What you'll learn
Microorganisms and biotechnology covers the industrial and commercial exploitation of microbial metabolism, from brewing and baking to antibiotic production, sewage treatment and single-cell protein. Microorganisms are useful because they grow rapidly, can be cultured on inexpensive substrates including waste materials, are readily genetically modified, and carry out reactions that would otherwise require high temperatures and pressures. At CAPE level you must also know how populations grow, how fermenters are designed and controlled, and how aseptic technique is maintained. By the end of this topic you should be able to describe the bacterial growth curve and its phases, explain fermenter design and the reasons for each feature, distinguish batch from continuous culture, describe the major applications, and explain aseptic technique.
Key terms and definitions
Biotechnology — the use of living organisms or their products in industrial processes
Fermenter — a vessel in which microorganisms are cultured under controlled conditions
Batch culture — culture in which a fixed quantity of nutrient is supplied and the process runs to completion
Continuous culture — culture in which nutrient is added and product removed continuously
Primary metabolite — a substance produced during normal growth, in proportion to the population
Secondary metabolite — a substance produced after the growth phase, not required for growth
Lag phase — the initial period when cells are adjusting and little division occurs
Log or exponential phase — the period of maximum growth rate when the population doubles at regular intervals
Stationary phase — the period when the rate of cell division equals the rate of death
Death or decline phase — the period when death exceeds division and the population falls
Aseptic technique — procedures preventing contamination of a culture or the environment
Single-cell protein — protein obtained from the biomass of microorganisms, used as food
Immobilised enzyme — an enzyme attached to or trapped within an inert support
Core concepts
Why microorganisms are used
Microorganisms have a short generation time, some dividing every twenty minutes, so large quantities of product accumulate quickly.
They can be grown on inexpensive substrates, often industrial or agricultural waste such as molasses or whey, which converts a disposal problem into a resource.
They grow at moderate temperatures and atmospheric pressure, so the energy costs are far lower than for equivalent chemical synthesis.
They are easily genetically modified, so they can be engineered to produce substances they would not normally make, such as human insulin.
They are not affected by climate or season, so production is continuous and predictable, and the process occupies little land.
The products are often produced in a pure form, simplifying extraction.
The bacterial growth curve
A population of bacteria cultured in a closed system shows four phases, and each must be explained rather than merely named.
In the lag phase the population size changes little. The cells are adjusting to the new conditions, synthesising the enzymes needed to metabolise the available substrate, and taking up water and nutrients. They are metabolically active but not yet dividing rapidly.
In the log or exponential phase the population doubles at regular intervals, giving a straight line when plotted on a logarithmic scale. Nutrients are plentiful, waste has not accumulated, and there is little competition, so the growth rate is at its maximum.
In the stationary phase the population size remains constant, because the rate of cell division equals the rate of death. This occurs because nutrients have become limiting, toxic waste products have accumulated, oxygen may be depleted, and space is restricted. Many secondary metabolites, including antibiotics, are produced during this phase.
In the death or decline phase the death rate exceeds the division rate and the population falls, as nutrients are exhausted and toxic products reach inhibitory concentrations.
Calculating the number of bacteria after a given time is a standard exercise: with a generation time of t, the number of divisions in a period is that period divided by t, and the final population is the initial population multiplied by 2 to the power of the number of divisions.
Primary and secondary metabolites
A primary metabolite is produced during normal growth and its production is proportional to the increase in population. Ethanol from yeast and amino acids are examples, and they are produced during the log phase.
A secondary metabolite is not required for growth and is produced after the growth phase has ended. Penicillin is the standard example: it is produced by the fungus Penicillium during the stationary phase, when growth has slowed.
This distinction determines the choice of culture method. Since secondary metabolites are produced in the stationary phase, a process making them must allow the culture to reach that phase, which batch culture does. A primary metabolite can be harvested continuously while growth continues.
Fermenter design
A fermenter is a sealed vessel providing optimum controlled conditions, and each design feature answers a specific requirement. Questions very often ask for the reason rather than the feature, so the pairs should be learned together.
A stirrer or paddle keeps the microorganisms in suspension so that all cells have equal access to nutrients, distributes heat evenly, and maintains contact between cells and oxygen.
An air inlet with a sparger supplies sterile air, providing oxygen for aerobic respiration, and the fine bubbles maximise the surface area for dissolving.
A water jacket surrounds the vessel. Microbial respiration is exothermic and generates considerable heat, which would denature enzymes if unchecked, so cold water circulating in the jacket removes excess heat and maintains the optimum temperature.
