6.2.1 Biotechnology

What is biotechnology?

Use of living organisms in industrial processes

Why are microorganisms used in biotechnology?

• Can grow well at low temperatures

• Have simple growth requirements

• Grow rapidly in favourable conditions

• Metabolites can be harvested

• Can be genetically engineered to produce specific products

• Can grow anywhere in the world

• Not dependent on climate

• Tend to generate more pure products

• Not many ethical issues

• Their food is cheap and readily available

• Don’t require a lot of space

What are the uses of microorganisms in biotechnology?

• Production of food

• Production of drugs

• Production of enzymes

• Bioremediation of waste products

What food production processes can microorganisms be used in?

• Brewing

• Baking

• Cheese making

• Yoghurt production

• Mycoprotein production

How are microorganisms used in brewing?

• Uses yeast species Saccharomyces cerevisiae

• Saccharomyces cerevisiae respires anaerobically using barley malt to produce ethanol and CO2

• Drinks with a higher alcohol content are fermented first and then distilled to concentrate the alcohol

How are microorganisms used in baking?

• Flour is mixed with yeast and other ingredients to make dough

• Yeast enzymes hydrolyse starch in flour to maltose

• Maltose is then hydrolysed to produce monosaccharides for aerobic respiration

• When oxygen runs out, yeast respires anaerobically

• This produces CO2 which causes the dough to rise

• Baking kills the yeast and causes the gas pockets in the dough to expand, so the bread rises more.

How are microorganisms used in cheese making?

• Milk pre-treated with a culture of bacteria (Lactobacillus)

• The bacteria digests the lactose, producing lactic acid

• Lactic acid lowers the pH of the milk

• The low pH causes proteins in the milk to denature leading to separation of curds (solid) and whey (liquids)

• Curds are pressed and processed into hard cheeses

How are microorganisms used in yoghurt making?

• Pasteurised milk is added to a culture of Lactobacillus bulgaricus and Streptococcus thermophilus bacteria

• The bacteria converts lactose to lactic acid and partially digest the milk

• Lactic acid denatures the proteins in the milk, causing them to coagulate (stick together)

• This produces yoghurt

• Flavours can be added

How are microorganisms used in mycoprotein production?

• e.g. Quorn

• Uses fungus, Fusarium venenatum

• Source of glucose is added and oxygen is supplied

• Fungi respires aerobically to maximise growth of hyphae

• Mycoprotein is low in fat and high in fibre, so it is a good substitute for meat.

What are the arguments for using microorganisms in biotechnology in food production processes?

• No animal welfare issues

• Doesn’t require much land

• Provides a good source of protein

• Production is independent of seasonal variations

• Can increase or decrease production due to demand

• Microorganisms can be easily genetically modified to adjust amino acid content of protein

What are the arguments against using microorganisms in biotechnology in food production processes?

• Protein has to be purified to ensure it is uncontaminated

• Amino acid profile may be different than traditional animal protein

• Risk of infection as conditions needed for microorganisms to grow are also ideal for pathogens

• Microorganisms need to be isolated from the material on which they grow

• Protein doesn’t have taste or texture of traditional sources

How are microorganisms used in bioremediation?

• Bioremediation is the breakdown of pollutants and contaminants in soil or water

• Natural organisms break down organic material, producing CO2 and water

• Genetically modified organisms break down matter they wouldn’t normally break down e.g. mercury

What conditions need to be controlled during commercial drug production using microorganisms?

• Temperature

• Available nutrients

• Oxygen levels

• pH

• Concentration of product

What drugs are produced using microorganisms in biotechnology?

• Penicillin

• Insulin

How are microorganisms used in production of penicillin?

• Penicillin is a secondary metabolite

• Manufactured by batch culture

• Fermenter is run for 6-8 days

• Culture is filtered to remove cells

• Potassium compounds are added to precipitate the penicillin

• The penicillin is mixed with an inert substance and prepared for administration

How are microorganisms used in production of insulin?

• Manufactured by continuous culture

• Gene for human insulin is inserted into a plasmid vector and into a bacteria

• Bacteria produces large amounts of insulin at a low cost

How was insulin retrieved before it was produced by microorganisms?

• From pancreas of animals

• Was expensive, hard to isolate and not as effective as human insulin

What are the 2 main methods used to culture microorganisms by fermentation?

• Batch fermentation

• Continuous fermentation

What type of metabolite does batch culture produce?

Secondary metabolites

How are microorganisms cultured in batch fermentation?

• Grown in batches

• Starter population mixed with a specific quantity of nutrient solution

• Microorganisms are allowed to grow for a fixed period before death phase begins

• Products are removed

• Fermentation tank is emptied and sterilised

What are the advantages of culturing microorganisms by batch culture?

• Closed culture

• Easy to setup and maintain

• useful for producing secondary metabolites

• Useful for maintaining culture in stationary phase

What are the disadvantages of culturing microorganisms by batch culture?

• Must know the end of the log phase and the end of the stationary phase

• Less efficient as fermenter is not in operation full time

What type of metabolite does continuous culture produce ?

Primary metabolites

How are microorganisms produced in continuous fermentation?

• Nutrients are added continually once exponential growth has been reached

• Products and waste are removed from the fermentation tank at regular intervals

• Volume in fermenter remains constant

What are the advantages of culturing microorganisms by continuous culture?

• useful for maintaining culture in log phase

• more efficient as fermenter operates continuously

What is a disadvantage of culturing microorganisms by continuous culture?

• Difficult to set up

What is the purpose of aspesis?

• Ensures sterile conditions are maintained

• Important as the conditions are often perfect for the growth of other unwanted microorganisms

• The unwanted microorganisms would compete for nutrients and space, reduce the yield, spoil the product, may produce toxic chemicals and may destroy wanted microorganisms.

