Biology › Gene regulation, genomics and biotechnology › Industrial biotechnology and fermentation
Industrial biotechnology and fermentation
Industrial biotechnology uses microorganisms because they grow quickly on fairly simple feedstocks and can be selected or modified to give a high yield. The lesson looks at microorganisms as human food, at the stirred-tank fermenter as a controlled environment, and at when to choose batch or continuous culture.
Key terms Every definition in gene regulation, genomics and biotechnology, lesson by lesson
Before this The closed-culture growth curve: lag, log, stationary, death · Enzyme inhibition and metabolic control – immobilised enzymes · Aseptic technique when culturing microorganisms · Factors affecting enzyme rate: temperature, pH and concentration
COMMON MISCONCEPTION
A fermenter is a big brewing vat: tip in the microorganisms and the sugar, seal the lid, and come back when it is done.
A fermenter monitors and controls asepsis, temperature, pH, aeration, mixing and nutrient supply. Cooling removes heat released by respiration, while sterile air and stirring maintain oxygen availability. Loss of control can reduce yield or contaminate the culture.
What you should be able to do
- Give the reasons industry grows microorganisms rather than farming a plant or an animal for the same product.
- Set out the arguments for and against using microorganisms as human food, with mycoprotein as the named example.
- Name the parts of a stirred-tank fermenter and explain why each condition inside it has to be controlled.
- Distinguish batch from continuous fermentation on the closed-culture growth curve, and state which part of the curve each uses.
- Match a product to the method that suits it, using primary and secondary metabolites.
- Describe broth, agar and selective media, and say what each is used for.
- Compare cell counts, dilution plating, mass and turbidity as measures of bacterial growth, distinguishing a total count from a viable count.
- Calculate an exponential growth rate constant and a mean generation time from two population sizes in the log phase.
- Follow the growth of bacteria in liquid culture by turbidity, and find the growth rate constant from the log phase.
- Isolate single species from a mixed culture by streak plating, working aseptically.
- Test plant extracts for antibacterial activity on a lawn of bacteria, working aseptically, and compare them by the area of the zone of inhibition.
Why industry cultures microorganisms
Microorganisms are used in industrial biotechnology because they grow rapidly, can be cultured at high density, use relatively simple feedstocks, and can be selected or genetically modified for a high product yield. Production in a fermenter allows temperature, pH, aeration, mixing, nutrients and contamination risk to be monitored and controlled. It is less dependent on season than agriculture, although energy, water, feedstock and infrastructure still affect cost and performance.
Generation time accounts for the rate. A cow doubles its numbers in years, a fungus in hours, and a bacterium such as E. coli, given warmth and nutrients, in about twenty minutes.
The remaining reasons are distinct from speed and from each other. Microorganisms use low-cost substrates. Many grow on by-products of other industries, such as whey from cheese-making, molasses from sugar refining, straw and paper pulp. These are cheaper than purpose-grown feedstocks, and some carry a disposal cost for the producer. Culture conditions are set inside the vessel, so production does not depend on season, though it does depend on local energy, water and infrastructure. The organisms raise no animal-welfare objections of the kind livestock farming does. And their genomes are small and readily altered. A strain can be improved by selection over a few weeks, or transformed with a human gene, which is how recombinant insulin is manufactured at scale.
- Biotechnology
- The use of living organisms, usually microorganisms, or of their components such as enzymes, in industrial processes that make useful products.
- Fermentation (industrial)
- The large-scale culture of microorganisms in a fermenter to make a product. The industrial word covers aerobic processes as well as the anaerobic pathway the respiration unit gave the same name.
The word 'fermentation' has two meanings. In respiration, fermentation is the anaerobic regeneration of NAD. In industry, a fermentation is any large-scale culture with a product at the end, and most of them, penicillin and mycoprotein included, are aerated. The industrial term identifies a vessel of microorganisms and does not indicate whether oxygen is involved.
The products are varied: yoghurt, cheese and bread from traditional fermentations, and ethanol for fuel as well as for drink. Enzymes include the proteases in washing powder and the lactase and glucose isomerase you met immobilised. Antibiotics and human proteins from transformed cells complete the list. Two of those categories, food and medicines, are treated in the sections below.
Microorganisms as human food
Most industrial biotechnology sells a product made by a microorganism. The microorganism itself is also sold, dried and flavoured, as protein-rich food, called single-cell protein whether or not the producer organism is single-celled.
The example on British shelves is mycoprotein: the hyphae of the fungus Fusarium venenatum. Grown continuously on glucose syrup with ammonia as the nitrogen source, it is harvested, heat-treated and pressed into a food sold since the 1980s. The fibrous hyphae matter as much as the protein. They give a texture close enough to muscle for the product to stand in for meat, which yeast or bacterial cells, being small and round, cannot do.
| For microorganisms as food | Against |
|---|---|
| Protein produced in days, in a small building, all year round | The product has little taste of its own, so flavour and colour must be manufactured into it |
| Grown on low-cost substrates, including by-products of other industries | Consumer acceptance is uneven, and accurate labelling of the organism and substrate can reduce sales |
| High in protein and fibre, low in fat, with no cholesterol | Microbial cells are rich in nucleic acid, which must be reduced during processing because its breakdown product, uric acid, can cause gout |
| No animal is reared or slaughtered, and far less land and water are used per kilogram of protein | The culture must be kept sterile and precisely controlled, so the plant is expensive to build and to run, and a contaminated batch is a food-safety failure rather than a nuisance |
- Single-cell protein
- Protein-rich food produced from cultured microorganisms, eaten by humans or fed to livestock.
- Mycoprotein
- Single-cell protein made from the hyphae of the fungus Fusarium venenatum, grown in continuous culture and textured to resemble meat.
