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MICRO-ORGANISMS AND BIOTECHNOLOGY Organisms can be classified into groups=kingdoms (5 in number); Plantae, animalia, Fungi, Protista, Monera. Three are micro-organisms (fungi, Monera and protista) Kingdom Fungi Structure -unicellular or - filamentous; fungal filament=hyphae network of hyphae=mycelium (hyphae not cellular but have many nuclei) -nucleus present - Cell wall made of chitin -food stored in form of glycogen Genetic code & Reproduction Genetic code=DNA Reproduction; -asexual reproduction by spores -sexual by conjugation budding Nutrition Heterotrophs; either -Parasitic; ring worm Athletes foot potato blight Saprophytes(decomposers) No chlorophyll External digestion=release enzymes to substrate which is digested outside fungal body before absorption 1. some are autotrophs (cyanobacteria=blue green algae; have chlorophyll) 2.heterotrophs -parasites; cause diseases like syphilis 3.saprophytes(decomposers) Note; are found in different habitats like, found in soil,

Monera (bacteria , cyanobact eria)

-unicellular; some occur single, in chains, or in groups Average; about 1-5 microns (plant &animal cells=10-100microns in diameter) Shape; spherical, rod, spiral, comma -cell wall made of proteins, fats and sugars -some have slime capsule which; 1.protects from drying and from being engulfed 2.food store Movement; some have flagella &Number Varies Forms; Coccus=round e.g pneumococcus streptococcus= filamentous staphylococcus Vibrio=coma cholera bacillus=rods Tuberculosis E. coli Tetanus Spirillum=spiral syphilis

Genetic code=DNA Asexual; binary fission -some transfer DNA in plasmids using pili

water, sewage, fruits ,animal intestines ,blood, and saliva

Protista

-mostly unicellular

Genetic code=DNA

Viruses

particle not made of cell(s) has protein coat around nucleic acid (either RNA or DNA ) Size very tiny =300nm or less Can only be seen using electron microscopes spherical, rod, or polyhedral(e.g. Pages)

-Genetic code=RNA or DNA Reproduction; do not undergo cell division but replicates in host cell, its different part synthesised separately

Some are autotrophs & have chlorophyll like euglena Some are parasitic like plasmodium, trypanosoma Parasitic; feed on living host cell cause diseases like polio, AIDs

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2 VIRUSES

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Components of viruses Two main; 1. nucleic acid (Nucleoid) = infective part of a virus and 2. capsid some may have in an envelope plus one or two enzymes A bacteriophage virus (phage)

Resemblance to living things Have organic molecules proteins and nucleic acids Presence of genetic materials Ability to multiply or reproduce in host cells They breed true to their type Some have vitamins and enzymes Ability to undergo mutation most mutable are RNA viruses lead by HIV then influenza viruses infectivity and host specificity They are killed by autoclaving and UV rays(Presence of enzyme and vitamins in some) No food store CLASSIFICATION OF VIRUSES Group 1. Plant viruses

Resemblance to non-living thins Lack protoplasm (=cytoplasm, cell membrane, organelles) Inability to reproduce out of a living cell Ability to get crystallised like polio virus Absence of respiration, growth, division;

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Genetic material Mostly RNA, DNA in a few

2. Animal viruses

Those parasites to lower organisms Viruses of fungi like in yeast Viruses in algae Bacterial viruses like; bacteriophage affect E. coli

Mostly DNA, (RNA in few like HIV, HERPES, Rabies, polio viruses) Commonly DNA like

Examples of diseases Mosaics like; tomato and potato mosaics leaf curls like; in tomato AIDS, Polio, measles

May Kill the organisms

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BACTERIA (Discovered by Leeuwenhoek) Gram staining characteristics Depends on cell wall structures; those that are stained by gram stain = gram positive; cell wall not covered by lipids thus easily attacked and killed by most natural antibiotics like penicillin Those that cannot (=gram negative like E. coli) have high lipid content covering cell wall thus resis some antibiotics like penicillin = Gram -ve LIFECYCLES OF SOME BACTERIA, FUNGI AND BACTERIOPHAGE VIRUS

