Microbial growth. Unit 2: Industrial Microbiology. Key terms

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1 Unit 2: Industrial Microbiology. 21 Microbial growth What do microorganisms need to grow? Think of all the environments in which you would expect to find microorganisms. Many different microorganisms are used in industrial microbiology processes and it is important to know their growth requirements and useful products. Prokaryotic cells: Cells with naked DNA not enclosed in a nucleus and with no membranebound organelles. Eukaryotic cells: Cells with their DNA organised into linear chromosomes and enclosed in a nucleus; also having membranebound organelles such as mitochondria, Golgi apparatus and endoplasmic reticulum. When carrying out practical investigations into microbial growth the microorganism being investigated is always treated as if it is pathogenic. Your use of aseptic technique should become automatic this protects the investigator and prevents contamination of microbial cultures. On successful completion of this topic you will: be able to investigate microbial growth (LO1). To achieve a Pass in this unit you will need to show that you can: carry out practical investigations in order to obtain data on microbial growth, using safe practices (1.1) construct graphs that provide information and data on microbial growth cycles and characteristics (1.2) interpret the experimental growth data relating to growth cycles and characteristics (1.3) explain the limits of growth in large-scale production of microorganisms (1.4). 1

2 Before you start If you need to check your understanding of anaerobic respiration in fungi and bacteria and your understanding of aerobic respiration and the stages of glycolysis, link reaction, the Krebs Cycle and the electron transport chain, you may find Unit 1 Module 4 of OCR A2 Biology (Sue Hocking, 2008) useful. If you need to check your understanding of eukaryotic and prokaryotic cells you may find Unit 1 Module 1 of OCR AS Biology (P Kennedy and F Sochacki, 2008) useful. Table 2.1.1: Table summarising growth requirements of microorganisms. 1 Factors that limit the growth of microorganisms Like all other living organisms, to synthesise complex organic compounds and to carry out metabolic reactions, microorganisms need water, nutrients and a source of energy. Table summarises their growth requirements. They also need a suitable temperature, ph and salinity (osmotic potential). Requirement Use Source carbon nitrogen non-metallic elements: sulfur phosphorus synthesis of all organic compounds for cellular structures and for metabolic activity to make amino acids (and therefore proteins), nucleic acids, ATP and co-enzymes such as NAD, NADP component of some amino acids component of nucleic acids, ATP, co-enzymes, phospholipids chemoautotrophs use inorganic carbon dioxide as the carbon source chemoorganoheterotrophs use organic substances, such as carbohydrate, lipids and amino acids some (Rhizobium, Azotobacter, Clostridium) can fix atmospheric nitrogen some use inorganic nitrogen compounds such as ammonium or nitrate salts some use organic compounds such as amino acids or polypeptides sulfates, sulfur, amino acids cysteine and methionine phosphate salts metallic elements: calcium zinc sodium potassium copper manganese magnesium iron cobalt co-factor (non-protein component needed for enzyme activity) for some enzymes inorganic component of some enzymes osmoregulation; and essential for photosynthetic bacteria, e.g. cyanobacteria co-factor for certain enzymes inorganic component of some enzymes co-factor for some enzymes component of chlorophylls, co-factor for enzymes involved in ATP synthesis component of carrier proteins in the electron transport chain component of vitamin B 12 traces of calcium salts in growth media traces of zinc salts in growth media traces of sodium salts in growth media traces of potassium salts in growth media traces of copper salts in growth media traces of manganese salts in growth media traces of magnesium salts in growth media traces of iron salts in growth media traces of cobalt salts in growth media Continued on next page 2