Set the argument out on both sides, and attach each point to a mechanism. 'It is unnatural' names no process; 'the nucleic acid content must be lowered because its breakdown product uric acid can cause gout' does.
The stirred-tank fermenter: a controlled environment
A stirred-tank fermenter is a stainless steel vessel, from bench size up to hundreds of thousands of litres. It holds a large volume of nutrient medium at the optimum conditions for one organism's enzymes while excluding other organisms.
Describe the drawing
A stirred-tank fermenter drawn in vertical section as a tall vessel with a motor-driven shaft entering from the top and turning an impeller in the middle of the culture. A sparger near the bottom releases a row of air bubbles that rise through the liquid. Inlets on the left carry sterile nutrient medium, steam for sterilising the empty vessel, and filtered air; three probes for pH, temperature and oxygen sit in the wall above them. A water-cooled jacket wraps the lower part of the vessel with cold water entering at the bottom and warm water leaving higher up, and the product is drawn off through a tap on the right.
Asepsis first. The vessel is sterilised with pressurised steam between runs, the medium is sterilised before it enters, and the air is passed through filters fine enough to strip bacteria and spores. The reason is competitive: a contaminating organism consumes the substrate, may out-grow the production strain, may degrade the product, and in a food or medicine makes the batch unsellable in any case. Aseptic technique at industrial scale uses the same principles as laboratory practice, with steam sterilisation in place of a flame.
The conditions controlled are those affecting enzyme rate. A dense culture respires rapidly and respiration releases heat. An uncooled fermenter warms past its own optimum and the enzymes denature. Water circulating through the cooling jacket carries that heat away, holding the culture at its optimum temperature. Metabolism also shifts pH, since carbon dioxide and organic acids accumulate, so a pH probe monitors the broth and alkali or acid is metered in to hold the optimum.
Aerobic cultures consume oxygen faster than it dissolves. Sterile air is forced in through a sparger, a ring of fine holes near the bottom. The impeller above it breaks the rising bubbles up and keeps organisms, nutrients, heat and oxygen evenly mixed, so no part of the tank becomes a stagnant corner running anaerobic. Nutrients are added, the exhaust gas escapes through a filter, and probes report continuously. Each variable is corrected as it departs from its set point, which is negative feedback.
- Fermenter
- A vessel in which microorganisms are cultured at large scale under controlled conditions of temperature, pH, oxygen and nutrient supply.
- Aseptic conditions
- Conditions from which unwanted microorganisms are excluded, achieved in a fermenter by steam-sterilising the vessel and medium and filtering the incoming air.
Batch and continuous fermentation
Both methods start from the closed-culture growth curve in populations and their limits. That curve has a lag phase while the population adjusts and synthesises the enzymes its new medium requires. An exponential (log) phase of doubling follows while nothing limits growth. In the stationary phase nutrients run short and wastes accumulate, so deaths balance divisions. In the death phase deaths exceed divisions. The two ways of running a fermenter operate at different points on that curve.
Describe the drawing
The growth curve of a microorganism in closed culture, plotted as number of living cells against time and rising through a flat lag phase, a steep exponential phase and a level stationary phase before falling in the death phase. An amber window is drawn over part of the exponential phase to mark where continuous fermentation holds the culture by adding fresh medium and drawing off product at matching rates, and a coral arrow points late in the stationary phase to mark where a batch fermentation is stopped and harvested in one go.
Batch fermentation is a closed culture. The vessel is filled with sterile medium, inoculated, sealed, and left to run through the curve. The whole tank is harvested in the stationary phase (late in it for a product such as penicillin, which is made as growth slows), then emptied, sterilised and set up again. Nothing is added during the run except air and pH correction, so the culture visits every phase the growth curve has. Industrial penicillin is made by fed-batch culture: sugar and a precursor are fed in slowly to keep growth limited, and nothing is drawn off until the harvest.
Continuous fermentation is an open one. In a chemostat, fresh medium enters and culture leaves at the same rate. The dilution rate is that flow rate divided by the culture volume. A limiting nutrient and the dilution rate control growth, allowing a steady state to be maintained without passing through a complete batch growth curve. Nutrient supply and waste removal are balanced against consumption and production, and the population grows at a constant rate set by the dilution rate for weeks or months.
Which to choose turns on when the product is made, and the pair of terms that captures it is examined by name. A primary metabolite is made during normal growth, as part of it: ethanol, biomass itself, most enzymes. A secondary metabolite is made when growth slows, typically as the culture enters the stationary phase; penicillin is one, an antibiotic that inhibits the mould's competitors under crowded, nutrient-limited conditions. A product made during growth suits a continuous process that maintains growth; a product made when growth slows is produced in batch, because a continuous culture in steady state does not reach the stationary phase.
Describe the drawing
Two graphs stacked on one shared time axis. The upper graph is the closed-culture growth curve, the same trace the growth-modes figure draws, with an amber window over part of its exponential phase for continuous culture and a coral arrow late in the stationary phase for a batch harvest. The lower graph plots how much product has accumulated. The cyan trace, a primary metabolite, climbs steeply through the exponential phase and is level from the moment growth stops, because it is made as part of growing. The amber trace, a secondary metabolite such as penicillin, lies flat along the bottom for the whole of the growth phase and only begins to climb where the biomass curve above it has gone flat, so the window a continuous culture is held in contains none of it.
| Batch | Continuous | |
|---|---|---|
| The culture | Closed: nothing added after the start | Open: medium in and culture out at matching rates |
| Phase used | The whole curve, harvested at stationary | Held in the exponential phase |
| Harvest | Once per run, then clean out and restart | Continuous draw-off, for weeks at a time |
| If contamination gets in | One batch is lost | A long production run is lost |
| Rate of production | Lower: the vessel spends time in lag, harvest and cleaning | Higher: the vessel is always at peak output |
| Suits | Secondary metabolites such as penicillin | Biomass and primary metabolites, such as mycoprotein |
Choosing the method from the product
A company makes two products: penicillin, and mycoprotein for food. State, with reasons, which fermentation method suits each.
