions, eg. drought. They are hen they settle on a suitable medium, they mycelia, Shae spread over the surface of the substratum grow). The bread mould growing on the imon sight. The hyphae secrete enzymes to Substances outside their bodies. This is body) digestion. The enzyme amylase , Maltase digests maltose to glucose. Proteases ® acids. Glucose and amino acids are absorbed Phae. Such activities of fungi enable them to yytes. They include fungi and certain bacteria, the soil. Although moulds and mushrooms osers are microorganism: that decomposers play an essential role in nature. Decomposers form the link between ving environment (carbon cycle, Figure 17.7 Some examples of fungi d and decaying organisms and their faeces, their food source such as a decaying leaf complex organic compounds (food), like s, into simple soluble or gaseous inorganic josers absorb only a small amount of jeir own use. Most of the energy is lost g nutrients are released into the ater. leased during decay include gases like iphide and water vapour, and salts like and potassium ions. These are reused thesis and other synthetic processes.Figure 17.8 (2) Bedok Sewage Treatment Works (b) Aeration tank: Microorganisms decompose biodegradable organic ‘matter in the waste water, {c) Final sedimentation tank: Sludge Seitles while treated water (final effluent) is discharged into the sea. The final effluent meets the Royal Commission standards for biochemical oxygen demand (see page 298), (0) Digester: Microorganisms act on the sludge under anaerobic conditions to break down the organic matter in It. The digested product is dried and used mainly as landfill material. & by. Product of this process is biogas which contain about 60-70% methane, we \y BIOLOGY: A Course for ‘0’ Level By enabling the recycling of nutrients, decomposers play an important role in maintaining life on earth. They aleg an unsightly accumulation of the r wastes of living organisms on earth's Decomposers in Sewage 1; Sewage is normally decomposed by saprophytic microorganisms, However, natural cycle takes a long time. In towng cities, sewage from households is drain large settling tanks. Bacteria present in ¢ sewage secrete enzymes to digest the soli organic matter into soluble, harmless substances and the gas, carbon dioxide, Bacteria also feed on such digested produ The liquid that results from this bacterial digestion is filtered and pathogens removed before it is discharged into the rivers or seas. The digested sol called sludge, is removed from the tank, dried and used as fertilizers. ee) Sue Biotechnology is not easy to define. We can s: scale industrial use of biolo, make substances or to prov “biotechnology” was coined o ut to produce genetically en, result, biotechnology is usual] production of the specific genetically engineered o; using the abi ay that it is the large- gical processes from microorganisms to services for Man. The term nly in the 1970s, inspired by work carried igineered organisms (Chapter 22). As a ly linked to genetic engineering, and the Products (e.g. enzymes and hormones) of *ganisms, Historically, however, we have been y of microorganisms to car Fermentation Products Yoghurt making Yoghurt is made by fermenting milk, usin certain bacteria like Lactobacillus bulgaricus and Streptococcus deomrphie Milk is incubated with these bacteria. The ‘Streptacaccus removes oxygen thereby creating n the absence of oxygen, the Lactobacillus Converts lactose (milk sugar) to lactic acid, Protein, casein, to curdle producing yoghurt, ired by adding sugar and fruit. which can then be flavou: Mi & 4 & 4 gz(b) Cheese produced by ugh to rise microbial fermentation fermentation ‘of cheese makes use of both bacteria and fungi to ferment milk sugar to lactic acid. The latter The curdled protein, together with the fats in the d acted upon by a mixture of bacteria and fungi to varying conditions, such as temperatures, and fures of bacteria and fungi, different types of cheese Making bread contains starch, protein and an ¢ flour is mixed with water to form a dough. Yeast the dough. Amylase digests the starch to sugar, | de the dough causes the yeast to respire menting the sugar to alcohol and carbon dioxide. de produced causes the dough to rise, thus pear in the bread. The alcohol produced evaporates oho! ing. Here, yeast is mixed with sugar found in food ‘brewing such as barley, grapes, et Jn the absence| t respires anaerobically to produce alcohol and the sugar. Examples of alcohols brewed in this .