3 Requirement Use Source vitamins water energy act as co-enzymes (organic compounds that act in conjunction with enzymes) in many metabolic pathways component of cytoplasm and provides a medium (in solution) for all metabolic reactions also provides hydrogen ions (some bacteria use proton-motive force for movement) and oxygen ions (final electron acceptor in aerobic respiration) for synthesis of molecules and structures for active transport for movement for signalling within and between cells to form biofilms (aggregation of microorganisms, where the cells stick to each other and to a surface) many microorganisms synthesise vitamins but some may need certain vitamins added to the media yeast extract is a good source of the B-group vitamins water both liquid and solid media such as nutrient agar contain a high percentage of water light: used by photoautotrophs used by photoheterotrophs, e.g. Rhodospirillum a purple non-sulfur bacterium oxidation of electron donors: chemoheterotrophs obtain energy by respiration (aerobic or anaerobic) from organic carbon compounds such as carbohydrates, lipids and amino acids chemoautotrophs (chemolithotrophs) obtain energy from inorganic energy sources such as chemical reactions involving sulfur, iron, nitrogen or manganese compounds. Archaea and bacteria at thermal oceanic vents are the producers in food chains where there is no light. Osmoregulation: Regulation of salt and water content of cells or body fluids. Chemoautotrophs: Organisms that obtain energy from chemical reactions involving sulfur, iron, manganese or nitrogen. Chemoorganoheterotrophs: Organisms that use organic substances, such as carbohydrates, lipids and amino acids, as their source of energy and carbon. Photoautotrophs: Photosynthetic organisms that use inorganic carbon dioxide as their source of carbon and light as the source of energy to synthesise organic molecules such as carbohydrates, lipids, amino acids, vitamins and nucleic acids. Photoheterotrophs: Organisms that use light as their source of energy and organic molecules such as carbohydrates, lipids and amino acids as their source of carbon. Archaea: Previously classified as prokaryotes and named Archaebacteria, Archaea are now classified as a separate domain as they have different biochemistry from bacteria. Some of their genes and metabolic pathways resemble those of eukaryotes and they appear to have a separate evolutionary pathway from bacteria. They use a greater variety of energy sources than bacteria and many, but not all, are extremophiles. Some methanogens inhabit ruminants intestines as commensals there are no known Archaea pathogens. Many are salt-tolerant and live in the oceans. Proton-motive force: Force generated by proton (hydrogen ion) gradient across a membrane; can be used to synthesise ATP (as in respiration) or, in bacteria, can move flagella. 3

4 Fastidious: Microorganisms (bacteria) that need specific nutrients added to their growth media. Enriched media: Growth media with added nutrients, such as certain vitamins, for fastidious microorganisms. Aerobes: Organisms that need to use free oxygen as the final electron acceptor for aerobic respiration. Obligate anaerobes: Organisms that respire anaerobically and must not be exposed to oxygen. Microaerophiles: Organisms that respire aerobically but need only small amounts of oxygen as they are killed by larger concentrations of oxygen. Facultative anaerobes: Organisms that can grow in the presence (using aerobic respiration) or absence (using anaerobic respiration) of oxygen. Mesophiles: Organisms that grow best at temperatures between 20 and 40 C. Thermophiles: Organisms that grow best at temperatures above 40 C. Psychrophiles: Organisms that grow best at temperatures below 20 C. Halophiles: Organisms that tolerate and grow in high saline (salt concentration) conditions. Activity Why do you think research into extremophiles such as the organisms in the Rio Tinto is important with respect to the question of whether there is, has been or could be life on Mars? Nutrients Some bacteria are fastidious because they need enriched media; for example Staphylococci need blood added to the medium. Other bacteria may need vitamins or plant or animal extracts added. These additions form the basis of differential and selective growth media that can be used to identify bacterial contaminants (see Topic guide 2.4). Oxygen Aerobes require free oxygen as the final electron acceptor in aerobic respiration but obligate anaerobes must have oxygen excluded as they lack the enzyme catalase and cannot deal with hydrogen peroxide produced in the presence of atmospheric oxygen. Microaerophiles need only small concentrations of oxygen as larger amounts kill them by inhibiting their oxidative enzymes. Facultative anaerobes can grow in the presence or absence of atmospheric oxygen. Temperature Temperature affects enzyme action and so influences the rate of metabolic reactions. All organisms have an optimum temperature range for their growth. Most microorganisms are mesophiles growing best at between 20 and 40 C. This includes pathogens as they have to live inside their hosts. Thermophiles grow best at temperatures above 40 C. Some are extreme thermophiles and can grow in hot springs or in thermal oceanic vents, where high pressures mean the water can be as hot as 250 C. Enzymes obtained from thermophiles are useful in biotechnology as they are heat-stable because they contain many disulfide bridges, and can be used in chemical reactions that also need heat, such as the polymerase chain reaction. Psychrophiles grow best at temperatures below 20 C. They can be found in deep oceans, in the Arctic and Antarctic regions, and in fridges and freezers. ph Most bacteria grow within the ph range of between 6 and 8. However, some grow at very low ph values. Helicobacter pylori causes stomach ulcers and can live at ph 1 2. In the red river, Rio Tinto, in southwest Spain, chemoautotrophs (also known as acidic chemolithotrophic bacteria) use the inorganic iron, copper and manganese compounds present to obtain energy and, in the process, some release sulfur that other chemoautotrophs oxidise to sulfuric acid. The ph of this river is between 1 and 3 (and the iron gives it the blood red colour). These bacteria survive the acidic conditions (and, surprisingly, so do some eukaryotes algae, protozoa and fungi). Salinity Most bacteria cannot live in high salt concentrations as water leaves their cells by osmosis. However, halophiles can grow in highly saline conditions. 4