Show the working
Penicillin is a secondary metabolite: the mould makes very little of it while nutrients are plentiful and growth is fast, and switches to making it as the culture crowds and enters the stationary phase. A continuous culture in steady state does not reach that phase, so it would produce mould biomass and little antibiotic. Penicillin is therefore made in batch: the culture grows, becomes nutrient-limited, the drug is harvested, and the vessel is cleaned and restarted.
Mycoprotein is the opposite case, because the product is the organism. Biomass accumulates fastest while the culture is in exponential growth, so the process should hold it there for as long as engineering allows: continuous culture, with hyphae drawn off at the same rate the fungus replaces them, running for weeks between shutdowns.
The criterion is when the product is made. If it is made during growth, use continuous culture; if it is made as growth slows, use batch culture.
Broth, agar and selective media
A culture medium supplies what a microorganism needs to grow: water, a carbon and energy source such as glucose, a nitrogen source such as amino acids or ammonium salts, mineral ions and, for some organisms, vitamins. Pearson Edexcel Biology B names three kinds, and the stirred-tank fermenter above is the first of them on an industrial scale.
Broth is liquid medium, in a test tube, a flask or a fermenter. The organisms spread through all of it, so broth is used to grow large numbers of cells and to follow growth over time, by taking samples or by measuring how cloudy it becomes. Individual organisms cannot be separated from one another in it.
Agar medium is broth set into a gel with agar, a polysaccharide extracted from seaweed that few microorganisms can digest. It is sterilised in an autoclave, poured into Petri dishes while molten, and left to set. A single cell on the surface divides where it lies and gives a visible colony of millions of its descendants, so agar is the medium for separating organisms, by streak plating, and for counting them.
A selective medium allows some kinds of microorganism to grow while preventing the growth of others. It may contain a substance that inhibits most organisms but not the wanted one, such as an antibiotic, a high concentration of salt or bile salts. Or it may lack a nutrient, or offer only one carbon source, so that only organisms able to make or use it can grow. Mannitol salt agar contains 7.5 per cent sodium chloride, which staphylococci tolerate and most other bacteria do not. The antibiotic agar used to pick out transformed bacteria in recombinant DNA work is a selective medium too: only cells carrying the resistance gene survive on it.
Some media are also differential: an indicator shows which colonies have carried out a particular reaction, so different organisms on the same plate look different. MacConkey agar is both. Its bile salts and crystal violet inhibit most Gram-positive bacteria, and among the Gram-negative bacteria that grow, those that ferment lactose produce acid and form pink colonies. To isolate one species from a mixed culture, as core practical 13 requires, a selective medium narrows the field and streak plating the survivors separates them into single colonies, each grown from one cell.
A medium designed to pick out a pathogen also concentrates it, so media for isolating pathogens are not used in school laboratories. Follow the CLEAPSS guidance and your school's risk assessment for any culture work, and treat every culture as potentially pathogenic.
- Broth
- A liquid culture medium, used to grow large numbers of microorganisms and to follow their growth over time.
- Selective medium
- A culture medium containing a substance, or lacking a nutrient, so that only certain kinds of microorganism can grow on it.
- Colony
- A visible mass of microorganisms on a solid medium, all descended from a single cell or a small clump of cells.
Measuring the growth of a bacterial culture
A growth curve plots population against time, and the numbers on its vertical axis have to be measured somehow. Pearson Edexcel Biology B names four methods: cell counts, dilution plating, mass and optical methods. They differ in one important way. Some count every cell, living or dead, which is a total count; others count only the cells that are alive and able to divide, which is a viable count.
Cell counts. A drop of culture is placed on a haemocytometer. This is a thick microscope slide with a grid ruled on it and a chamber of known depth under the coverslip, so each square of the grid holds a known volume. The cells in a sample of squares are counted under the microscope, and the mean per square is scaled up to a number per cm³. A dead bacterium looks like a live one, so this is a total count. With yeast, methylene blue separates the two: living cells decolourise it and dead cells stay blue. The method is quick, but it needs a dense culture, and small bacteria are hard to see and count accurately.
Dilution plating. A culture holds far too many cells to count as colonies, so it is first diluted in a serial dilution. Put 1 cm³ of culture into 9 cm³ of sterile water and mix, then 1 cm³ of that into a further 9 cm³, and so on, each step diluting tenfold. A known volume from several of the dilutions, often 0.1 cm³, is spread over an agar plate and incubated. Each living cell gives one colony. So the colony count divided by the volume spread and multiplied by the dilution factor gives the number of living cells per cm³ of the original culture.
This is a viable count, since dead cells form no colonies. Plates with between about 30 and 300 colonies are used: fewer gives a large sampling error, and more crowd and merge. Two cells lying together give one colony, so the answer is strictly in colony-forming units. It takes a day or more of incubation to obtain.
Describe the drawing
A row of six tubes: the original culture and five tenfold dilutions labelled ten to the minus one through ten to the minus five, with arrows showing one cubic centimetre carried from each tube into nine cubic centimetres of sterile water in the next. From the last tube an arrow leads to an agar plate on which 0.1 cubic centimetres was spread and 32 colonies grew. The working beneath reads 32 divided by 0.1 is 320 per cubic centimetre of the last tube, and 320 times ten to the five is 3.2 times ten to the seven living cells per cubic centimetre of the culture.
