OCR A2 F215 UNIT 2

MODULE 2 BIOTECHNOLOGY

Specification:

a) State that biotechnology is the industrial use of living organisms (or parts of living organisms) to produce food, drugs or other products

b) Explain why microorganisms are often used in biotechnological processes

c) Describe, with the aid of diagrams, and explain, the standard growth curve of a microorganism in a closed culture

d) Describe how enzymes can be immobilised

e) Explain why immobilised enzymes are used in large-scale production

f) Compare and contrast the processes of continuous culture and batch culture

g) Describe the differences between primary and secondary metabolites

h) Explain the importance of manipulating the growing conditions in a fermentation vessel in order to maximise the yield of product required

i) Explain the importance of asepsis in the manipulation of microorganisms

Definition of Biotechnology: this is the large scale, industrial use of living organisms, particularly micro-organisms, and enzymes to produce useful products such as food and drugs

1

Table to show examples of Products from Biotechnology

FOOD and DRUG PRODUCTS DETAIL

Cheese and yoghurt Lactobacillus bacteria ferment lactose in milk producing lactic acid that clots milk protein. In yoghurt this thickens the product and in cheese production, it produces the curd that is matured to form cheese

Mycoprotein (brand named Quorn) A meat substitute produced from a fungus called Fusarium

Wine, beer and cider Products of yeast fermentation of sugar producing ethanol

Antibiotics such as Penicillin Antibiotics are released from some species of fungi. Penicillin is produced by the fungus Penicillium. Antibiotics are used to kill pathogenic bacteria

Products such as insulin or growth hormone Human products of genetically engineered yeast or bacteria

Pectinase enzyme production Pectinase is an enzyme extracted from a fungus. It hydrolyses pectin in plant cell walls and is used commercially to clarify and increase the yield of fruit juice

Gasohol production A fuel for cars combining ethanol (from yeast fermentation) with petrol

Biogas production Methane produced by the bacterial fermentation of waste organic molecules. Biogas is used as an alternative energy source, to produce electricity. It is carbon neutral (does not result in an increase in CO2 in the atmosphere)

2

Why use micro-organisms?

Many biotechnological processes use microorganisms, including bacteria, archeans and fungi. Micro-organisms have several features that make them particularly useful in large scale industrial processes.

Under suitable conditions, they reproduce rapidly; the generation time (time for a doubling of numbers) of some microbes is 20-30 min. this means that large populations build up quickly

Microbes can be genetically engineered to produce useful products such as human insulin

Grow well at lower temperatures, making the industrial processes safer and cheaper - saving money in fuel costs

Some can be grown on waste products from industry that would otherwise have no use such as whey (a waste product of cheese production)

The useful products are often released into the culture medium and can be separated easily. There is little downstream processing (the processes involved in separating and purifying useful products from a culture medium)

Have very simple and specific growth requirements so they can be grown in fermenters under controlled conditions, with very little attention, anywhere in the world

More Definitions

A culture is a nutrient medium (either liquid or agar gel) containing specific micro-organisms. If one species is grown, it is a pure culture

The nutrient medium/broth would contain a carbohydrate source ( eg glucose), amino acids, vitamins and mineral ions

A closed culture is one in which nutrients are added only at the beginning of the fermentation. No waste products are removed. A fermenter is a vessel used for growing micro-organisms. A nutrient broth is mixed with a starter culture of the organism. Fermenters may be laboratory size or industrial size

Fermentation refers to the metabolic processes whereby micro-organisms produce products. These processes may be aerobic or anaerobic

3

Aseptic technique refers to the procedures taken when handling micro-organisms, to avoid contamination of the culture and product

The Standard Growth Curve

When a growth medium is prepared in a fermenter and a known number of bacteria are added, the population density of the closed culture can be measured over time.

The graph shown below is a typical growth curve that would result for a bacterial population in a closed culture.

A small number of cells are introduced to the fermenter at time 0 and then samples are taken at intervals to estimate the number of cells in the culture medium

The growth curve shows four main phases A, B, D and E

A. The Lag Phase

Very slow or no cell division (check the graph that you asked to describe in an examination)

The cells are adjusting to the new culture medium Genes are switched on So that new enzymes can be synthesised

4

B. The Exponential or Log Phase

Rate of cell division/cell reproduction is at a maximum and very fast The population doubles per unit time – this is exponential growth No limiting factors to exponential growth – the cells have plenty of

nutrients and oxygen (if aerobically respiring)

D. Stationary Phase

The number of cells produced = number of cells dying Due to accumulation of waste products/lack of nutrients/lack of oxygen There is more competition The culture has reached its carrying capacity (the maximum population

density that can be supported by the environment}.

E. Death Phase

Number of cells dying is greater than the number of cells produced There is more competition and a more significant lack of nutrients/lack of

oxygen and accumulation of waste products.

5

FERMENTERS

Fermenters are vessels used to grow micro-organisms in a culture medium.

Commercially, an industrial fermenter is a huge tank that holds thousands of litres of culture medium.

