OCR A Level Biology A: Microorganisms in biotechnology and immobilised enzymes (H420)

This study guide covers learning outcomes 6.2.1(e) to 6.2.1(i) of the OCR A Level Biology A (H420) specification, Version 4.1 (April 2026), for first assessment in 2025. It teaches the use of microorganisms in biotechnology, culture and aseptic technique, the growth curve and N = N0 × 2^n, factors affecting growth, and immobilised enzymes. Use the revision notes for quick recall and the practice questions to test yourself. The course hub, the checklist and the free diagnostics show the wider course.

What this guide covers

Outcome	What you must be able to do
6.2.1(e)	Explain the use of microorganisms in biotechnological processes: why they are used, economic considerations, short life cycle, growth requirements
6.2.1(f)	Give advantages and disadvantages of using microorganisms to make food for humans, with bacterial and fungal sources
6.2.1(g)(i)	Culture microorganisms effectively using aseptic techniques, including serial dilutions and agar plates
6.2.1(g)(ii)	Explain why growing conditions are manipulated in batch and continuous fermentation to maximise yield
6.2.1(h)(i)	Describe the standard growth curve in a closed culture and use N = N0 × 2^n
6.2.1(h)(ii)	Plan and analyse practical investigations into factors affecting growth, using broth and serial dilutions
6.2.1(i)	Describe methods of enzyme immobilisation and evaluate the use of immobilised enzymes

Biotechnology is the industrial use of living organisms, or parts of them, to produce food, drugs or other products. You may need to apply these ideas to unfamiliar examples, so learn the reasons behind each fact.

6.2.1(e) Why use microorganisms?

Microorganisms include bacteria, yeasts and other fungi. They suit industrial processes for these reasons.

• Short life cycle. Many bacteria divide in under an hour in good conditions, so a large mass of cells is made quickly and a process can be repeated often.
• Simple growth requirements. They need a carbon source, a nitrogen source, minerals, water, a suitable temperature and pH, and oxygen if they are aerobic. Cheap carbohydrate, including waste from other industries, can be the substrate.
• Economic advantages. Fermenters can run all year, in any climate, on a small area of land. The product is often made in large quantity from cheap inputs.
• Fewer ethical objections. Using microorganisms avoids the welfare issues of using animals, and most people have no concerns about growing them.
• Genetic modification. Their genetic material is simple, so they can be engineered to make a product they would not normally make, such as human insulin.

Costs also include sterilising equipment, energy for heating, cooling and stirring, and separating the product. A product released into the medium is cheaper to collect than one kept inside the cells.

6.2.1(f) Microorganisms as food

Bacterial sources. Lactobacillus and Streptococcus thermophilus ferment lactose in milk to lactic acid. The falling pH denatures milk protein, which curdles to make yoghurt and cheese.

Fungal sources. Saccharomyces cerevisiae (yeast) makes carbon dioxide for bread and ethanol for beer and wine. Mycoprotein is made from the fungus Fusarium venenatum, grown in continuous culture on a carbohydrate source with a nitrogen source added. The mycelium is harvested, heat-treated to lower its RNA content and processed into meat substitutes.

Advantages	Disadvantages
High in protein and fibre, low in saturated fat, and no cholesterol	Some people find the texture or taste unappealing, so acceptance is low
Suitable for vegetarians; no animals are used	A few people have allergic reactions or digestive upset
Fast production from cheap or waste substrates	RNA content must be reduced; flavour and texture usually need to be added
Needs less land than farming animals	Sterile fermenters are expensive to build, heat and cool
Production does not depend on weather	Contamination by other microorganisms can spoil a whole culture

6.2.1(g)(i) Culturing and aseptic technique

Aseptic technique stops unwanted microorganisms entering the culture and stops your culture escaping. Contamination ruins results and can be a health risk.