Mass. The cells in a measured volume of culture are separated by filtering or centrifuging, washed, and dried in an oven until their mass stops changing. Dry mass measures biomass directly, which is the figure an industrial process making biomass needs, but it includes dead cells, needs a large sample, and destroys it. Wet mass is quicker to obtain but varies with how much water is left with the cells.
Optical methods (turbidity). Cells in suspension scatter light, so a culture becomes cloudier, or more turbid, as it grows. A colorimeter or spectrophotometer, zeroed with sterile broth, measures how much less light passes through a sample, read as absorbance. A reading takes seconds, so a culture can be sampled often, which suits following a growth curve in core practical 12. Absorbance does not count anything by itself: it must be calibrated against counts made by another method, it includes dead cells, and in dense cultures it stops rising in proportion to the number of cells, so samples are diluted first.
| Method | What it measures | Living cells only | Main drawback |
|---|---|---|---|
| Haemocytometer count | Number of cells per unit volume | No (yeast can be stained) | Needs a dense culture; small cells are hard to count |
| Dilution plating | Colony-forming units per unit volume | Yes | A day or more before the answer; clumps count as one |
| Dry mass | Biomass per unit volume | No | Needs a large sample, which is destroyed |
| Turbidity | Absorbance of the suspension | No | Must be calibrated; not proportional in dense cultures |
A viable count from a dilution series
A culture is diluted in six tenfold steps, and 0.1 cm³ of the final dilution is spread on an agar plate. After incubation the plate has 47 colonies. Calculate the number of living bacteria per cm³ of the original culture, and give one reason why the true number may be higher.
Show the working
Six tenfold steps give a dilution factor of 10⁶, so each cm³ of the final tube holds a millionth of the cells in a cm³ of the culture.
The plate received 0.1 cm³ and grew 47 colonies, so 1 cm³ of the final dilution held 47 ÷ 0.1 = 470 living cells.
The original culture held 470 × 10⁶ = 4.7 × 10⁸ living bacteria per cm³.
The true number may be higher because two or more cells lying together on the agar grow into a single colony, and because some living cells may fail to grow on that medium at that temperature. A count from one plate also carries a large sampling error, so replicate plates are spread and the mean used.
- Total count
- A count of all the cells in a sample, living and dead, as made with a haemocytometer.
- Viable count
- A count of the living cells in a sample, as made by dilution plating, where each living cell grows into one colony.
- Serial dilution
- A series of dilutions in which each step dilutes the previous one by the same factor, usually tenfold.
- Turbidity
- The cloudiness of a suspension, measured as absorbance in a colorimeter, which rises as a culture grows.
Calculating the exponential growth rate constant
In the log phase every cell divides at the same rate, so the population doubles at regular intervals. Over n generations a population N0 becomes N = N0 × 2n. Taking logarithms to base 10 turns this into log10 N = log10 N0 + n × log10 2, and log10 2 = 0.301. So the number of generations in a time interval can be found from two population sizes, and the number of generations per unit time is the exponential growth rate constant, k.
k = (log10 Nt − log10 N0) ÷ (0.301 × t)N0 and Nt are the population sizes at the start and end of a time t within the log phase; k is in generations per unit time, and dividing by 0.301 is the same as multiplying by 3.32
The mean generation time, the time the population takes to double, is 1 ÷ k. Both population sizes must come from the log phase: a point in the lag or stationary phase gives a rate that belongs to no phase. That is why growth data are plotted with log10 N on the vertical axis. On that scale the log phase is a straight line, the two points are taken from it, and the gradient of the line is 0.301k.
Describe the drawing
A graph of log to base ten of the number of bacteria per cubic centimetre against time in minutes from 0 to 360. The line is flat in a short lag phase, then rises as a straight line through the log phase, then levels off in the stationary phase. Two points on the straight section are marked, at 60 minutes with a log value of 4.301 and at 210 minutes with 5.806, with dashed lines showing a run of 150 minutes and a rise of 1.505. The working beside it reads 1.505 divided by 0.301 is 5 generations, and k is 5 divided by 2.5 hours, 2.0 generations per hour.
Some books use natural logarithms and write the constant as (ln Nt − ln N0) ÷ t. That gives the specific growth rate, which equals 0.693 × k. Use the form a question gives you; where none is given, use the base-10 form and state the unit.
Growth rate constant and mean generation time
A broth culture is sampled during its log phase. At 60 minutes it holds 2.0 × 10⁴ bacteria per cm³, and at 210 minutes 6.4 × 10⁵ per cm³; the figures are constructed for this example. Calculate the exponential growth rate constant in generations per hour, and the mean generation time.
Show the working
Take logarithms: log10 (6.4 × 10⁵) = 5.806 and log10 (2.0 × 10⁴) = 4.301. The difference is 1.505.
Divide by 0.301 to find the number of generations: 1.505 ÷ 0.301 = 5.0 generations. As a check, 2⁵ = 32, and 2.0 × 10⁴ × 32 = 6.4 × 10⁵.
The time is 210 − 60 = 150 minutes, which is 2.5 hours, so k = 5.0 ÷ 2.5 = 2.0 generations per hour.
The mean generation time is 1 ÷ k = 1 ÷ 2.0 = 0.5 hours, which is 30 minutes.
- Exponential growth rate constant
- The number of generations per unit time in the log phase: k = (log₁₀ N − log₁₀ N₀) ÷ 0.301t, where N₀ grows to N in time t.
- Mean generation time
- The time a population takes to double during the log phase, equal to 1 ÷ k.
