Questions on industrial biotechnology and fermentation

Question 1

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.

Accepted Answer

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 2

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.

Accepted Answer

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 3

Compare batch fermentation with continuous fermentation, referring to the closed-culture growth curve in your answer.

Accepted Answer

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

Question 4

A company cultures a bacterium that secretes a new antibiotic only once the culture stops dividing. It also sells the bacterial biomass itself as animal feed. Suggest which fermentation method suits each product, and suggest why.

Accepted Answer

B1 the antibiotic is a secondary metabolite, made only as growth slows and the culture enters the stationary phase B1 it must therefore be made in batch culture, because a continuous culture is held in the exponential phase and never experiences the conditions that switch production on, so it would grow bacteria and make almost no antibiotic B1 the biomass is the product of growth itself, a primary product that accumulates fastest while the culture is growing exponentially B1 it therefore suits continuous culture, with cells drawn off as fast as they are replaced, so the vessel stays at peak output instead of losing time to harvesting and cleaning

Question 5

A local authority proposes to replace part of the meat served in its school meals with mycoprotein. Evaluate this proposal.

Accepted Answer

B1 in favour: mycoprotein is produced in days in a small building all year round, on cheap substrates, and uses far less land and water per kilogram of protein than livestock, with no animal reared or slaughtered B1 in favour: the hyphae of Fusarium venenatum give a fibrous texture close enough to muscle to stand in for meat, and the product is high in protein and fibre, low in fat and free of cholesterol B1 against: the product has little taste of its own so flavour and colour must be manufactured into it, the cells are rich in nucleic acid whose breakdown product uric acid can cause gout so this must be reduced during processing, and some people are unwilling to eat a fungus grown on industrial glucose B1 a judgement supported by those points, for example that the nutritional and environmental case is strong provided the nucleic acid content is reduced and the food is labelled honestly, while accepting that the sterile, precisely controlled plant is expensive to build and a contaminated batch is a food-safety failure

Question 6

Describe how mycoprotein is produced from Fusarium venenatum, from culture to finished food.

Accepted Answer

B1 the fungus is grown in continuous culture on glucose syrup, with ammonia supplied as the nitrogen source B1 the fungal hyphae are harvested continuously as the culture grows B1 the harvested biomass is heat-treated, partly to reduce its nucleic acid content B1 it is then pressed into a textured food product, the fibrous hyphae giving it a texture close enough to muscle to stand in for meat

Question 7

Describe the four phases of the closed-culture growth curve that a batch fermentation passes through, in order.

Accepted Answer

B1 a lag phase, during which the population adjusts to the new medium and synthesises the enzymes it requires, so numbers barely rise B1 an exponential, or log, phase, during which nothing yet limits growth and the population doubles at a constant rate B1 a stationary phase, when a nutrient runs short or a waste product accumulates, so the rate of division comes to balance the rate of death and numbers level off B1 a death phase, when deaths exceed divisions and the population falls

Question 8

In a continuous fermenter, fresh medium flows in and culture flows out at the same rate, so the volume stays constant. The dilution rate is the flow rate of medium divided by the culture volume. A continuous fermenter has a working volume of 8000 dm³ and is run at a dilution rate of 0.15 per hour. Calculate the flow rate of fresh medium into the fermenter in dm³ per hour, and calculate how long it takes for a volume of medium equal to the whole vessel to pass through.

Accepted Answer

M1 rearranging dilution rate = flow rate ÷ volume gives flow rate = dilution rate × volume A1 0.15 × 8000 = 1200 dm³ per hour M1 time for one working volume to pass through = volume ÷ flow rate A1 8000 ÷ 1200 = 6.7 hours, which is about 6 hours 40 minutes

Question 9

A small start-up proposes cutting costs by skipping the steam sterilisation of its fermenter medium, relying only on filtering the incoming air. Evaluate this proposal.