5 Chemicals and radiation Some chemicals, short wavelength ionising radiation (UV, gamma and X-rays), as well as heat can be used to kill bacteria when necessary see Topic guide Growth cycles Metabolites: Products of metabolism. Primary (metabolites): Produced during log phase. Secondary (metabolites): Produced during stationary phase. For a large-scale process to be properly managed with optimum population growth and product yield, you need a clear understanding of microbial growth kinetics. It is also important to know whether the desired product is a primary or secondary metabolite as these are produced during different growth phases. Once a sterile nutrient medium has been inoculated with the microorganism that is to make a useful product, the four phases of a typical microbial growth curve occur (see Figure and the explanations of the phases shown in Table 2.1.2). Figure 2.1.1: Graph showing phases of the microbial growth curve. Log cell number Lag phase Log phase Stationary phase Death phase Table 2.1.2: Microbial growth phases and growth characteristics. Time Phase of microbial growth curve Lag phase Log phase Stationary phase Death phase Explanation A period before growth when microorganisms adapt to their new environment. Genes are switched on and the required enzymes are synthesised. The transfer to a new medium may cause a change in ph, increase in available nutrients and reduction of growth inhibitors. Length of lag phase can vary if the inoculum is taken from a culture during its log phase then there may be no lag phase, but if it is taken from a culture in its stationary phase then the lag phase may be quite long. Cells have adapted to the new conditions and can now double their number (filamentous organisms such as fungi or Streptomyces double their biomass) per unit time, giving an exponential growth rate. Growth curves are plotted on logarithmic graph paper giving a straight line. The specific growth rate depends on concentration of substrate (S), maximum growth rate (µ m ) and a substrate specific constant (K s ). K s is equivalent to the Michaelis constant (K m ) in enzyme kinetics and is the substrate concentration where half the maximum specific growth rate occurs (see Unit 1: Biochemistry of Macromolecules and Metabolic Pathways). The maximum specific growth rate is very important in industrial processes. Each depends on the type of organism and the conditions of fermentation. Metabolites produced during the log phase are called primary metabolites. Growth slows due to the substrate having been completely metabolised or to the accumulation of toxic by-products. The biomass remains constant during this phase as number of cells produced = number of cells dying. However, as dead cells lyse (split), carbohydrates, lipids or proteins are released, which are new substrates and energy sources for the remaining surviving cells. Metabolites produced during the stationary phase are called secondary metabolites. The length of the stationary phase depends on the organism and on the process being used for its culture. Cells die at an exponential rate, giving a straight line on logarithmic graph paper. They die because they have exhausted all their energy reserves. In commercial and industrial processes the fermentation is usually interrupted before the end of the log phase, or before the death phase begins. 5