Core practical 12: the rate of growth of bacteria in liquid culture
Edexcel B CP12
Pearson Edexcel Biology B (9BI0) sets Core Practical 12: Investigate the rate of growth of bacteria in liquid culture taking into account the safe and ethical use of organisms. It puts statements 6.1 iv and 6.1 v to work: a broth culture is followed over several hours by turbidity, and the growth rate constant is calculated from the log phase.
The apparatus: a culture of a bacterium that your school's risk assessment approves, from a recognised supplier. You also need sterile nutrient broth in a conical flask closed with a cotton wool plug or foil, and sterile pipettes or syringes. Use a colorimeter set at a wavelength the broth itself absorbs little, usually red light at about 600 nm, and cuvettes with lids. Add an incubator or water bath, ideally one that shakes, a Bunsen burner, and a beaker of disinfectant for used pipettes and cuvettes.
| Step | What to do | Why |
|---|---|---|
| 1 | Warm the flask of sterile broth to the incubation temperature, which is below body temperature and set by the school's risk assessment | Growth starts at the temperature being tested |
| 2 | Working beside a Bunsen flame, flame the neck of the flask, add a measured volume of the culture with a sterile pipette, flame the neck again and replace the plug | Aseptic technique keeps other microorganisms out of the broth and keeps the culture in |
| 3 | Swirl, then take a sample aseptically into a cuvette, close it, and read its absorbance against a blank of sterile broth. This is time zero | The blank sets the reading for broth with no cells in it to zero |
| 4 | Incubate, swirling or shaking the flask, and take a sample the same way every 30 to 60 minutes for six to eight hours | Shaking keeps the cells suspended and the broth aerated |
| 5 | When the absorbance becomes high, dilute each sample with sterile broth by a known factor before reading it, and multiply the reading by that factor | In a dense culture absorbance stops rising in proportion to the number of cells |
| 6 | Incubate a flask of broth that was never inoculated alongside, and read it at the start and the end | If it stays clear, the broth and the technique were sterile |
To investigate a factor, such as temperature within the range the risk assessment allows or the glucose concentration of the broth, make that the independent variable. The dependent variable is the absorbance, standing in for the number of cells. Control the volume and age of the culture added, the volume and recipe of the broth, the rate of shaking, and the temperature.
Plot absorbance against time to see the lag, log and stationary phases, and log10 absorbance against time to find the log phase as a straight line. While absorbance is proportional to the number of cells, the ratio of two absorbances equals the ratio of the two populations, so the growth rate constant can be calculated from absorbances directly, using the equation in the section above.
A growth rate from absorbance readings
These readings are invented to show the processing, and are already corrected for any dilution. The absorbance of a broth culture was 0.040 at 0 h, 0.042 at 1 h, 0.050 at 2 h, 0.141 at 3 h, 0.400 at 4 h, 0.620 at 5 h, 0.660 at 6 h and 0.670 at 7 h. Identify the phases, and calculate the growth rate constant and the mean generation time from the log phase.
Show the working
From 0 to 2 h the absorbance barely changes: the lag phase. From 2 to 4 h it rises by a factor of about 2.8 each hour, which is the log phase. After 5 h it levels off: the stationary phase.
Using 2 h and 4 h: log10 0.400 − log10 0.050 = −0.398 − (−1.301) = 0.903.
Number of generations = 0.903 ÷ 0.301 = 3.0 in 2 hours, so k = 1.5 generations per hour.
Mean generation time = 1 ÷ 1.5 = 0.67 h, which is 40 minutes.
| Source of error | Effect | Improvement |
|---|---|---|
| Contamination by other microorganisms | Growth that is not the culture's own | Strict aseptic technique; the uninoculated flask shows whether it happened |
| Cells settle between samples | A reading too low or too high | Swirl the flask before every sample |
| Absorbance includes dead cells | The death phase does not show | Make viable counts by dilution plating at some of the same times |
| Readings in a dense culture | Absorbance underestimates the number of cells | Dilute samples by a known factor |
| The flask cools while samples are taken | Growth slows | Take samples quickly and return the flask at once |
Safe and ethical use. A broth culture holds very large numbers of bacteria, so spills and splashes are the main risk. Treat every culture as a possible pathogen, use only the organism the school approves, and never culture organisms from yourself or your surroundings. Incubate below body temperature so that organisms suited to the human body are not favoured. Use a pipette filler, never your mouth. Keep cuvettes closed, and put used pipettes and cuvettes straight into disinfectant. Cover a spill with disinfectant and tell the teacher. Every culture is killed by autoclaving or pressure-cooking before disposal, so that nothing grown in the laboratory is released. Check the CLEAPSS guidance and your school's risk assessment before starting.
- Inoculum
- The small measured volume of culture added to a sterile medium to start a new culture.
Core practical 13: isolating individual species by streak plating
Edexcel B CP13
Pearson Edexcel Biology B (9BI0) sets Core Practical 13: Isolate individual species from a mixed culture of bacteria using streak plating taking into account the safe and ethical use of organisms. It is printed in section 6.1, Microbial techniques, and uses the media described above.
A loopful of culture holds millions of cells. Streak plating spreads them thinner and thinner across the surface of an agar plate, in a series of areas, until in the last areas single cells are left well apart. Each grows into a separate colony, all descended from that one cell, so each well-separated colony is a single species. Moved to a fresh plate it gives a pure culture.