What are some general aseptic techniques that should be used when culturing microorganisms?

• Wash hands thoroughly before and after

• Disinfect the working area

• No food or drink allowed in the lab

• Should have a nearby Bunsen burner

• Avoid removing petri dish lid entirely

• Sterilise any equipment in bunsen burner before making contact with microorganism

• Place neck of any open test tubes over bunsen burners

Why is it important to work close to a lit Bunsen burner in aseptic technique?

• Warms air

• Creates a convection current

• Causes airborne microbial contaminants to rise away from work area

Why is it important to place the neck of any open test tubes over bunsen burners in aseptic technique?

• Prevents bacteria in surrounding air from entering the vessel

Describe the process of aseptic technique used to culture microorganisms

Sterilisation

• Agar and equipment must be sterile

• Use autoclave at 121C for 15 minutes

• All organisms must be killed

• Keep lid on the petri dish to maintain sterility

Inoculation

• Introduce microorganisms to sterile medium

• Use a streaking technique with a inoculating loop to drag the sample in a zig-zag across the agar

• Sterilise the inoculation loop again

• Tape the lid of the petri dish at intervals

Incubation

• Petri dish placed in warm environment of 25C in school lab

• Placed upside down

• Do not open the petri dish

• Observe culture growth after 24 - 48 hours

• Sterilise petri dish after use

Why is the inoculating loop passed through a hot flame before it is used?

• Sterilise it

• Kills any microorganisms on the loop to prevent contamination

Why is the petri dish opened as little as possible

• Decreases the risk of microorganisms contaminating the dish

Why is the petri dish stored upside down after inoculation?

• Prevents drops of condensation falling on surface of agar

Why is the petri dish incubated at 25C?

• Restricts growth of harmful pathogens which are more likely to grow at higher temperatures

What are the 2 types of growth medium?

• Broth

• Agar

What does the standard population growth curve of a microorganism look like?

What are the 4 phases in a standard population growth curve of a microorganism?

What happens in the lag phase?

• Cells increase in size

• Population remains fairly constant

• No cell division

• Gives time for population to acclimatise to ensure it is a stable environment

What happens in the log phase?

• Exponential growth due to high metabolic activity and reproduction

• Microorganisms have adjusted to environment

• Population doubles in size with each generation

What happens in the stationary phase?

• Nutrients levels decrease

• Toxic metabolites build up

• Growth rate slows

• Population stabilises

• The number of organisms dying = number being reproduced by binary fission

What happens in the decline phase?

• Lack of nutrients

• Increased level of toxic metabolites

• Death rate exceeds reproduction rate

What is the formula used to calculate the number of individual microorganisms after a certain number of divisions

N = N₀ x 2ⁿ

N₀ = the initial number of bacteria

n = no. of divisions

What is meant by serial dilutions and the purpose of this?

Repeated dilutions of a solution in order to reduce its concentration

How is dilution factor calculated?

initial volume / final volume

left as a fraction

What is the purpose of a haemocytometer?

• Used to count total cells counts

What is the general method of using a haemocytometer?

1. drop of microbial culture placed on the grid and a special cover slip is suspended above it

2. The cover slip traps a known depth of liquid above the grid

3. The dimensions of the grid squares are already known, so the volume of fluid over each square is calculated

4. Direct counting gives use the number of cells in the volume of culture

How can viability be determined from a haemocytometer?

• identifies living cells from dead cells

• add a viability die

• can calculate viability as a %

• e.g. 83% viability means 83% of cells are living

What are counting conventions used to make total cell counts in a haemocytometer?

• Count cells touching top and left side of square

• Don’t count cells touching right and bottom of square unless if more than 50% of the cell is inside the square

What are the advantages of calculating the number of individual microorganisms by total cell counts in a haemocytometer?

• Quick and accurate when done by experienced workers

• More accurate than other methods such as turbidity

What are the disadvantages of calculating the number of individual microorganisms by total cell counts in a haemocytometer?

• Dead cells cannot be distinguished from living ones

• Only dense suspensions can be counted

• Highly viscous solutions don’t get drawn in by capillary action

What are immobilised enzymes?

Enzymes that are fixed and unable to move freely throughout a solution

Why are immobilised enzymes used in biotechnology?

• Can be removed from the microorganisms themselves to simplify biotechnological processes

What are the general advantages of using immobilised enzymes?

• Enzymes don’t mix with product, so lower purification cost

• Enzymes can be reused

• Used to replace cells containing enzymes, so don;t need to worry about cellular demand for nutrients or release of waste products

• Enzymes are fixed within a immobilising matrix, so is protected from harsh environments e.g. high temp and extreme pH

What are the general disadvantages of using immobilised enzymes?

• Expensive to set up

• Immobilised enzymes are less active than free enzymes, so slower process

What are the 4 methods of immobilising enzymes?

1. adsorption

2. covalent bonding

3. entrapment

4. membrane barrier

How are enzymes immobilised by adsorption?

• Enzymes bound to clay surface by hydrophobic interactions and ionic links

• Has weak bonds so some enzymes break loose

How are enzymes immobilised by covalent bonding?

• Enzymes bound to clay surface by covalent bonds

• Strong bonds due to sharing of electrons

• This can reduce enzyme action

• can be expensive

• Enzymes unlikely to break free into the reaction mixture

How are enzymes immobilised by entrapment?

• Enzymes trapped in matrix

• Enzymes are fully active

• Substrate diffuses into matrix

• Product diffuses out of matrix

How are enzymes immobilised by a membrane barrier?

• Semi-permeable membrane around enzymes

• Substrate small enough to pass through membrane to meet enzymes

• Limits rate of reaction