6 Link Find out more about enzyme kinetics in Unit 1: Biochemistry of Macromolecules and Metabolic Pathways. Activity: Length of substrate molecules The fungus Fusarium graminearum doubles its mass in 2.48 hours when grown at 30 C with glucose as the source of carbon and energy. At the same temperature, its doubling time with maltose (a disaccharide) as the source of carbon and energy is 3.15 hours and with maltotriose (a trisaccharide) it is 3.85 hours. Suggest why the doubling time (also known as the generation time) for microorganisms grown on long-chain substrates, such as polysaccharides, is longer than if they are grown on simple small-molecule substrates such as mono- or disaccharides. (For explanations of monosaccharides, disaccharides and polysaccharides see Unit 1, Topic guide 1.5: Carbohydrates.) Activity: Primary and secondary metabolites The table gives data for the industrial production of a metabolite, ergotamine, used obstetrically to stimulate labour in childbirth by causing the uterus to contract, by a culture of fungal microorganism, Claviceps purpurea, supplied with a substrate rich in amino acids, and grown at 30 C over 12 days. Time from start (days) Residual substrate (mg dm 3 ) Dry biomass of microorganisms (mg dm 3 ) Cumulative metabolite production (mg dm 3 ) Graph these data on a single set of axes. 2 Describe and explain the growth stages of this fungus. 3 Calculate the rates of (a) dry biomass production and (b) metabolite production over (i) the first 3 days and (ii) over the final 5 days. 4 Calculate the ratio of dry biomass production to metabolite production (a) over the first 3 days and (b) over the final 9 days. 5 Is the product a primary or secondary metabolite? Explain your answer. Aseptic technique: Technique used when carrying out practical work with microorganisms. Involves disinfecting surfaces, using sterilised equipment, and sterilising all cultures before disposal. Some procedures may take place in special laminar flow cabinets where air is filtered to exclude any unwanted microorganisms. Turbidity: Cloudiness of a liquid culture (due to presence of microorganisms) that can be used to assess microbial growth. The measurement can be taken by using a turbidimeter or a colorimeter (see Figure 2.1.4). 3 Setting up the process Screening In order to determine the optimum growth conditions of the useful microorganism, researchers grow it in small (200 cm 3 ) laboratory flasks. They may investigate the optimum nutrient, temperature, oxygen and ph requirements of the organism and may also need to find out if it is fastidious. They use aseptic technique and, in order to determine growth rate, can use dilution plating (serial dilution, see Figure 2.1.2) and viable count, direct count (haemocytometer, see Figure 2.1.3) or turbidity. Dilution plating and viable count This method counts only living cells. The solution being investigated is diluted, plated onto nutrient media and incubated. Colonies are counted and, as each colony has arisen from a single cell, the number of cells in the original solution can be estimated. This method is time-consuming and heavy on apparatus. 6

7 Figure shows the method used to make a serial dilution for dilution plating. The contents of the dilution tubes are mixed and, using sterile micros, of each of the tubes 10-4,10-5,10-6 and 10-7 is placed in separate sterile Petri dishes and warm (just above setting point) sterile nutrient agar added. The lids are replaced and the dishes swirled. When the agar has set these plates are taped and then incubated, inverted, at 30 C. After 2 days the number of colonies is counted, using a colony counter, on plates that have between 30 and 200 colonies. This number can be multiplied by the dilution factor to give the original number of cells per ml. Figure 2.1.2: Method used to make a serial dilution for dilution plating. Transfer with sample Direct count (haemocytometer) Cells, both living and dead, are observed under a microscope and counted. This method is fairly time-consuming. In Figure (a) the central grid contains 25 small squares each subdivided into 16 smaller squares. As the grid is lower than the rest of the slide, when the coverslip is firmly in place the depth of the chamber above the grid is 0.1 mm. The area of the central grid is 1 mm 2. Cells in the four corner squares and the central square of the central grid are counted. Any cells on the bottom or right-hand lines are not counted. This gives the number of cells in 0.02 mm 3. Multiplying this number by 50 gives the number of cells per ml and if the sample was diluted, then this number must be multiplied by the dilution factor. This method counts both living and dead cells. 7

