Biotechnology

1. Biotechnology

2. B. BIOLOGICAL FUELS 1 The need for biological fuels 2 Raw materials These include wastes and crops; wastes Dry Wastes Wet wastes Crops In the future, crops may be grownspecially for energy production, perhaps on landunsuitable for growing foodstuffs. Sugar cane is already being grown in Brazil for this purpose.

3. A. ETHANOL PRODUCTION 1- Substrates include sugar cane, cassava roots, cellulose waste and corn. Cassavaroots contain starch which must be hydrolysed to sugars, and cellulosewaste, such as timber and straw, needs quite complex pre-treatment with ligno-cellulaseenzymes or chemicals. 2- At present, alcohol production is similar to the traditionalprocess but much research is taking place.

4. It is hoped that more efficient, genetically engineered M.O.s will be developed and that newer fermentordesigns and immobilizedenzyme technology will improveefficiency. 3- Distillationcosts can be reduced by using a cheapfuel, and bagasse (the waste from sugar cane) has proved to be an economicalfuel for raising steam for the process by combustion.

5. 4- A range of M.O.s have been used in the production of ethanol, using many different carbohydrates as substrate. Traditionally, ethanol production has relied upon the use of yeasts, mostly Saccharomyces species. 5- Zygomonasmobilishas been used in SouthAmerica for many years in the production of tequila, and in Indonesia and Africa to make palm wine.

6. However, its use in the western world is quite new. Recentresearch into Zygomonas has shown that it is moreefficientthanyeasts in converting sugar to ethanol. 6- A technique has been developed to produce ethanol using Zygomonas in a continuous culture process, ratherthan the more traditionalbatch culture methods.

7. 6.4 The production of methane (1) Sewage (2) Urbanwaste, landfillgas (3) Biogasfermentors However, while this is a useful small-scale process, it is unlikely to be commerciallyavaible on a large scale because: methane can be produced far more cheaply from coal at present;

8. naturalgas is cheaper than microbially produced methane. • There are many natural sources of methane • Gas is expensive to store, transport and distribute at present. • It is expensive and difficult to liquefy.

9. (4) Agricultural wastes Some farms now place animal manure and other crop residues into anaerobic digestion tanks. Here, the waste is fermented by M.O.s and the methane produced is collected, liquefied and used topowerfarmmachinery. In some cases it may be used to fire boilers, which heat glasshouses and produce early crops of tomatoes, peppers and other vegetables.

10. C. Pharmaceuticals produced by M.O.s: • 1. Dextrans Dextrans are polysaccharides produced by lacticacidbacteria, in particular members of the genus Leuconostoc (e.g. L. dextranicus and mesenteroides) following growth on sucrose.

11. 2- Vitamins, amino acids and organic acids • 1. Vitamins Vitamin B2 (riboflavin) is a constituent of yeast extract and incorporated into manyvitaminpreparations. Vitamin B2 deficiency is characterized by symptoms which include an inflamed tongue, dermatitis and a sensation of burning in the feet.

12. 2. Amino acids Amino acids find applications as ingredients of infusionsolutions for parenteralnutrition and individually for treatment of specificconditions. They are obtained either by fermentation processes similar to those used for antibiotics or in cell-freeextracts employing enzymesisolated from bacteria.

13. 3. Organic acids Examplesoforganicacids (citric, lactic, gluconic) producedbyM.O.s. Citric and lactic acids also have widespread uses in the food and drink and plastics industries, respectively. Gluconicacid is also used as a metal-chelatingagent in, for example, detergentproducts.

14. 3 Iron-chelating agents Growth of many M.O.s in iron-deficient growth mediaresults in the secretion of lowmolecularweightiron-chelatingagents called siderophores, which are usually phenolate or hydroxamate compounds. -The therapeuticpotential of these compounds has generatedconsiderableinterest in recentyears.

15. 4 Enzymes • 1- Streptokinase and streptodornase Mammalian blood will clot spontaneously if allowed to stand: however, on further standing, this clot may dissolve as a result of the action of a proteolyticenzyme called plasmin. Plasmin is normally present as its inactiveprecursor, plasminogen.

16. Streptokinase is administered by intravenous or intra-arterialinfusion in the treatment of thrombo-embolicdisorders.

17. 2 - L-Asparaginase • L- Asparaginase, an enzyme derived from E.coli or Erwiniacarotovora, has been employed in cancerchemotherapy where its selectivity depends upon the essential requirement of some tumors for the amino acid L-asparagine . - Normaltissues do to require this aminoacid and thus the enzyme is administered with the intention of depletingtumor of asparagine by converting it to aspartic acid and ammonia.

18. 3 - Neuraminidase • Neuraminidase derived from Vibriocholeraehas been used experimentally to increase the immunogenicityoftumourcells. -It is capable of removingN-acetylneuraminic (sialic) acid residues from the outer surface of certain tumor cells, thereby exposingnewantigens which may be tumorspecific together with a concomitantincrease in their immunogenicity.