The apparatus: a mixed broth culture of two species whose colonies look different, prepared by the technician from cultures the school's risk assessment approves. You also need sterile plates of nutrient agar, or of a selective medium. Add a wire inoculating loop, a Bunsen burner, adhesive tape, a marker pen, an incubator and disinfectant.
| Step | What to do | Why |
|---|---|---|
| 1 | Disinfect the bench and wash your hands. Label the base of the plate round its edge with your name, the date and the culture | The base is labelled because lids can be swapped; the edge keeps the writing off the colonies |
| 2 | Heat the loop in the Bunsen flame until it glows red along its length, then hold it near the flame for a few seconds to cool | Heat kills anything on the loop; a hot loop would kill the cells it picks up |
| 3 | Remove the cap of the culture bottle, flame the neck, take a loopful, flame the neck again and replace the cap | Flaming the neck stops microorganisms from the air falling in |
| 4 | Lift the lid of the plate only far enough to get the loop in, and streak backwards and forwards over a small area at one edge, lightly so as not to cut the agar | This is area 1, which holds most of the cells |
| 5 | Flame and cool the loop, turn the plate a quarter turn, drag the loop once through area 1 and streak area 2 without going back into area 1 | Only the few cells picked up from area 1 reach area 2 |
| 6 | Repeat to streak area 3 from area 2, and area 4 from area 3, flaming the loop between each | Each area receives fewer cells than the one before |
| 7 | Flame the loop. Tape the lid with two or four short strips, invert the plate, and incubate it below body temperature for two to three days, as the risk assessment sets | Not sealing all round keeps the plate aerobic; inverting stops condensation dripping on the colonies |
| 8 | Examine the plate through the closed lid: describe the colonies in each area by colour, size, shape, edge and surface | Different species usually form different-looking colonies |
A single, well-separated colony is the starting point for a pure culture: it is picked with a sterile loop and streaked onto a fresh plate, which should then grow colonies that all look alike. Whether a grown plate may be opened for this depends on the organisms and the level of work your school's risk assessment allows. Where it does, the teacher sets the conditions; where it does not, the plate stays taped shut.
The streak plate is not a quantitative investigation. Its control is a sterile plate, taped and incubated alongside without being inoculated, which should grow nothing; growth on it means the agar or the technique was contaminated.
Reading a streak plate
This description is invented to show how a plate is read. Area 1 is covered by continuous growth. Area 2 has dense, overlapping colonies. Area 3 has 46 colonies, many touching. Area 4 has 11 separate colonies: 8 small, cream, round with a smooth edge, and 3 larger, yellow, round and glossy. Decide which colonies to use, and say what the counts in area 4 do and do not show.
Show the working
Use area 4. Its colonies do not touch, so each grew from a single cell or a small clump of one kind and contains one species. Colonies in areas 1 to 3 may have merged with their neighbours.
There are two colony types, consistent with the two species in the mixed culture. One cream and one yellow colony would be picked to subculture.
8 of the 11 colonies in area 4, 73%, are cream, but this does not give the proportion of the two species in the original culture. The number of cells carried into each area is not measured, and the two species may grow at different rates. Counting needs dilution plating.
| Source of error | Effect | Improvement |
|---|---|---|
| The loop is still hot when it touches the culture | Cells are killed and little grows | Cool it near the flame, or touch it on the agar at the edge first |
| The loop is not flamed between areas, or goes back into an earlier area | Too many cells are carried on, so no single colonies form | Flame between areas and cross the previous area only once |
| The agar surface is cut | Growth along the groove, not separate colonies | Hold the loop flat and press lightly |
| Microorganisms from the air | Colonies that are not from the culture | Keep the lid open as little as possible, work near the flame, and include the sterile control plate |
| Two cells land together | A colony contains two species | Subculture a single colony and streak it again |
Safe and ethical use. Work only with the cultures the school approves and never with organisms from yourself or the surroundings. A loop carrying culture can spit when it goes straight into a hot flame, so heat it in the cooler part of the flame first and then in the hottest part. Tie long hair back, and keep ethanol away from the Bunsen burner. Once incubated, plates are not opened unless the risk assessment allows it. Used plates and cultures are killed by autoclaving or pressure-cooking before disposal, so that nothing grown is released. Disinfect the bench afterwards, wash your hands, and check the CLEAPSS guidance and your school's risk assessment before starting.
- Streak plate
- An agar plate on which a culture has been spread in a series of areas, each with fewer cells than the last, so that single colonies grow in the final areas.
- Pure culture
- A culture containing only one species, grown from a single isolated colony.
Core practical 9: investigate the antimicrobial properties of plants, including aseptic techniques for the safe handling of bacteria
Edexcel A CP9
Pearson Edexcel Biology A (9BN0) sets Core Practical 9: Investigate the antimicrobial properties of plants, including aseptic techniques for the safe handling of bacteria, beside statement 4.14. An extract of each plant is soaked into paper discs, which are laid on a lawn of bacteria. A clear zone of inhibition round a disc shows that something from it diffused into the agar and stopped growth. Aseptic technique is as for streak plating above.
The apparatus: fresh plant material such as garlic, ginger or mint, a pestle and mortar, filter paper and ethanol. You also need sterile paper discs of one size and a broth culture of a bacterium that your school's risk assessment approves, from a recognised supplier. Add sterile nutrient agar plates, a sterile pipette and spreader, sterile forceps and a prepared antibiotic disc. A Bunsen burner, tape, a ruler, an incubator and disinfectant complete the list.