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Economic importance of bacteria Harmful activities; Some spoiling food (rotting of food) Some cause food poisoning Some degrade things like leather and wooden articles Denitrifying bacteria convert soil nitrates to gaseous nitrogen Diseases like syphilis, typhoid and gonorrhoea

Useful activities; (99% of all bacteria are helpful.) There are more microbes on your body than there are humans on the entire planet. decomposition of organic matter leading to nutrient cycling and waste removal manure and methane gas formation from dung and farm wastes nitrogen fixation ; conversion of atmospheric compounds to nitrogen compounds in soil Ammonification bacteria reduce amino acids formed during decay to ammonia Nitrification bacteria convert ammonia to nitrites then to nitrate in soil Production of vitamins like E. coli in human intestine produce vitamin K and B complex Bacteria help with digestion especially in ruminants help destroying harmful organisms within our bodies. making helpful vaccines. Scientists estimate that bacteria produce nearly half the oxygen found in the atmosphere. A number of human growth hormones, blood clotting agents (factor VIII), insulin & potential anti-cancer agents have been produced in bacteria. Treatment of tea, tobacco and coffee leaves to develop required characteristics Cleaning hides during leather processing Production of single cell protein (biomass production) Biological control agents (bio-pesticides);e.g. bacterium

Bacillus thuringiensis - produces a toxin which kills certain species of caterpillars

FERMENTATION PRODUCTS Fermented milk, yoghurt and cheese Microbe secondary metabolites (i) Enzyme production like insulin (ii) Vinegar (acetic acid=ethanoic acid) production by acetic acid bacteria (iii) Alcohols; can be mixed with gasoline=gasohol and used as fuels like in Brazil (iv) Antibiotics; organic secretions produces by micro-organisms that kill or prevent growth of other micro-organisms (mostly pathogens)

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Decomposition; 1. Decomposers like bacteria and fungi ; organisms that break down dead organic matter to simpler substances (are also called saprophytes) 2. Their digestive enzymes break down complex organic substance like proteins, lipids and carbohydrates to; glucose, amino acids, fatty acids and glycerol. 3. Inorganic salts are also released from the organic matter

Uses Humus formation Fermentation

Putrefaction Sewage disposal

Mode of action Dead matter in soil decay to form black, sticky liquid If oxygen is lacking and decomposition is not complete, peat is formed Anaerobic decomposition of carbohydrates Yeast decomposes glucose to ethanol Bacteria decomposes glucose to alcohol, lactic avcid (for making butter, yogurt) and acetic acid (vinegar) Anaerobic decomposition of protein Leads to production of nitrogen and sulphur compounds which smells bad Sewage reduced to harmless substances by aerobic bacteria and aerobic fungi

Sample question;
Describe part plaid by micro-organisms in production of any two of the following: Bread, alcohol, cheese, yoghurt

Baking bread

Flour mixed with water to form dough Yeast added to dough The flour has starch, protein and amylase Amylase digest starch to sugar Lack of oxygen in dough cause yeast to respire anaerobically; (=alcohol fermentation converts the sugar to alcohol and CO2, & heat Yeast use the energy to provide adequate temperature for the enzymes thus multiply The CO2 trapped in the dough causes it to rise leaving small spaces in bread as the CO2 escapes

Note; -The alcohol produced disappear during baking - Use warm water to provide adequate temperature for yeast enzymes - very hot can kill yeast and very cold can inactivate yeast enzymes
- if oxygen is present in the dough initial yeast respiration may be aerobic then after oxygen is finished it is anaerobic