| ; wine, sake and whiskey. Beer is made from and wine from grape juice. The Japanese sake i entation of rice. In spirit-based drinks, like wl grated from the fermented liquors or mashes (e: lation, Making beeril BIOLOGY: A Course for ‘0’ Level ‘So was Fleming just lucky or was he creative? Sometimes discoveries involve luck and creativity. The creative side of science involves making, the mental connections to take advantage of chance observations. A classic example of this involves Alexander Fleming In 1928, Fleming left a culture dish of a disease-causing bacterium, Staphylococcus, ‘uncovered in his laboratory by ‘chance. Other microorganisms rapidly colonized the dish, ‘contaminating the culture of Staphylococcus he was growing, Usually such contaminated plates are thrown away, but just before he did this Fleming noticed several clear areas on the plate Where the bacteria were not growing. This observation Suggested to Fleming that some ‘contaminant on the dish had stopped the bacteria from growing. This is how Fleming “accidentally” discovered penicillin. Was Fleming just lucky? Possibly ‘but he would not have discovered penicillin if he had not been very ‘observant and been able to ‘ecognize the importance of what he saw. Most people would not even have recognized the clear areas on the plate, let alone ‘thought them worth investigating. ‘Such observations are crucial to science end Fleming's discovery is a good example of a famous Antibiotics i a roduced by microorgani ibiotics are complex substances pr mere 3 eaten they excrete. They are used mainly in the of bacterial diseases. sate, in antibiotic producers. The best i! bacteria and fungi are the main ant The eee tere acai ‘produced by the fangus Pilon Oy, examples include streptomycin and tetracyclines produced by the Streptomyces bacteri Commercial production ‘The large scale production of antibiotics involves the use of giant fermenters (refer to page 264). conheiat r= 50" erage, Penicillin production i For penicillin production, the fermenter size\is usually 100 000 to | 150 000 litres, The fermenter is filled with a nutrient broth (culture | medium) for the growth of the fangus Penicillium. The main fermentable carbohydrate source in the culture medium is lactose. Other nutrients include an organic nitrogen source, such as corn-steep liquor, and mineral salts. The pH is adjusted to between 5 and 6 and temperature during the penicillin production stage is maintained at 26 °C. As aeration is important for optimal growth of Penicillium, air is blown into the fermenter and the medium is stirred constantly. Then the relevant Penicillium strain is added to the culture medium. The medium conditions are maintained for the rapid growth of the fungus, There is little penicilin production at this stage. As the growth slows down, penicillin production begins. Conditions in the fermenter Sterile conditions are essential throu, essentic ighout this process. If microorganisms contaminate the ee medium, the be disrupted, incurring heavy financial losses, '® WROlE Process will In the production of penicillin, growth oft conan volume of culture medium, This mediacs iH ee a by the fungus. When the desired amount of prada eS medium, the process is stopped. The fermenter is cles iol Prooss is repeated This is hnown a the batch operadores ooo, In the batch operation system, unproductive time is i t 5 is spent in clean and sterilizing the fermenters after each batch. In this kind! of ace operation, a cooling jacket (Figure 17.12) for the fermenter leaves its inside relatively uncluttered, making it easier to clean the ferment ‘The advantage of this system is that each batch of product can be identified and checked-an important aspect in antibiotic production,| Microorganisms and Their Applications in Biotechnology at : microorganisms, such as bacteria and yeast, are Stay proteins (SCP). Yeast has been used in making n making single- | jag cow Disease Through the late part of the 4980s and the early part of the 1990s, cows in dairy herds in Hs, need alot of protein in their diet ifthey are to | Gfastares end new iiness, jounts of milk. To solve the problem of low levels | whieh became known as mad cow pal feed, the chemical company ICI decided to use | disease. However, when proteins for animal feed. These are allowed to biologists examined the brains of in methanol, a petroleum by-product. They are | tes® cows ey found that the rain ti large noles in it, d and squashed. This protein-rich food was prise be ICI. It was used'to feed animals like cattle, Spongiform Encephalopathys. of producing this product was much higher than | found in sheep, where it is called ging protein for animal feed, such as from the scrapie, and humans, where it is danimals. Consequently, Pruten production called CrevtzfeldtJakob disease. xxample