| Step | What to do | Why |
|---|---|---|
| 1 | Crush the same mass of each plant with the same volume of ethanol for the same time, and filter off the liquid | Only the plant differs between extracts |
| 2 | Soak discs in each extract, and some in ethanol alone. Dry them well away from any flame until the ethanol has evaporated | Ethanol is flammable and kills bacteria itself |
| 3 | Disinfect the bench, wash your hands and label the base of each plate. Working beside a lit Bunsen burner, or as your school's practice directs, flame the neck of the culture bottle, take the volume the method gives with a sterile pipette, and flame the neck again. Lift the lid only far enough to add the culture, and spread it over the whole surface | The flame is usually said to keep airborne microorganisms away from the open plate |
| 4 | With sterile forceps, place one disc of each extract, an ethanol-only disc and the antibiotic disc well apart, and press each down gently | Spaced discs give zones that do not merge |
| 5 | Tape the lid with two or four short strips, never all the way round. Invert and incubate for two to three days at the temperature the school's risk assessment sets, well below body temperature | Sealing all round can make the plate anaerobic; a low temperature avoids favouring organisms that grow in people |
| 6 | Without opening the plate, measure each clear zone across its disc, twice at right angles | Zones are rarely perfect circles |
The independent variable is the plant. The dependent variable is the area of the zone of inhibition. Control the mass, part and freshness of the plant material, the volume of ethanol, the disc size, the volume of culture spread, the agar depth, and the incubation temperature and time. The ethanol-only disc is the negative control: a zone round it would mean the solvent stopped growth. The antibiotic disc is the positive control: its zone shows that a zone could form. Use at least three plates. Halve each mean diameter for the radius r, and calculate the area as πr². Where bacteria grow up to a disc's edge, record no zone.
Zones round three plant extracts
These results are invented. On three plates the mean zone diameters were 16, 18 and 17 mm for garlic, 11, 13 and 12 mm for ginger, and 8, 10 and 9 mm for mint. The ethanol-only disc had no zone on any plate. Calculate the mean area for each extract, and compare garlic with mint.
Show the working
Mean diameters are 17.0, 12.0 and 9.0 mm, so the radii are 8.5, 6.0 and 4.5 mm. Areas: garlic π × 8.5² = 227 mm²; ginger π × 6.0² = 113 mm²; mint π × 4.5² = 63.6 mm².
Garlic's zone has 227 ÷ 63.6 = 3.6 times the area of mint's, though less than twice the diameter, because area goes as the square of the radius.
The plate does not show which plant has the most potent compound. Zone size also depends on how much compound the disc carried and how fast it diffuses through agar, and a crude extract has an unknown concentration.
| Source of error | Effect | Improvement |
|---|---|---|
| Ethanol not fully evaporated | A zone caused partly by the solvent | Dry discs for a set time; the ethanol-only disc reveals it |
| Discs take up different volumes of extract | Zones vary between discs of one extract | Pipette the same small volume onto each disc |
| Microorganisms from the air or hands | Foreign colonies | Open the lid as little as possible; incubate an uninoculated plate alongside |
Risk assessment. Treat every culture as a possible pathogen, use only the culture your school's risk assessment approves, and never culture organisms from yourself or your surroundings. Ethanol is highly flammable: make the extracts and dry the discs with the Bunsen burner off or well away. Plant extracts may irritate skin or eyes, and some people are allergic to plants or antibiotics: wear eye protection, handle discs with forceps, and declare any allergy beforehand. Once incubated, the plate stays taped and is examined through the lid, never opened. Every plate and culture is killed by autoclaving or pressure-cooking before disposal. Wash your hands, and check the CLEAPSS guidance and your school's risk assessment before starting; where they differ from this page, follow them.
- Zone of inhibition
- The clear area round a disc on a lawn of bacteria, where a substance diffusing from the disc has stopped the bacteria growing.
- Bacterial lawn
- An even layer of bacteria grown over the whole surface of an agar plate from a spread liquid culture.
In the exam
- Give distinct reasons for using microorganisms: fast growth, low-cost or by-product substrates, conditions set inside the vessel rather than by season, no animal-welfare issues, and ease of genetic modification.
- Attach a reason to every fermenter feature: the jacket removes the heat respiration releases, the impeller distributes oxygen and nutrients, the sparger supplies oxygen for aerobic respiration, and asepsis excludes competing organisms.
- Give the reason temperature and pH are controlled: each enzyme has an optimum, and departures from it reduce the rate or denature the enzyme.
- Answer batch against continuous on the growth curve: batch harvests at the stationary phase, continuous holds the culture in the exponential phase at a rate set by the dilution rate. Name the phases.
- Penicillin is made in batch because it is a secondary metabolite; mycoprotein is made continuously because the product is the growing biomass.
- In an evaluation of microorganisms as food, argue both ways and give a mechanism for each point: nucleic acid content must be lowered because its breakdown product uric acid can cause gout.
- Say whether a method counts living cells only. Dilution plating is a viable count; a haemocytometer, dry mass and turbidity include the dead.
- In a dilution plating calculation, divide the colony count by the volume spread before multiplying by the dilution factor, and give the answer per cm³.
- Take both population sizes for a growth rate constant from the log phase, and give k with its unit, such as generations per hour.
- In a streak plating answer, say that the loop is flamed between areas so that each area receives fewer cells, and that single colonies form in the last areas.
- For a zone of inhibition, halve the mean diameter before using πr², and name the solvent-only disc as the control showing that the plant, not the solvent, stopped growth.
Check yourself
A manager proposes converting the company's penicillin plant from batch to continuous fermentation, arguing that continuous culture produces more product per litre per day and wastes no time on emptying and cleaning. Evaluate the proposal, and state what would happen to penicillin yield if it went ahead.
Answer
The manager's general premise holds but does not apply to this product. Continuous culture does keep a vessel at high output with no downtime, and for that reason mycoprotein is made that way.
But that output is biomass. Penicillin is a secondary metabolite: the mould synthesises it in quantity only as growth slows, when the culture is crowded and nutrients are running short, at the approach to the stationary phase. A continuous fermenter is operated so that the culture does not reach those conditions: medium is supplied and culture removed at matching rates, and the population is held in exponential growth at the dilution rate.