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Wine Grape juice extracted from grapes The juice has glucose and fructose and wild yeast from grape skins Yeast ferments the sugar into ethanol for wine Beer Barley grains allowed to start germination where enzymes; Converts starch to sugars (maltose) Sugar is extracted from germinating barley grains; Procedure; Grain dried then milled Water added ;the mixture=wort Wort boiled and cooled Yeast added to convert all the sugar to ethanol=fermented liquor/marshes The mixture filtered, bottled and pasteurised In spirit drinks, like whisky, the alcohol is separated from the liquor by distillation Cheese or yoghurt Fermentation involves bacteria not yeast Yoghurt Milk pasteurised then bacteria added 1. Lactic acid bacteria (Lactobacillus bulgaricus) which; Ferments; by converting milk sugar, lactose, to lactic acid Lactic acid coagulates/curdle milk protein (casein) to yoghurt Which is thick creamy s with slight sour taste it also has milk fat in it Cheese; both lactobacillus bacteria (ferment milk sugar to lactic acid which coagulate milk protein, casein) and fungi are used) (this Initial procedure is similar to that of yogurt) then; Rennet(rennin/chymosin enzyme) is added to precipitate casein protein in the milk-bacteria mixture=casein and liquid called whey separate The Whey (liquid) is drained away Curd are packed into moulds(fungi) and pressed It is then allowed to ripen in the ripening room Different cheese types are produced by; Fruit flavours added Using different conditions such as temperature and different mixtures of bacteria and fungi

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Biotechnology
Use of living things or substances from them to make useful products
Biotechnology can be divided into 4 main areas: 1. Production of biomass 2. Fermentations: 'life/respiration in the absence of oxygen'. Today, the term is taken to mean: 'biological processes that occur in the dark & do not involve respiratory chains with oxygen or nitrate as electron acceptors'. 3. Harvesting microbial secondary metabolites (i) like insulin hormone to treat diabetes and (ii) penicillin antibiotic to treat some bacterial infections like syphilis. 4. The use of genetically manipulated organisms (GMOs) (i) Use bacterial plasmids (ii) Bacteriophage Fermenters

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FERMENTERS Closed steel cylinders tanks that keep favourable environment for particular biological processes. Allow large scale production of micro-organisms and their products. Genetic engineering also allows us to genetically change microbes to produce gene products that we need like insulin hormone and penicillin antibiotic Use of fermenters for single cell protein (SCP) SCP are proteins made from microbes like bacteria and yeast Example= Mycoprotein= human food made from fungus Fusarium Its production; in specially designed fermenters Gown in glucose and mineral salts at 30 degrees Celsius Unlike in antibiotics it is the fungal cells which are harvested The culture medium is continually circulated around fermenter Culture Medium is added when necessary Then the fungus is filtered off Remaining liquid is returned to fermenter The fungus is dried and processed This method is for growth-associated process & is called continuous operating system

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9 Advantages of single cell protein

Soofia English Medium School Disadvantages of single cell protein

1. Much quicker and efficient to produce protein; (microbes have a high rate of multiplication(algae: 2-6hours, yeast: 1-3 hours, bacteria: 0.5-2 hours) 2. Use less space land requirements are low . 3. Natural microbes can be selected to produce best quality amino acid 4. The microbes can be genetically modified to produce required amino acid composition. 5. Very rich in proteins 6. Has sufficient fibre 7. Has low fat content 8. They can use many types of raw materials as carbon sources, including waste products. 9. This also helps in the removal of pollutants. 10. It is not dependent on climate 11. Quality is consistent since the growth is not dependent on seasonal and climatic changes.

1. Very expensive to produce therefore the SCP product may cost more than usual food product. There are also some technical problems, such as:

2. Needs high technique to produce 3. Needs very high level of sterility needed 4. Many microorganisms produce some toxins (we have to be sure the SCP doesnt have toxins). 5. May lead to indigestion or allergies re 6. The high level of nucleic acid may lead to kidney stones or gout, poor digestion. ( Normally some of the amino acids are removed during processing)

ANTIBIOTICS

The history of antibiotics The early work done by Fleming, who discovered the effect of penicillin, and Florey and Chain, who developed it further. Development of this and other antibiotics such as actinomycin and aureomycin and other tetracyclines solved many problems during the second world war. These are defined as substances produced by some micro-organisms which are in some way lethal to other micro-organisms. It is thought that these substances give the organisms that produce them (usually Fungi (moulds) or actinomycetes - which grow slowly) some sort of advantage in competition with other micro-organisms (usually bacteria - which grow fast) in the same habitat.