19. -In lab animals administration of neuraminidase-treatedtumourcells was found to be effectiveagainst a variety of mouseleukaemias.

20. 4 β-Lactamases - β-Latamase enzymes, whilst being a considerable nuisance because of their ability to conferbact.resistance by inactivatingpenicillins and cephalosporins are useful in the sterilitytesting of certain antibiotics and, prior to culture, in inactivating various β-lactams in blood or urine samples in patientsundergotherapy with these drugs.

21. - One other importanttherapeuticapplication is the rescue of patients presenting symptoms of a severe allergicreaction following administration of a β-lactamase - sensitivepenicillin.

22. 3- Applications of M.O.s in the partial synthesis of pharmaceuticals: • 3.1 Production of antibiotics Alexander Fleming's accidental discovery of penicillin in 1929 is well known. He found the mould Penicillium notatumcontaminating a Petri dish of pathogenic bacteria and inhibiting their growth.

23. He isolated penicillin but it was not until the SecondWorldWar that it was successfullyproduced on a largescale. At first, it was grown in static liquid culture in flasks, shallow pans and bottles, but this processwasinefficient and it was notpossible to produceenoughpenicillin to meetdemand.

24. Two theories have been proposed to explain antibiotic production. 1- Antibiotics are secondary metabolites, so they may be produced to keepenzymesystemsoperative when the microbe has runout of nutrients and celldivision is nolongerpossible. Normally, when the substrate has been usedup, the enzymes of that particularpathway would be brokendown.

25. Then, if a newnutrient supply was found, there would be a delaywhile the necessaryenzymeswereproduced. • It has been suggested that making a secondarymetabolitekeeps the enzymesactive, so that the microbe canquickly take advantage of any newfoodsupply. • 2- Some scientists think antibioticproduction is for ridding of the celltoxicmetabolicwaste.

26. - Although not toxic to the organismproducing them, these substances couldstill be highlytoxic to otherM.O.s. • If the toxinphenylaceticacid is added to a culture of Penicillium, penicillin production is increased. This observation supports this theory. - It is of course, possible that both theories are correct since they are not contradictory.

27. The industrial production of antibiotics; • PENICILLIN PRODUCTION 1- M.O. the organism used for production of penicillin was Penicillium notatum, but the mostly common used is P. chrysogenus. 2- InoculumPreparation; a pure inoculum in sufficient volume and in the fast growing (logarithmic) phase so that a high populationdensity is soon obtained.

28. 3- The fermenter; A typicalfermenter is closed, vertical, cylinderical, stainlesssteelvessel with convexlydishedends and 25 - 250 m3 capacity. The height is usually two to three times its diameter. 4- Oxygensupply; Penicillin fermentation needoxygen, which is supplied as filteredsterilisedair from a compressor.

29. 5- Temperaturecontrol; The production of penicillin G is verysensitive to temperature, the tolerance being less than 1 C. Heat is generated both by the metabolism of nutrients and by the powerdissipated in stirring, and has to by removed by controlled cooling. 6- Defoamingagents; The fermenter system stirredvigorously and aerated usually foam, so provision has to made for adding defoamingagents.

30. 7- Instrumentation; The vessel is fitted with severalprobes to detect foaming, temperature, pH, O2-tension and exhaustgas. 8- Media; Not all the nutrients required during fermentation are initially provided in the culture medium. Provision is therefore made to add these while the fermentation is in progress. The media used is cornsteepliquor (CSL).

31. 9- Transfer and samplingsystems; Appropriate pipework is provided to transfer the inoculum to the vessel, to allow taken routinesample and to transfer the finalcontent to the extractionplant. 10- The optimumtemperature and pH for growth are not those for penicillinproduction they must be changed during the process.

32. 11- The production phase begin with the addition of phenylacetic acid (PAA). 12- PAAsupplies the sidechain of penicillinG. 13- PAA is toxic for the M.O so it must be supplied in smallquantitieswithoutapproaching the toxic level. 14- Termination; The harvest is carried out shortly after the firstsigns of faltering in the efficiency of conversion of the most costly rawmaterial to penicillin.

33. 15- Extraction: A- Removal of the cell; penicillinG is extracellular the firststep is to remove the cells by filtration. B- Isolation of penicillin G; Penicillin G is veryunstable, so it must be quickly extracted by organicsolvent (amyl acetate) from the acidified aqueous solution. C- Treatment of crude extract; first formation of an appropriate salt, charcoal treatment toremovepyrogens and sterilization by using dryheat.

34. Interferons are antiviralchemicals, which also have sometumour­ inhibitingproperties. These used to be extracted from human fibroblast cells, but yields were minute. RecombinantDNAmethods have now been used to synthesizeinterferons using a suitable bacterium, such as Escherichiacoli. Some otheranti-tumour pharmaceuticals are also mademicrobiologically. An example is bleomycin, a glycopeptide, made by Streptomycesverticillus. This drug has the ability to disrupt the DNA and RNA of tumourcells.