The converted plant would therefore produce a high yield of mould biomass and very little penicillin.
The recommendation is to reject the proposal for penicillin while retaining the principle behind it. Continuous methods suit products made during growth; batch methods suit products made as growth slows. The criterion is when the product is made.
Questions
Question 15 marksEdexcel B
The table shows the number of bacteria in a broth culture over six hours; the figures are constructed for this question. Use the values at 2 h and 4 h to calculate the exponential growth rate constant, k, in generations per hour, and the mean generation time in minutes. Explain why the values at 0 h and 6 h would not be suitable for this calculation.
| Time / h | Bacteria per cm³ |
|---|---|
| 0 | 1.0 × 10⁵ |
| 1 | 1.2 × 10⁵ |
| 2 | 4.8 × 10⁵ |
| 3 | 1.9 × 10⁶ |
| 4 | 7.7 × 10⁶ |
| 5 | 1.5 × 10⁷ |
| 6 | 1.6 × 10⁷ |
Mark scheme
- M1 log10 (7.7 × 106) − log10 (4.8 × 105) = 6.886 − 5.681 = 1.205
- M1 1.205 ÷ 0.301 = 4.0 generations in 2 hours
- A1 k = 4.0 ÷ 2 = 2.0 generations per hour
- B1 mean generation time = 1 ÷ k = 0.5 h = 30 minutes
- B1 0 h lies in the lag phase and 6 h in the stationary phase, where the population is not doubling at a steady rate, so k must be found from two points within the log phase
Question 24 marksOCR A
Describe how the conditions inside a stirred-tank fermenter are held at the optimum for the production organism, naming the part of the fermenter responsible in each case.
Mark scheme
- B1 a dense culture respires hard and respiration releases heat, so water circulating through the cooling jacket carries that heat away and holds the optimum temperature
- B1 metabolism produces carbon dioxide and organic acids, so a pH probe monitors the broth and acid or alkali is metered in to hold the optimum pH
- B1 an aerobic culture uses oxygen faster than it dissolves, so sterile air is forced in through the sparger near the bottom of the vessel
- B1 the impeller breaks up the rising bubbles and keeps organisms, nutrients, heat and oxygen evenly mixed, so no part of the tank becomes a stagnant corner running anaerobic
Question 34 marksOCR A
Compare batch fermentation with continuous fermentation, referring to the closed-culture growth curve in your answer.
Mark scheme
- B1 batch culture is closed, with nothing added after inoculation, whereas continuous culture is open, with sterile medium flowing in and culture flowing out at matching rates
- B1 a batch culture passes through the whole curve and is harvested once, in the stationary phase, whereas a continuous culture is held permanently in the exponential phase and never reaches stationary, because fresh medium replaces the limiting nutrient and removes wastes as fast as they are made
- B1 batch production is slower overall, because the vessel spends time in lag phase, harvesting and cleaning, whereas a continuous vessel is always at peak output and runs for weeks
- B1 contamination costs a batch process one run, whereas it costs a continuous process a long production run
All 24 questions with mark schemes, the other 21 set out in full on the question page.
Worth remembering
- Industry cultures microorganisms because they grow fast on low-cost substrates under conditions set inside the vessel, without animal-welfare objections, and are readily modified.
- Mycoprotein: fungal hyphae grown continuously, protein-rich and meat-textured. Argue the food case on both sides, with mechanisms.
- A fermenter controls temperature, pH, oxygen and nutrients at an organism's optima, under aseptic conditions, with probes feeding back to each control.
- Batch culture runs through the whole growth curve and harvests at the stationary phase; continuous culture holds the exponential phase by matching inflow to draw-off, with growth set by the dilution rate and a limiting nutrient.
- Primary metabolites are made during growth and suit continuous culture; secondary metabolites, including penicillin, are made as growth slows and are produced in batch.
- Broth grows cells in bulk, agar separates and counts them, and a selective medium lets only some kinds grow.
- Dilution plating gives a viable count; haemocytometer counts, dry mass and turbidity include dead cells.
- k = (log₁₀ N − log₁₀ N₀) ÷ 0.301t, both values from the log phase; the mean generation time is 1 ÷ k.
CHECK YOUR PROGRESS
Rate how confident you are with each objective for this lesson. Ratings are saved in this browser, on this device, unless you sign in.
- Give the reasons industry grows microorganisms rather than farming a plant or an animal for the same product.
- Set out the arguments for and against using microorganisms as human food, with mycoprotein as the named example.
- Name the parts of a stirred-tank fermenter and explain why each condition inside it has to be controlled.
- Distinguish batch from continuous fermentation on the closed-culture growth curve, and state which part of the curve each uses.
- Match a product to the method that suits it, using primary and secondary metabolites.
- Describe broth, agar and selective media, and say what each is used for.
- Compare cell counts, dilution plating, mass and turbidity as measures of bacterial growth, distinguishing a total count from a viable count.
- Calculate an exponential growth rate constant and a mean generation time from two population sizes in the log phase.
- Follow the growth of bacteria in liquid culture by turbidity, and find the growth rate constant from the log phase.
- Isolate single species from a mixed culture by streak plating, working aseptically.
- Test plant extracts for antibacterial activity on a lawn of bacteria, working aseptically, and compare them by the area of the zone of inhibition.
Open the full revision checklist to see every objective in the curriculum in one place.
Practise this lesson
WORKBOOK
The written questions from this topic, on paper with room to work, and a separate book of mark schemes. The question that is set on a diagram stays on this page, where the diagram can be drawn. Free to use; please do not redistribute or sell.