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Penicillin production a fungus=Penicillium (Penicillium chrysogenum) is used Fermenter filled liquid nutrient broth (culture medium) for fungal growth Main Carbon=food source is lactose Others =organic nitrogen source like corn-steep liquor and mineral salts pH between 5-6 Temperature 26 degrees Celsius during penicillin production Air blown in and medium constantly starred The selected penicillium added Conditions needed maintained for rapid growth During growth there is little penicillin produced As growth slows down rapid penicillin production begins Right conditions adjusted at this stage After some time conditions become unsuitable for the fungus thus penicillin production cannot be continued endlessly The nutrient broth containing the antibiotic is filtered and penicillin extracted usually by crystallisation

Usually these fermenters are operated in a batch process (batch operating system) fungal growth occur in fixed volume of culture medium After a certain amount of time for fungal growth, followed by gradual production of antibiotic, the contents are removed and processed to extract the antibiotics, then, Fermenter is cleaned, sterilised and the process is repeated. Penicillin extraction
After 6-8 days of batch culture, the liquid medium is pumped out, filtered and concentrated. The basic antibiotic - benzyl penicillin - is precipitated as crystals when potassium compounds are added.

This antibiotic may t be modified by the action of other micro-organisms or by chemical means, before being mixed with inert substances and pressed into tablets or converted into syrup or injectable form. There are several versions of Penicillin, produced by different strains of Penicillium, or using different culture media and methods.

These other types of penicillin differ in the efficacy and ease of use in different applications, e.g. some are better in injectable form, or less likely to be broken down by enzymes of host or bacterial origin.

Various other antibiotics have also been developed with different modes of action, e.g. interfering with bacterial protein synthesis. Penicillin production relies on fungal fermentation based on biological principles, although modern strains are much more productive than the early strains. This has been achieved
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through screening programmes involving isolates from different sources, and treatment to encourage mutations.
Sample quiz

What does the term productive mean? Producing large quantities of something useful like in this case, antibiotic. What is meant by an isolate? Getting a pure culture (a single species) of an organisms from a particular source How might mutations be encouraged? By chemicals or radiation (ultra-violet, x-rays etc)
radiation (ultra-violet, X-rays etc) or chemicals

What is meant by a screening programme? Many experiments, testing lots of strains of microbes see if they are the best needed
What nutrients should the liquid medium contain?

C=Carbon source; glucose (sugars) N = nitrogen source NH4+ / NO3- /amino acids other mineral salts Why is the liquid stirred? -To dissolve oxygen, -Uniform distribution of; nutrients, fungus, wastes, heat, heat) For what purpose is air pumped in? -For oxygen to dissolve so aerobic respiration occurs. -Leading to efficient growthssolv Why must the air be sterile? So that no other microbes get in to contaminate the products (Before use, fermenters must be sterilised, usually with superheated steam.) Why is the temperature monitored? To prevent excess acid which may prevent growth by denaturing enzymes? How is the temperature controlled? By pumping cold water into the jacket and controlling out let of the hot-water jacket that carry away heat
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Why is the pH monitored? To prevent build up of excess acid, that can denature enzymes and stop fungal growth &production How is the pH controlled? Addition of base or acid Why is stainless steel considered to be an ideal material for fermenter construction? Does not rust, withstand heat, allow heat to pass through, strong and withstands stirring strain What is meant by the term bacterial lawn? Even layer of bacteria growing on the surface of agar or within agar Why do antibiotics kill bacteria and not (usually!) humans? Antibiotics attack cell walls, Bacteria have cell walls and they die without the cell walls which humans cells dont have all penicillin type antibiotics operate by stopping the production of cell walls by bacteria, preventing bacteria growth. Bacteria lose water which kills the bacteria, due to osmotic damage because they are not protected by their outer wall. (Bacterialcidal) Antibiotic Resistance
Some strains of bacteria produce enzymes which break down and thus inactivate antibiotics such as penicillin, so they can still grow in its presence. These are said to be resistant strains to these antibiotics. What problem would this cause to an individual person infected with a resistant strain of bacteria Ans. Infection cannot be controlled by usual antibiotic, so disease may worsen. Or take alternative drugs which may be more expensive, not readily available and more difficult to handle and take
This means that the infection could not be controlled by the usual methods,