35. Steroid biotransformation Since steroidhormones can only be obtained in smallquantities directly from mammals, attempts were made to synthesize them from plantsterols which can be obtainedcheaply and economically in largequantities. However, all adrenocorticalsteroids are characterized by the presence of an oxygen at position 11 in the steroidnucleus.

36. More recentadvances involving the employment of M.O.s in biotransformationreactions utilize immobilizedcells (both livinganddead). • Immobilization of microbialcells, usually by entrapment in a polymer gel matrix, has severalimportantadvantages.

37. Chiral inversion Several clinicallyuseddrugs, e.g. salbutamol (a β-adrenoceptoragonist), propranolol (aβ-adrenoceptorantiagonist) and the 2-arylpropionic acids (NSAIDs) are employed in the racemic form. - It has thus been suggested that the enantiomerically pure S(+) form could be administeredclinically to give a reduceddosage and possibleless toxicity.

38. 4- use of m.o.s and their products in assays • Microbiological assays In microbiological assays the response of a growing population of M.O.s to the antimicrobial agent is measured. The usual methods involve agar diffusion assays, in which the drug diffuses into agar seeded with a susceptible microbial population and produces a zone of growth inhibition.

39. In the commonest form of microbiological assay used today, samples to be assayed are applied in some form of reservoir (porcelain cup, paper disc or well) to a thin lay of agar seeded with indicator organism. The drug diffuses into the medium and after incubation a zone of growth inhibition forms, in this case as a circle around the reservoir.

40. Vitamin and amino acid bioassays • The principle of microb. bioassays for growth factors such as vitamins and amino acids is quite simple. • Unlike antibiotic assays which are based on studies of growth inhibition, these assays are based on growth exhibition. - All that is required is a culture medium which is nutritionally adequate for the test M.O. in all essential growth factors except the one being assayed.

41. If a range of limiting concentrations of the test substance is added, the growth of the test M.O. will be proportional to the amount added. • Carcinogen and mutagen testing • A carcinogen is a substance which causes living tissues to become carcinomatous (to produce a malignant epithelial tumor). • A mutagen is a chemical (or physical) agent which induces mutation in a human (or other) cell.

42. The Ames test • The Ames test is used to screen a wide variety of chemicals for potential carcinogenicity or as potential cancer chemotherapeutic agents. • The test enables a large No. of compounds to be screened rapidly by examining their ability to induce mutagenesis in specially constructed bacterial mutants derived from Salmonella typhimurium.

43. Use of microbial enzymes in sterility testing - Sterile pharmaceutical preparations must be tested for the presence of fungal and bacterial contamination before use. • If the preparation contains an antibiotic, it must be removed or inactivated where membrane filtration is the usual recommended method. • However, this technique has certain disadvantages. Accidental contamination is a problem, as is the retention of the antibiotic on the filter and its subsequent liberation into the nutrient medium.

44. 6 Insecticides - Like animals, insects are susceptible to infections which may be caused by viruses, fungi bacteria or protozoa. - The use of M.O.s to spread diseases to particular insect pests offers an attractive method of bio-control, particularly in view of the ever-increasing incidence of resistance to chemical insecticides. - However, any M.O. used in this way must be highly virulent, specific for the target pest but non-pathogenic to animals, man or plants. - It must be economical to produce, stable on storage and preferably rapidly acting. Bacterial and viral pathogens have so far shown the most promise.

45. MICROBIAL DEGRADATION - Biodegradation and biodeterioration The use of M.O.s to break down substances is usually called biodegradation. However, M.O.s often break down substances in a way that is not beneficial to humans, for example in causing food spoilage. This activity is generally called biodeterioration. Sewage Sewage is composed of the following:-

46. a- Human waste made up of human excreta mixed with waste household water. This contains many M.O.s including potential pathogens. A major pollutant from waste household water is detergent, which causes persistent foam and has high levels of phosphates. b- Industrial wastes which are variable in nature, depending on the industry.

47. Some can be very toxic to M.O.s and must undergo pretreatment so that they do not kill or inhibit the M.O.s which degrade the sewage. Many industries are required to treat their own sewage, either wholly or partially. c- Road drainage consists of rain water together with grit and other debris which enters the sewers from roadside gutters.

48. Sewage treatment Sewage is treated in two or three stages as follows. • Primary treatment. Materials which will settle out are removed. The sedimented solids pass on to a digester for further treatment, while the liquid (effluent) continues into the secondary treatment stage .

49. Secondary treatment. Aerobic M.O.s are used to break down most of the organic matter in the effluent. Any sludge produce in this process is passed on to anaerobic digesters. Tertiary treatment This involves chemical and biological treatment which renders the sewage effluent fit for drinking. However, this is a very expensive treatment, so it is only carried out when absolutely necessary.

50. There are two main reasons for treating sewage. Firstly, sewage can contain pathogens which cause diseases, such as Salmonella typhi (typhoid), pathogenic Escherichia coli (gastroenteritis) and Ascarislumbricoides (roundworm). Secondly, by treating sewage, pollution of the environment can be avoided.