Accepted Answer

B1 for: filtering the incoming air does remove airborne bacteria and spores from that one contamination route, so some protection against contamination would remain B1 against: the growth medium itself, and the vessel and pipework, are further routes by which contaminating organisms can enter, and skipping medium sterilisation leaves those routes open B1 against: a contaminating organism can compete for the substrate, out-grow or degrade the product, and in a food or medicine renders the whole batch unsellable, so the saving from skipping one step could easily be outweighed by the cost of losing an entire batch B1 judgement: the proposal is not defensible as it stands, since the saving is small and the failure mode is a complete batch loss, though validating a shortened rather than an eliminated sterilisation cycle could be worth investigating

Question 10

A yeast culture was diluted 1 in 10 and placed on a haemocytometer. Each square counted is 0.2 mm × 0.2 mm and the chamber is 0.1 mm deep. A total of 120 cells was counted in 5 squares. Calculate the number of cells per cm³ of the undiluted culture. (1 cm³ = 1000 mm³)

Accepted Answer

M1 the volume of one square is 0.2 × 0.2 × 0.1 = 0.004 mm³ M1 the mean is 120 ÷ 5 = 24 cells per square, so 24 ÷ 0.004 = 6000 cells per mm³ of the diluted sample M1 6000 × 1000 = 6.0 × 10⁶ cells per cm³ of the diluted sample A1 multiplying by the dilution factor of 10 gives 6.0 × 10⁷ cells per cm³

Question 11

In an investigation of the growth of bacteria in liquid culture, the absorbance of the broth was 0.06 at 1.5 hours and 0.48 at 3.5 hours, both in the log phase; the readings are invented for this question. Absorbance is proportional to the number of cells over this range. Calculate the exponential growth rate constant, k, in generations per hour, and the mean generation time in minutes.

Accepted Answer

M1 log₁₀ 0.48 − log₁₀ 0.06 = 0.903, or the population increased by a factor of 8 M1 0.903 ÷ 0.301 = 3.0 generations in 2 hours A1 k = 1.5 generations per hour B1 mean generation time = 1 ÷ 1.5 = 0.67 h = 40 minutes

Question 12

In streak plating, the loop is flamed and then cooled before each new area of the plate is streaked. Explain why the loop is flamed, why it is cooled, and why single colonies are expected only in the last areas streaked.

Accepted Answer

B1 flaming kills the cells left on the loop, so the only cells carried into the next area are those picked up by crossing the previous area B1 a hot loop would kill the cells it picks up, so it is cooled first B1 each area therefore receives fewer cells than the one before, so the culture is diluted across the plate B1 in the last areas cells are deposited far enough apart that each grows into a separate colony from a single cell, giving one species per colony

Question 13

A student tested ethanol extracts of plants for antibacterial activity on an agar plate spread with bacteria. Explain why the discs were left to dry before use, why a disc soaked in ethanol only was included, why the lid was held on with two short strips of tape rather than sealed all round, and why the plate was incubated well below body temperature.

Accepted Answer

B1 ethanol kills bacteria, so it must evaporate or a zone could be caused by the solvent rather than the plant B1 the ethanol-only disc is a negative control: no zone round it shows that any zone round an extract disc was caused by the plant extract B1 sealing the lid all round could make the plate anaerobic, favouring the growth of anaerobic bacteria, some of which are pathogens B1 a lower temperature reduces the chance of growing organisms that are adapted to the human body and could cause disease

Question 14

Explain why a fermenter must be run under aseptic conditions, and explain how those conditions are achieved at industrial scale.

Accepted Answer

B1 a contaminating organism competes for the substrate and may out-grow the production strain or destroy the product, and in a food or a medicine it renders the whole batch unsellable B1 the vessel is sterilised with pressurised steam between runs and the medium is sterilised before it enters B1 the incoming air is passed through filters fine enough to remove bacteria and spores, and every other inlet is treated as a contamination route and sterilised

Question 15

A fermenter is inoculated with 5.0 × 10⁴ bacteria of a strain whose doubling time under these conditions is 20 minutes. Calculate the number of bacteria present after 4 hours of exponential growth.

Accepted Answer

M1 the number of divisions is the time divided by the doubling time, so 240 ÷ 20 = 12 M1 the number present is the starting number multiplied by 2 raised to the number of divisions, so 5.0 × 10⁴ × 2¹² A1 2¹² is 4096, giving 2.0 × 10⁸ bacteria (2.048 × 10⁸ unrounded)

Question 16

Explain why microbial cells intended as human food have their nucleic acid content reduced during processing.