It is thought that in a large population of bacteria there are always a few bacteria with resistance to some antibiotics. The few bacteria with this ability can grow and multiply greatly when the other bacteria - "sensitive" to the antibiotic - have been killed. Bacteria can also pass on the antibiotic resistance to other unrelated bacteria. In effect, these resistant strains of micro-organisms are showing rapid evolution based on the currently accepted principles of evolution - natural selection favouring some sections within populations which normally show variation, most of which is due to mutation, i.e. chance alteration of DNA. In some cases, other ("second line") antibiotics are available which work in a different way, and may therefore bring infections under control. This is an area of constant application by microbiologists.

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Nowadays there are, unfortunately, frequent instances of multiple resistance to antibiotics. Bacteria with this multiple resistance are extremely hazardous and threaten to put the clock back to times before the introduction of antibiotics. Cause of antibiotic resistance In the past, antibiotics have been added to animal feed, because they cause increased growth rates (by modifying the balance of bacteria in the animals' gut) and hence gave greater profitability. However, this practice has been banned because it led to the development of resistance which could be passed to other bacteria. When taking antibiotics for medical reasons, it is also important to complete the course of -+ antibiotics prescribed, in order to maximise the benefit of the treatment, and not to allow survival of some bacteria exposed to a sub-lethal concentration.

Other infections which cannot be controlled by antibiotics

It is said that infections due to viruses cannot be cured by the use of antibiotics, unless there is a secondary infection by bacteria. Explain why viral diseases are not controlled by antibiotics. Most antibiotics damage cell walls of bacteria, which viruses dont have. Bacteria are also metabolically active, so can be easily hit by interfering with their metabolism Viruses are not metabolically active unless inside host cells Once virus is in host cell the cell will die anyway and virus activities inside cells can only be stopped by killing the cell
Most antibiotics damage cell walls of bacteria, which viruses do not

Testing bacteria for sensitivity to antibiotics Individual bacterial strains can be tested against a variety of antibiotics (or vice versa) by growing the bacteria as "lawns" on agar in the presence of different concentrations of a single antibiotic, or several different antibiotics may be tested at the same time.

Under what circumstances might individual bacterial strains be tested against a variety of antibiotics? To see if a bacterium causing a disease can be controlled by a specific antibiotic or to find the best antibiotic for a particular patient Under what circumstances might individual antibiotics be tested against a variety of bacterial strains?To see if an antibiotic is likely to work in a particular situation like control
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a certain disease
to see if an antibiotic under development by a drug company is likely to

This testing may be achieved either by either of 2 methods: - placing antibiotic liquid into wells or ditches which have been cut into the agar, or - applying discs containing mea

sured amounts of antibiotics, which will diffuse out.

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The size of the zone of inhibition - in which bacteria will not grow - gives an indication of the sensitivity of the strain involved, i.e. how easily the bacterial strain will be controlled by the particular antibiotics. In a medical context, the prescription by a doctor of an appropriate antibiotic or dose rate for a particular patient may thus be confirmed from these laboratory tests. How long would these laboratory tests take? Explain why. About 24 hours ( or at least overnight) to give culture time to grow (or be killed)
about 24 ho

Genetic engineering (modification of organisms genes to produce needed products)

Genetic engineering involves gene transfer using bacterial plasmids or viruses

number of enzymes used in genetic engineering are from bacteria -like Escherichia coli and Agrobacterium tumefansiens NOTE; BACTERIAL CELL STRUCTURE :