Probes monitor temperature, pH and dissolved oxygen continuously, and a computer adjusts conditions automatically to keep each at the optimum.
An acid or alkali inlet allows pH to be corrected, since metabolic products such as organic acids would otherwise change the pH and denature enzymes.
A nutrient inlet supplies the carbon and nitrogen sources and any required minerals and growth factors.
The vessel is made of stainless steel, which can be sterilised with superheated steam, does not corrode and is easy to clean.
An outlet allows the product to be removed, after which downstream processing separates and purifies it.
Batch and continuous culture
In batch culture, a fixed quantity of nutrient is added at the start and nothing further is supplied. The culture passes through all four growth phases, the product is harvested at the end, and the fermenter is then emptied, cleaned and sterilised before the next run.
Its advantages are that it is well suited to producing secondary metabolites, since the culture reaches the stationary phase; contamination affects only one batch; and it is easier to manage. Its disadvantages are that the fermenter stands idle between runs, so it is less efficient, and the growth rate is not constant.
In continuous culture, nutrient is added and culture medium containing product is removed continuously, so the population is maintained in the log phase indefinitely.
Its advantages are a constant high growth rate, continuous product formation and more efficient use of the vessel. Its disadvantages are that it is suitable mainly for primary metabolites, that maintaining steady conditions is technically demanding, and that contamination affects the whole continuous process rather than a single batch.
Aseptic technique
Contamination must be prevented for two reasons: a contaminating organism competes with the culture and reduces yield, and it may produce toxic or harmful substances.
In the laboratory, aseptic technique involves sterilising equipment and media by autoclaving at high temperature and pressure; flaming the neck of culture bottles to create an upward convection current preventing airborne organisms entering; sterilising the inoculating loop by heating to red heat and allowing it to cool; working near a Bunsen flame, which creates an updraught; opening Petri dishes as little as possible and at an angle; sealing plates but not completely, so that anaerobic conditions favouring pathogens do not develop; and incubating cultures at 25 degrees Celsius rather than at body temperature, to reduce the risk of culturing organisms pathogenic to humans.
Industrially, the fermenter and all inlets are sterilised with superheated steam before use, and incoming air is filtered.
Applications
Brewing uses yeast to ferment sugars from malted barley to ethanol and carbon dioxide under anaerobic conditions. Anaerobic conditions are essential, since in the presence of oxygen the yeast would respire aerobically and produce carbon dioxide and water rather than ethanol.
Baking uses the same organism for the opposite product. Yeast ferments sugars in the dough and the carbon dioxide produced is trapped by the gluten network, raising the bread; the ethanol evaporates during baking.
Yoghurt and cheese production uses bacteria that ferment lactose to lactic acid. The acid lowers the pH, denaturing milk proteins so that they coagulate, and also inhibits spoilage organisms, which preserves the product.
Penicillin production uses the fungus Penicillium in batch culture, harvesting the antibiotic produced as a secondary metabolite during the stationary phase.
Single-cell protein is produced from fungal or bacterial biomass grown on cheap substrates. It has a high protein content, can be produced independently of climate and land, and grows far faster than any crop or animal. Its disadvantages are a high nucleic acid content requiring processing, the risk of contamination, and consumer reluctance.
Sewage treatment uses aerobic bacteria to break down organic matter in effluent and anaerobic bacteria to digest sludge, producing methane that can be burned as fuel.
Biogas production uses anaerobic digestion of organic waste to produce methane, which is important in rural areas and converts a waste problem into an energy source.
Bioremediation uses microorganisms to break down pollutants such as oil spills and pesticides.
Immobilised enzymes in industry
Enzymes may be used instead of whole organisms, and immobilising them on an inert support such as alginate beads or a membrane brings substantial advantages.
The enzyme can be recovered and reused, greatly reducing cost. The product is not contaminated with enzyme, simplifying purification and making the product suitable for food and pharmaceutical use. Immobilised enzymes are more stable to changes in temperature and pH, because the support holds the tertiary structure. The process can run continuously and be stopped simply by removing the beads.
The disadvantages are a somewhat lower rate, because substrate must diffuse to the enzyme and the support may partially obstruct the active site, and a higher initial setup cost.
Applications include lactase to produce lactose-free milk and glucose isomerase to convert glucose to the sweeter fructose.
Worked examples
Example 1: Explaining the stationary phase (4 marks)
Explain why a bacterial population in a closed culture enters a stationary phase.
During the log phase the population grows exponentially because nutrients are abundant and waste has not accumulated.