Accepted Answer

B1 microbial cells are proportionately much richer in nucleic acid than the cells of a plant or animal food source, because they grow rapidly and so contain many RNA-rich ribosomes B1 nucleic acid consumed in the diet is broken down, and one breakdown product is uric acid B1 a high intake of uric acid can cause gout, so the nucleic acid content of the harvested biomass must be lowered before it is sold as food

Question 17

Compare the generation time of a bacterium such as Escherichia coli with that of a fungus and with that of a farm animal such as a cow, and compare the consequence each has for industrial production.

Accepted Answer

B1 a bacterium such as Escherichia coli can double its numbers in about twenty minutes given warmth and nutrients, whereas a fungus doubles over hours and a cow doubles its numbers over years B1 industrial biotechnology exploits this difference by culturing microorganisms rather than farming plants or animals, since a much larger yield can be obtained from a given starting culture in a much shorter time B1 generation time is one of several distinct reasons for using microorganisms, working alongside low-cost substrates, conditions set inside the vessel and not by season, no animal-welfare objections and ease of genetic modification

Question 18

Explain why the temperature and pH probes in a fermenter are described as providing negative feedback.

Accepted Answer

B1 each probe continuously measures a condition, temperature or pH, so that a departure from the optimum set point is detected B1 the fermenter's control system responds by correcting the departure, for example running more coolant through the jacket if the temperature rises, or metering in alkali if the pH falls B1 the correction acts to return the condition towards its original set point rather than push it further away, so the control is negative feedback

Question 19

Mannitol salt agar contains 7.5% sodium chloride. Explain how it can be used, together with streak plating, to isolate staphylococci from a mixed culture of bacteria.

Accepted Answer

B1 it is a selective medium: the high salt concentration prevents most bacteria from growing, while staphylococci tolerate it B1 so after incubation the colonies on the plate are mostly staphylococci, the other species having been inhibited B1 a colony is then streaked onto a fresh plate so that cells are spread thinly enough to give single colonies, each grown from one cell and so containing one species

Question 20

A sample of a bacterial culture was diluted in five tenfold steps. 0.1 cm³ of the final dilution was spread on an agar plate, and 63 colonies grew. Calculate the number of living bacteria per cm³ of the original culture, giving your answer in standard form.

Accepted Answer

M1 the dilution factor is 10⁵ M1 63 ÷ 0.1 = 630 colony-forming units per cm³ of the final dilution A1 630 × 10⁵ = 6.3 × 10⁷ bacteria per cm³

Question 21

A student followed a closed bacterial culture for several days, measuring both its absorbance in a colorimeter and its viable count by dilution plating. During the death phase the viable count fell steeply while the absorbance stayed almost unchanged. Explain these results.

Accepted Answer

B1 dilution plating gives a viable count: only living cells grow into colonies, so the count falls as cells die B1 absorbance measures turbidity, which is caused by all the cells in suspension, living or dead, and by cell debris B1 dead cells that have not broken down still scatter light, so the absorbance stays high although fewer cells are alive; turbidity is a total measure, not a viable one

Question 22

In an investigation of the antimicrobial properties of plants, discs soaked in plant extracts were placed on a lawn of bacteria. The zone of inhibition round the garlic disc measured 18 mm and 20 mm across at right angles, and the zone round the mint disc measured 10 mm and 12 mm; the results are invented for this question. Calculate how many times greater the area of the garlic zone is than the area of the mint zone.

Accepted Answer

M1 mean diameters 19 mm and 11 mm, so radii 9.5 mm and 5.5 mm M1 areas π × 9.5² = 283.5 mm² and π × 5.5² = 95.0 mm² A1 283.5 ÷ 95.0 = 3.0 times; accept 2.98 to 3.0

Question 23

Apart from their rapid growth, state two reasons why industry grows microorganisms rather than farming a plant or an animal for the same product.

Accepted Answer

B1 they are cheap to feed, because many grow on waste from other industries such as whey, molasses or straw B1 production is independent of climate and season, because the environment is made inside the vessel; or there are no welfare objections; or the genome is small and easily altered, so a strain is quickly improved

Question 24

Name the fungus used to produce mycoprotein, and name the two substances supplied as its main carbon and nitrogen sources during culture.

Accepted Answer

B1 the fungus Fusarium venenatum B1 glucose syrup as the carbon source and ammonia as the nitrogen source

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