1. Wipe the bench with disinfectant.
2. Sterilise glassware, agar and broth, usually in an autoclave at about 121 °C, before use.
3. Sterilise the inoculating loop in a hot flame until it glows red, then let it cool without touching anything.
4. Keep Petri dish lids on as much as possible. Lift the lid only as far as you need, and work close to a Bunsen flame so rising air carries contaminants away.
5. Briefly pass the neck of a bottle through the flame before and after pouring.
6. Seal the dish lid with tape in two or three places only. A fully sealed dish could allow anaerobic pathogens to grow.
7. Incubate the dish upside down, at a temperature that suits the organism, but below human body temperature in school work to reduce the chance of growing human pathogens.
8. Autoclave all used cultures before disposal.

A broth is a liquid nutrient medium. Growth makes it cloudy, and a colorimeter can measure the turbidity. An agar plate is a solid medium, so each living cell can grow into a visible colony that you can count.

Serial dilution and viable counts

A culture is often too dense to count. A serial dilution reduces it in equal steps. Transfer 1 cm³ of culture into 9 cm³ of sterile diluent: this is a tenfold dilution, 10⁻¹. Repeat from tube to tube to reach 10⁻², 10⁻³ and so on, using a fresh sterile pipette each time. Spread a known volume of a suitable dilution over an agar plate, incubate, then count colonies.

Worked example. A student spreads 0.1 cm³ from the 10⁻⁵ tube and counts 46 colonies.

• Colonies per cm³ of the 10⁻⁵ tube = 46 / 0.1 = 460.
• Multiply by the dilution factor, 10⁵: 460 × 10⁵ = 4.6 × 10⁷ viable cells per cm³ of the original culture.

Only living cells that can divide form colonies, so this is a viable count. Colony counts are most reliable when a plate carries a countable number of colonies (many sources use 30 to 300). Repeat the count on several plates and use the mean.

6.2.1(g)(ii) Batch and continuous fermentation

A fermenter is a large vessel where conditions are kept at their best for the organism or product.

Feature	Batch fermentation	Continuous fermentation
Nutrients	Added once at the start	Added steadily throughout
Product removal	Harvested at the end	Culture drawn off at the same rate as nutrient is added
Growth phase	Passes through the whole growth curve	Held in the exponential phase
Typical use	Secondary metabolites, such as penicillin	Primary products, such as mycoprotein
Contamination	One batch is lost, then the vessel is cleaned	Harder to keep sterile over a long run
Equipment	Simpler, but idle time between batches	Needs careful monitoring and control

Manipulating conditions to maximise yield:

• Temperature. Respiration releases heat, so cooling is needed to stay at the optimum for enzymes.
• pH. Probes detect changes, and acid or alkali is added to keep the optimum.
• Oxygen. Sterile air is bubbled in and stirred. Without oxygen, aerobic respiration, and so growth, would slow.
• Nutrients. A balanced supply of carbon and nitrogen prevents any one nutrient from limiting growth.
• Stirring. It spreads nutrients, oxygen and heat evenly.

6.2.1(h)(i) The growth curve

In a closed culture no nutrients are added and nothing is removed. A plot of the logarithm of cell number against time has four phases.

1. Lag. Cells adjust to the medium. They make enzymes and take up nutrients, so there is little increase in number.
2. Exponential (log). Nutrients are plentiful and cells divide at a constant rate. The number doubles each generation, so a log scale gives a straight line.
3. Stationary. Nutrients run low, toxic waste builds up and pH changes. The rate of division equals the rate of death, so the number is steady.
4. Death (decline). Deaths exceed divisions and the number falls.

Calculating numbers: N = N0 × 2^n

N is the number of organisms after n generations, and N0 is the starting number. This applies only to exponential growth in which each cell divides every generation.

Worked example 1. A culture starts with 40 cells and the generation time is 30 minutes. How many cells after 3 hours?

• n = 180 / 30 = 6 generations.
• N = 40 × 2⁶ = 40 × 64 = 2560 cells.