8 Figure 2.1.3: (a) Haemocytometer slide and central area with grid. The cells marked x are used for counting cells. (b) Cell count on a haemocytometer grid. Five squares (0.2 x 0.2 mm) are used and cells within are counted. (a) 40 (b) top 0.2 mm 1 mm 1 mm left 0.05 mm 1 mm 1 mm bottom Turbidity The amount of microorganisms can be estimated based on cloudiness of the solution. This method is quick and easy. When using a colorimeter (as shown in Figure 2.1.4), the device is usually zeroed between each reading by placing an appropriate blank sample to reset the 100% transmission/0% absorption. In this case, the blank used would be uninoculated liquid medium. Colour filters are often used for greater accuracy. In this case, a green filter would be used. Figure 2.1.4: Using a colorimeter. Light source Cuvette (contains sample) Photoelectric cell Display (may give a digital reading) Portfolio activity (1.1) Plan and carry out a practical to find the optimum growth requirements of one of the following bacteria: Bacillus subtilis, Escherichia coli (E. coli) or Staphylococcus epidermidis and, using the three different methods of estimating bacterial growth: write up your plan carry out the investigation, collect and tabulate data write up your report evaluate the three different methods of obtaining data. 8

9 Pilot plant The microorganism is now cultured in a small-scale fermenter with a volume of up to 200 dm 3 to see if its growth requirements remain the same as when grown on a small scale. Link You will find out more about the fermentation process in Topic guide 2.2. Figure in Topic guide 2.2 shows an industrial fermenter. Scaling up Now the microorganism is grown in a large industrial fermenter, which can be thousands of litres in volume. Problems of large-scale production In large fermenters, as the surface area to volume ratio is small, heat generated from respiration of the microorganisms may not be dissipated. Temperature must be monitored and a cooling water jacket prevents temperature increases that could kill the microorganisms by denaturing enzymes and other proteins. Batch fermentation This is where the microorganisms are grown within a nutrient medium in a closed fermenter. During the exponential growth (log) phase nothing is added to or removed from the fermentation vessel, although waste gases are vented out. At the end of the growth cycle the product is separated from the mixture. Fed-batch process The fed-batch process is used if secondary metabolites, such as penicillin, are being produced. Substrate (nutrients) is added in low concentrations at the beginning and then continuously during the production process. This is to overcome the inhibitory effect of large amounts of nutrients, which the microorganism would metabolise first, on the production of secondary metabolites from other nutrients present in the culture medium. Continuous fermentation Key term Conidia (conidiospores): Reproductive spores of fungi, produced asexually. This involves an open system. Sterile nutrient solution is continuously added to the bioreactor and at the same time an equivalent amount of converted nutrient solution with microorganisms is removed. Steady state growth is maintained by adjusting the concentration of one substrate, such as oxygen concentration, salts, nitrogen compound or carbohydrate, which acts as a limiting factor. Turbidity is measured to monitor biomass (cell) concentration and the rate of addition of nutrient solution is adjusted to maintain maximum growth rate in order to obtain maximum yield in the smallest fermenter in the shortest time. Maintaining growth rate is important. For example, if Penicillium chrysogenum is grown at low growth rate it forms conidia (conidiospores) and does not make penicillin. Downstream processing This is how the product is obtained from the rest of the contents of the fermenter (bioreactor). It involves separating cells from the medium by centrifugation or filtering. The medium containing the product needs to be concentrated by removal of water however, if the product is a protein, heat cannot be used. 9