Major parts of a bacterium; Bacterial chromosome (Nucleoid= single DNA), plasmids, pili and flagella in some. Plamismids; Small ring of DNA in cytoplasm, not part of the Nucleoid and Can replicate independent of the Nucleoid DNA. Can temporarily bind to Nucleoid DNA. They can pass from one bacterium to another and then becomes permanent part plasmid DNA of recipient bacteria Have genes which may or may not be useful to the bacteria; May help in bacteria survive in certain environments like offering antibiotic resistance but are not involved in basic metabolism. Plasmids genes (2 to 250); 1. F-plasmid (fertility factor); bacteria with this can transfer it together with nucleoid genes to other bacteria 2. Col-plasmid (colicinogenic factor); responsible for production of toxins called colicins (bateriocins) which kill some other intestinal bacteria R-plasmid; (resistance factor) ; carry genes for resistance to some antibiotics like tetracyclin, streptomycin www.soofiaems.org

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Gene cloning; process that lead to formation of many copies of a particular gene Aims; to study the DNA or to use the DNA as a tool like in production of insulin Starting materials=1. Vector DNA (bacteria plasmid or virus) and 2. Gene of interest ( Chromosomal DNA which carries the gene of interest) Obtained by opening the cell, removing and purifying the DNA by Chromatography or centrifugation Insulin production Diabetes mellitus can affect nearly all body system and leads to early death Type 1 diabetes= lack of enough insulin production by pancreas; appears in early during childhood. Signs; frequent thirst, fatigue, increased hunger, weight loss and nausea. Insulin helps in uptake of glucose in fat and muscle cells Treatment=insulin injection; initially insulin was removed from cattle pancreas but some people were allergic to this and for them, insulin was removed from other animals like pigs and dead human people which was expensive Today human insulin made recombinant bacteria (transgenic E. coli) Transgenic E. coli formation (genes for human insulin synthesis are inserted in the bacterium plasmid) 1. E. coli plasmid removed by by gel electrophoresis 2. Restriction enzyme open them up the ring of DNA , 3. Other enzymes used to insert needed genes for insulin into the gap 4. The plasmid is put back into bacteria (now= genetically engineered bacteria) which 5. then reproduce and pass on the foreign genes to all its off-springs 6. Because bacterial reproduction is asexual, soon the genetically engineered bacteria give rise to large volumes of this similar cell and their products in fermenters, harvested and purified.

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Other transgenic bacteria are used to produce Hepatitis B vaccine, human growth hormones, enzyme used in cheese production e.t.c.

Genetic engineering in agriculture Agrobacterium tumefansiens used to make transgenic plants (GMOs) Production of transgenic plants like those resistant to herbicides or drought It is easier than animals because plant somatic cells can easily regenerate than animals Genetically modified organisms=GMOs, include crops like Bt corn, named so because it carries a gene from the bacterium Bacillus thuringiensis which leads to production of toxin that kills larvae of European corn borer a major pest of corn. Initially farmers used to spray the bacterium in corn fields directly but the effect of single spray didnt last long (biological pest control) or they used chemical pesticides

Other GMO animals and crops are like golden rice , carry genes to produce a which our body turn to vitamin A. lack of vitamin A is a common cause of permanent blindness in many parts of the world May be in future we may; for example, produce spider silk in animal milk like goat milk. Spider silk is one if the strongest yet most flexible natural material. It is used to make bulletproof cloths or just cloths for people Short list other applications of genetic engineering (in pencil)

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EFFECTS OF GENETIC ENGINEERING ADVANTAGES Enables development of desirable organisms and their products like pest resistant crops Can lead to development and production of drugs against diseases like diabetes Can lead to faster food production thus reducing malnutrition

DANGERS and CONCERNS ABOUT GENETIC ENGINEERIG Unwanted genes can be accidentally transferred to unintended organisms like herbicide resistance in weeds or antibiotic resistant harmful bacteria Developing pesticide resistant crops may mean more chemicals will be sprayed on crops in the knowledge that their crop will not suffer and these may pollute environment Possible health risk for people who eat GMO food

Write notes on; (in pencil) Causes of antibiotic resistance caused by human beings Importance of not abusing (drug abuse of) antibiotics

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