Worked example 2. A culture grows from 2.0 × 10³ to 5.0 × 10⁶ cells, with a 20 minute generation time. How long does it take?

• 2^n = 5.0 × 10⁶ / 2.0 × 10³ = 2500.
• n = log₂ 2500 = log 2500 / log 2 = 11.29 generations.
• Time = 11.29 × 20 = 226 minutes, about 3.8 hours.

6.2.1(h)(ii) Investigating factors affecting growth

You might test temperature, pH, nutrient concentration or antibiotic concentration. A reliable plan has these features.

• One independent variable, with at least five values.
• The same volume of broth, same starting inoculum and same incubation time in every tube or plate.
• Aseptic technique, a sterile control with no inoculum, and repeats for each value.
• A dependent variable such as colorimeter reading, viable count from serial dilution, or width of a clear zone.

Antibiotic sensitivity. Filter paper discs soaked in different antibiotics, or concentrations, are placed on a lawn of bacteria. The bacteria cannot grow near an effective antibiotic, so a clear zone of inhibition forms. Compare areas, not diameters, using area = πr².

Worked example. Zones of 12 mm and 18 mm diameter.

• Area for 12 mm: π × 6² = 113 mm².
• Area for 18 mm: π × 9² = 254 mm².
• The larger zone is 2.25 times the area (254 / 113), although the diameter is only 1.5 times larger.

Use non-pathogenic strains and do not open incubated plates.

6.2.1(i) Immobilised enzymes

An immobilised enzyme is held in or on an inert support, so it stays in place as substrate flows past.

Method	How it works	Points to note
Adsorption	Enzyme sticks by weak bonds to a surface, such as porous glass or resin	Simple, but the enzyme may wash off
Covalent bonding	Enzyme joined to a support by covalent bonds	Held firmly, but the active site may be distorted
Entrapment	Enzyme trapped in a gel, such as alginate beads	Substrate and product diffuse through the gel, which slows the reaction
Membrane separation	Enzyme kept behind a partially permeable membrane	Small substrate and product molecules pass through; enzyme cannot

Advantages:

• The enzyme is not mixed with the product, so purification costs are lower.
• It can be reused many times, saving money.
• It works in continuous flow reactors.
• It is often more stable to heat and pH changes because the molecule is held in shape.

Disadvantages:

• Equipment and immobilisation cost more at the start.
• Reaction rates may be lower, because substrate must diffuse to the enzyme.
• Some enzyme may be lost or its activity may fall with reuse.
• Contamination in a column is hard to remove.

Examples include glucose isomerase (glucose to fructose, a sweeter sugar), lactase (lactose to glucose and galactose for lactose-free milk), penicillin acylase (semi-synthetic penicillins), aminoacylase (pure L-amino acids) and glucoamylase (dextrins to glucose). You do not need to recall them, but you may be asked to apply the ideas to them.

Worked example (bead size). Spherical beads have surface area 4πr² and volume (4/3)πr³, so the ratio is 3/r.

• Diameter 2 mm, r = 1 mm: ratio = 3.0 mm⁻¹.
• Diameter 4 mm, r = 2 mm: ratio = 1.5 mm⁻¹.
• Smaller beads have twice the ratio, so substrate reaches more of the enzyme and the reaction is faster. They may pack tightly and slow flow in a column.

Common errors

• Saying cells in lag phase are dormant, when they are making enzymes.
• Explaining the stationary phase as “all nutrients used up” when the cause is usually a limiting factor plus toxic waste.
• Using N = N0 × 2^n for a culture that is in lag phase.
• Forgetting the dilution factor or the volume plated when calculating viable cell numbers.
• Saying immobilised enzymes “work faster” without qualifying that diffusion may slow them.

Official syllabus

OCR Advanced GCE in Biology A (H420), Version 4.1 (April 2026), for first assessment in 2025, published by OCR.