10 Water can be removed by osmosis, pressure, adsorbent columns or electrical fields. The product is then purified. Trophophase: Stage in growth cycle where the organism is increasing its numbers/growth phase. Idiophase: Stage in growth cycle where the organism is producing metabolites/product formation phase. Take it further In some microorganisms their product formation (idiophase) is distinct from their growth phase (trophophase). They cannot be grown in a continuous fermentation. Find out how they are grown. Activity Explain why: 1 sterile air is introduced into fermentation vessels 2 there are stirrers to mix the solution 3 the ph has to be monitored and adjusted throughout the fermentation process. Case study Omar Amin works in quality control for a pharmaceutical company producing antibiotics where batch and continuous fermentation processes are used. He is responsible for analysing samples from each fermentation and for making sure that staff always follow correct procedures. Some products in the plant are made using batch fermentation and some are made using continuous fermentation. Omar needs to understand when batch or continuous fermentation is most appropriate. He oversees trainees who carry out many of the screening investigations to find out the optimum growth requirements of microorganisms to be used. The advantages of batch fermenters are: if a culture does become contaminated only one batch of product has been lost, minimising cost of wastage it is easy to set up and to monitor the factors that limit growth of the microorganism vessels can be used for different products at different times. The advantages of continuous fermentation are: as the microorganisms are maintained in their log phase, smaller vessels are used the process is more productive. However, there are more problems with continuous fermentation: foaming is more likely (anti-foaming agents need to be added to the mix) microbial cells may clump or block the inlets it is more difficult to monitor all the parameters and, if the process goes wrong, there is considerable expense and loss. Why do you think it is more costly to a biotechnology company if production using continuous fermentation goes wrong, than if production using batch fermentation goes wrong? 10

11 Checklist In this topic you should now be familiar with the following ideas: all investigations and industrial processes involving growth of microorganisms must be carried out adhering to strict guidelines on aseptic technique for safety and to prevent contamination the growth requirements of microorganisms from which we need to obtain products must be understood small-scale investigations into the growth requirements of microorganisms may involve dilution plating and viable count, haemocytometer and direct count, or turbidity the growth cycle of microorganisms must be understood so that their growth can be maintained in the correct phase to obtain primary or secondary metabolites large-scale industrial production of microbiological products involves batch or continuous fermentation. Each has specific advantages or disadvantages but both have to be made to high specifications and be able to be steam sterilised. Further reading Annets, F. (2010) BTEC Level 3 National Applied Science Student Book, London: Pearson Education. Case, C. Funke, B. and Tortora, G. (2012) Microbiology: An Introduction (11th edition), London: Pearson. Kennedy, P., Hocking, S. and Sochacki, F. (2008) OCR AS Biology Student Book, Oxford: Heinemann. Kennedy, P., Hocking, S. and Sochacki, F. (2008) OCR A2 Biology Student Book, Oxford: Heinemann. Acknowledgements The publisher would like to thank the following for their kind permission to reproduce their photographs: Corbis: Photoquest Ltd / Science Photo Library 1 All other images Pearson Education We are grateful to the following for permission to reproduce copyright material: Pearson Education Ltd for figures on pages 7 and 8 from BTEC Level 3 National Applied Science Student Book by Frances Annets, Edexcel 2010, copyright Pearson Education Ltd; Cambridge University Press for the figure Haemocytometer slide, on page 8, from Microbiology and Biotechnology, 2nd edition by Pauline Lowrie and Susan Wells, Cambridge University Press, pp. 31, 32, copyright Cambridge University Press Reproduced with permission of Cambridge University Press and the authors. In some instances we have been unable to trace the owners of copyright material, and we would appreciate any information that would enable us to do so. 11