As the population becomes large, nutrients such as the carbon and nitrogen sources become limiting, so not all cells can obtain enough to divide. Toxic waste products of metabolism accumulate and inhibit growth, and in aerobic cultures dissolved oxygen may be depleted faster than it can be replaced. Space also becomes restricted.
As a result the rate of cell division falls until it exactly equals the rate of cell death, so the total population size remains constant even though individual cells continue to divide and die.
Example 2: Justifying a choice of culture method (4 marks)
Penicillin is produced industrially by batch culture rather than continuous culture. Explain why.
Penicillin is a secondary metabolite, meaning it is not required for the growth of the fungus and is produced only after the main growth phase, during the stationary phase.
Continuous culture maintains the population in the log phase by continually adding nutrient and removing product. Since the culture never enters the stationary phase, little penicillin would be produced.
Batch culture supplies a fixed quantity of nutrient and allows the culture to pass through all the growth phases, including the stationary phase in which penicillin is synthesised. The product is then harvested at the end of the run.
A further advantage is that if contamination occurs, only a single batch is lost rather than an entire continuous process.
Example 3: Explaining fermenter features (5 marks)
Explain why a large industrial fermenter requires a cooling water jacket and a stirrer.
The respiration of the microorganisms is exothermic, and in a large vessel with a high population density a great deal of heat is generated. Without removal, the temperature would rise above the optimum, and the enzymes of the microorganisms would be denatured as their tertiary structure was disrupted, reducing the growth rate and the yield. Cold water circulating through the jacket absorbs this excess heat and maintains the optimum temperature.
The stirrer keeps the microorganisms evenly suspended rather than settling, so that all cells have equal access to nutrients and oxygen. It distributes heat uniformly throughout the vessel, preventing local hot spots that could denature enzymes, and it breaks up air bubbles, increasing the surface area for oxygen to dissolve.
Both features therefore maintain the optimum conditions throughout the vessel, which is what allows a high and uniform rate of growth and product formation.
Common mistakes and how to avoid them
The most frequent error is stating that the population stops dividing during the stationary phase. Division continues, but is balanced by an equal rate of death.
Students often say that the lag phase occurs because the bacteria are not yet alive or are dormant. They are metabolically active, synthesising enzymes and taking up nutrients, but not yet dividing rapidly.
Another common slip is confusing primary with secondary metabolites, and therefore choosing the wrong culture method. Secondary metabolites require the stationary phase and therefore batch culture.
Many candidates list fermenter features without giving reasons. The reason is where the mark lies, and heat removal in particular must be linked to enzyme denaturation.
In brewing questions, answers frequently omit that anaerobic conditions are essential. With oxygen present the yeast would respire aerobically and produce no ethanol.
Finally, candidates often state that cultures are incubated at 37 degrees Celsius. In school laboratories 25 degrees is used to reduce the risk of culturing human pathogens.
Exam technique for "Microorganisms and biotechnology"
Describe each growth phase in terms of the balance between division and death, and give the limiting factors responsible for the transition into the stationary phase.
For fermenter questions, pair every feature with its reason, and where the reason involves temperature or pH, take it through to enzyme denaturation.
When asked to choose between batch and continuous culture, identify whether the product is a primary or secondary metabolite first. That determines the answer.
For aseptic technique, give the procedure and what it prevents. Flaming the bottle neck creates an updraught; heating the loop sterilises it; incubating at 25 degrees avoids human pathogens.
In application questions, name the organism and the conditions as well as the product, since all three are typically credited.
Quick revision summary
Microorganisms are used industrially because they have short generation times, grow on cheap waste substrates at moderate temperatures, are easily genetically modified and are unaffected by climate. A closed culture shows a lag phase of enzyme synthesis, an exponential log phase, a stationary phase where division equals death because nutrients are limiting and waste has accumulated, and a decline phase. Primary metabolites are made during growth and secondary metabolites, including penicillin, during the stationary phase, which is why batch culture is used for the latter and continuous culture for the former. Fermenters have a stirrer for even suspension and heat distribution, a sparger supplying sterile air, a cooling water jacket to remove exothermic heat and prevent enzyme denaturation, probes and automatic control of temperature, pH and oxygen, and a sterilisable stainless steel vessel. Aseptic technique prevents contamination through autoclaving, flaming, working near a flame and incubating at 25 degrees Celsius. Applications include brewing and baking with yeast, yoghurt and cheese using lactic acid bacteria, penicillin from Penicillium, single-cell protein, sewage treatment, biogas and bioremediation, alongside immobilised enzymes that can be reused and leave an uncontaminated product.