of a clever piece of techn sch_| The new form was called Bovine ele olaceres peae ology which ‘Spongiform Encephalopathy or BSE for short. But where had this new frm come trom? The answer was terrifying. Dairy éliet form, to help them produce Tot of milk. These pellets are made from the bodies of dead dd is rich in protein, low in fat and has a sufficient | animals. Were the bodies of pared to meat, it is a much healthier protein sheep infected with scrapie and needs, cows infected with BSE used for making these pellets? Did this mean that the cows were catching BSE from their food? It seemed so. The use of this form of protein was banned and millions of cows have been slaughtered to eliminate BSE. groduction of mycoprotein, the fungus is grown in enters similar to that shown in Figure 1712. Unfortunately, it now appears possible that humans who ate infected cows, for example in the form of hamburgers, may also have caught the illness. More than twenty people have now died from a new variant of Creutzfeldt- | Jakob disease which, when the brains of these dead people were _ examined, looked like BSE. The Figure 17-10 Mycoprotein foods ‘agent which causes this disease is unknown but it seems to be a sing! molecule, called a Cos it passes to a separation stage where the fungus is liquid is returned to the fermenter. The culture medium is maintained by topping up of the ‘The filtered fungus is dried and processed. continuous operation system. This is suited d process involved in SCP production. |Biological processes: from ‘small scale to large scale Industrial biotechnology involves large-scale production of the ‘microorganism and its product. ‘The small-scale production of the ‘microorganism and its product in the laboratory are easier to Control, e.g. mixing and aeration are done easily in a flask, ‘Therefore, when transferring 3 biological process from the laboratory flask to the ‘commercial fermenter, many factors have to be considered This is the work of the ‘biochemical engineer. have resulted in a Increase in yield before the era neering, BIOLOGY: A Course for ‘0’ Level Industrial Biotechnology Fermenters A fermenter is ® agiant steel cylindrical tank closed at both ends, and designed to keep its inside environment favourable for the biological process to operate (Figure 1712). Most industrial processes use fermenters for the large Scale, rapiq production of microorganisms and their products. Present, technology allows us to manipulate microorganisms genetically tp produce a desired product, eg. production of human insulin (Chapter 22). Using fermenters to produce the gene products of such microorganisms is profitable, especially in the pharmaceutical industries, Fermenters may be designed for anaerobic ‘most industrial processes are aerobic, or aerobie processes. Since features of this type of fermenter. we shall outline the important Cooling system Removal of heat during microbial activities is vital for successil : air is passed into the ferm Eg Ac then meer nd: diffuses into the nutrient broth. 7 ane Figure 17.44 A fermenter i iN an industrial plant| oF a h exhaust es hI te |/L | cls | water out ry +— sterile air | is a stirring device. It mixes the air bubbles well broth. This ensures that oxygen and nutrients are of the microbes. mixed with a nutrient broth (culture medium) the oxygen concentration, pH, temperature mutrients are carefully monitored by a computer. used in industrial processes must produce ey should also possess the following features: enter ively cheap culture medium manipulated by genetic engineers loped luce toxic productsSOUOUOY SP Yononpoid Joyoo|e + ingesn Auew eW asao4o Buryew asaat . uo» ‘sonpoid ued SwwSIUERIO0I01UN Bupew yinysoK « Payipow. eves . yononpoid uisjoud j[99-9)8UIS « 198 uononpoid onoignue « = | eee] Vousoduooop I [mee] (eters | (mae i Auge mosd s9us1H ‘SWISIUESI0 J4]0 Jaijoue 0} saiseds auo Woy SOUAR Jo 19)SUEN aly UI J0}2an © se Buyeaulue ojaues uy = 40 ankydoudes aq Kew uniuo Jo apew sjreM jeonInW 40 JeInIjao1uN ‘onfydoides aq Kew « suWUIMs 40} e1jaBey ney AEW « (euesquiow Jeajonu ou Jo) snajanu any ou « ‘@uiosowosyo 42]NDs19 U0 Uy WSIURBIO pa|jao-2U0 + sasnuin UeU} 49812) episul luo eonpoides + onisered skemje + doo: luon99)9 28pun Aju 9} quasge wsejdoy adojenua ulajoid e Aq pepunouins ioe o!ajonu eu0 jo apew nowy peonposd. €q ued swsiuesi0010) ‘ui JO pouad = son@uad jo UOYS e ULM aje9s 2812} z ‘pms 04) ul Joo) yoieasale se « ® uo psonpoid aq ueo : ee — ‘swis|ue3s00101 njesn sseulnjesn, : Buisn Jo seZequenpy < é “syonpoud 4194) 2 pie ee $ ee = = | sesnuin UO é sonoignue Aq palin aque * yaya OU BAeY SonoIqnUe » : ‘aiydonoyne 40 onisesed 1129 1804