4.4 Fermenters and Penicillin Production – Booklet 2 S. Preston 1

A2 Unit BY4: Metabolism, Microbiology and Homeostasis Name: Date: Topic 4.4 Microbiology – Booklet 2 Fermentation and the Production of Penicillin Completed 1. Read page 2

Recall information about population growth curves. • Describe how adding nutrients to a growing culture might affect the

population growth is they were added in: a. the lag phase b. the stationary phase • Explain why the production of primary metabolites in a

microorganism matches the overall growth of the microbial population.

• Explain why secondary metabolites such as antibiotics are only produced after the main growth phase of the microorganism.

Complete the graph on page 2

2. Read p2-4 and complete all the questions 3. Read p5 and then go onto read the BioFactsheet about Penicillin production. 4. Complete the questions on p6-7 5. Read WJEC p38-39 6. Look at past paper questions on the wikispace

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Fermentation and fermenters The term fermentation was originally applied to the use of anaerobic respiration to produce substances, in particular, the product of ethanol through the anaerobic respiration of yeast. These naturally produced fermentation products are by-products of anaerobic respiration pathways. Fermentation now also refers to the culturing of microorganisms both aerobically and anaerobically in fermentation tanks. The substances generated by growth of the microorganism culture are separated and treated to produce the final useful product. Metabolism is a process and metabolites are the products Metabolism refers to the sum total of all the reactions that go on within an organism. These processes will produce

• New cells and cellular components • Chemicals such as hormones and enzymes • Waste products, these will depend on the organism involved and will vary from carbon dioxide and

oxygen to soluble products such as urea, ammonia and nitrates

Primary and Secondary metabolites The terms primary and secondary metabolites are often used when referring to the metabolic processes of microorganisms.

• Primary metabolites are substances produced by an organism as part of its normal growth; they include amino acids, proteins, enzymes, nucleic acids, ethanol and lactate. The production of primary metabolites matches the growth of the population of organisms.

• Secondary metabolites are substances produced by an organism that are not part of its normal growth. The antibiotic chemicals produced by a number of microorganisms are almost all secondary metabolites. The production of secondary metabolites usually begins after the main growth period of the organisms and so does not match the main growth in population of the organism.

**It is important to note that whilst ALL microorganisms produce primary metabolites (they need to in order to be able to grow) only a relatively small number produce secondary metabolites. Draw a population growth curves on the x-axis is time and on the y-axis is population size. On each graph plot three lines, one that shows the change in population and the other 2 which show the production of primary and secondary metabolites.

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Industrial Scale Fermenters Commercial applications of biotechnology often require the growth of a particular microorganism on an enormous scale. An industrial scale fermenter is essentially a huge tank, which may have the capacity for tens of thousands of liters. The growing conditions within it can be manipulated and controlled in order to ensure the best yield of the desired product.

The precise growing conditions depend upon the microorganism being cultured and on whether the process is designed to produce a primary or secondary metabolite. They are:

• Temperature – monitored and adjusted to provide optimum growth conditions for the microbe being cultured.

• Type and time of nutrient – sources of carbon, nitrogen, macro and micronutrients. The timing is manipulated depending on whether the desired product is a primary or secondary metabolite.

• Oxygen – the majority of commercial fermenters want microbes grown in aerobic conditions. A lack of oxygen would leas to unwanted products of anaerobic respiration and a reduction in growth rate. Aerators are often used and these also help to mix the culture and improve contact with the nutrients.

• pH 0 changes in pH will affect the activity of enzymes and consequently affect production, this needs to be monitored.

Looking at the diagram above explain the need for a water jacket: Aseptic techniques are essential, as unwanted organisms would lead to contamination of the product, produce a toxic by product and compete with the micorbe for resources A pure culture is needed in order that a pure product can be harvested. Before use the fermentation vessel will need to be sterilized and a sterile growth medium used.

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Looking at the diagram on the previous place what other features can you see on the fermentation vessel that helps to prevent contamination: Why do you think fermenter vessels are usually made from polished stainless steel? Industrial scale fermenters can be used in two ways as batch cultures and as continuous cultures. Continuous Cultures Nutrients are added continuously and the product is removed at intervals. The aim is to maintain the organisms in the exponential /log growth phase. Can continuous culture be used for the production of primary or secondary metabolites or both? Explain your answer: Examples include the production of GM Insulin from Escherichia coli. Batch Culture Nutrients are only added at the start and the product is only removed once the fermentation product has stopped. At the end the product is removed and the fermentation tank is emptied. Penicllin is produced using batch culture of the Penicillium fungus. Read the handout BioFactsheet Fermentation Made Simple and summarise the advantages and disadvantages of batch and continuous culture.

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Commercial Production of Penicillin Penicillin is a narrow spectrum antibiotic. Antibiotics are made when the growth of the producer organism is slowing down rather than when it is at its maximum. They are produced as secondary metabolites. And their production takes longer than primary metabolites, it also means that continuous culture is not suitable and they have to be produced using batch culture. In penicillin manufacture, a stirred tank fermenter is inoculated with a culture of Penicillium notatum or Penicillium chrysogenum and the fungus is grown under optimum conditions of 24oC, a good oxygen supply and a slightly alkaline pH. Penicillin production usually commences after about 30 hours, reaching a maximum after 4 days. Productions ceases after 6 days, at which point the contents of the fermenters are drained off. The antibiotic is an extracellular product, the fungal mycelium are filtered off, washed and discarded. The filtrate containing penicillin is purified using solvents to leave a crystalline salt.

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