The precise features of the fermenter depend upon the microorganism being grown and the nature of the product

Basic Principles of the Fermenter

Temperature Control

This is important to maintain the optimum temperature for the activity of microbial enzymes

As the organisms respire, they release thermal energy that heats up the culture medium

Industrial fermenters have a water jacket around them through which cold water flows, to cool down the culture medium

An electronic temperature probe monitors the culture temperature

pH Control

6

Changes in pH can alter the activity of enzymes and change growth rate. Extreme pHs would denature enzymes. Therefore it is important to maintain an optimum pH

A buffer (maintains pH) is added to the culture at the start

An electronic pH probe monitors the pH

There may be inlet tubes to add acid or alkali during the process

Stirring

Fermenters have a motorised stirrer with impellers as mixing blades

This ensures that the nutrients and micro-organisms are continually brought into contact and mixed evenly

Oxygen Supply

Most commercial processes involve the growth of organisms under aerobic conditions

. Oxygen will be required for aerobic respiration and a shortage will limit

growth rate and cause unwanted products from anaerobic respiration

The fermenter will have an inlet tube for sterile air. The tube will be fitted with a filter to ensure that the air is sterile

Air is often bubbled into the fermenter via a sparger - a ring with holes allowing small bubbles of air into the tank. This helps to mix the air with the culture medium

An oxygen probe monitors the oxygen concentration in the fermenter

Nutrient Supply

All micro-organisms require a nutrient supply including an energy source, nitrogen and carbon sources, vitamins and minerals

Glucose and other carbohydrates are the main carbon and energy sources. Amino acids or ammonia provide nitrogen.

Nutrients will be added to the medium at the start of fermentation

7

The timing of nutrient addition after this depends upon the type of fermentation (no further addition of nutrients in a closed fermenter)

There will be a sterile inlet for nutrients

Venting/Removing Waste Gases

Waste gases such as CO2 will be produced during fermentation

To avoid pressure build up in the fermenter and explosion, there will be an outlet, with a sterile filter, for venting these gases

Product Removal

Product is removed via an outlet tap at the base of the fermenter

Useful products are separated from the culture medium and purified during downstream processing

Stainless Steel Fermenters

Fermenters are often made of stainless steel. This has a smooth surface making it less likely that residues will remain in the fermenter after the process

Stainless steel fermenters can also be cleaned by very hot water or steam to maintain sterile conditions

Stainless steel does not corrode

Stainless steel is strong and withstands pressure build up in the fermenter

– these fermenters are safer and not likely to explode

Aseptic Techniques in Industry

Disinfecting and steam cleaning of fermenters and pipes when not in use kills micro-organisms and removes culture medium residues

Stainless steel fermenters have smooth surfaces that prevent accumulation of residues and allow the use of steam for cleaning

Filters on inlet and outlet pipes prevent microbes entering or leaving the fermenter in gas flow

8

Sterilisation of nutrient media before adding to the fermenter

Primary and Secondary Metabolites

Primary metabolites are the products of essential metabolic reactions of an organism during its normal growth, such as respiration

Enzymes, proteins, amino acids, ethanol (yeast) and carbon dioxide are examples of primary metabolites

The production of the primary metabolite matches the growth of the organism as shown in the graph on page 10

Graph to Show the Growth Curve of Yeast and its Yield of Ethanol – a Primary Metabolite

Secondary metabolites are products of metabolic reactions that are not essential for growth. It is the metabolic processes that are not essential but the secondary metabolites are often beneficial

The greatest production of secondary metabolites usually occurs at the end of the exponential growth phase and during the stationary phase, when the nutrients are declining

Most of the antibiotic products of fungi are secondary metabolites

9

Graph to Show the Growth Curve of Penicillin and Mould – a Secondary Metabolite

All organisms produce primary metabolites but not all produce secondary metabolites

Batch and Continuous Culture

BATCH CULTURE CONTINUOUS CULTURECarried out in a closed fermenter Carried out in an open fermenter

No nutrients added during the process Nutrients are added during the process

No products are removed during the process. Products are removed at the end of the process. Waste gases are vented off during the process

Products are removed during the process. Waste gases are vented off

Exponential phase is short since nutrients are used up – lower productivity

Culture is maintained in the exponential phase since nutrients are added frequently – higher productivity

Easier to set up and control More difficult to control than the batch processFermenters can be used for different purposes at different times

Smaller fermenters can be used for the same yield

Only one batch lost if culture is contaminated Losses are greater if culture contaminated since productivity is greater

Can be used to produce primary and secondary metabolites

Only useful for primary metabolites

Penicillin is produced by batch fermentation Human insulin is produced by continuous culture

10

Refer to the graph on page 11. How would the curves for lactose, ammonia and fungal biomass differ if the penicillin was being produced by continuous culture?

………………………………………………………………………………………………

………………………………………………………………………………………………

………………………………………………………………………………………………

………………………………………………………………………………………………

………………………………………………………………………………………………

………………………………………………………………………………………………

Immobilised Enzymes

Definition:

Immobilisation of enzymes refers to techniques whereby enzymes are attached to an inert, insoluble support

Features of immobilised enzymes

The enzymes molecules are not free to move within the reaction mixture

The substrate molecules can still move freely and can bind to enzyme active sites.

The products are released back into the reaction mixture, leaving the enzyme attached to the inert support

11

Methods of enzyme immobilisation:

Adsorbed onto an insoluble support such as clays and resins via ionic and hydrophobic interactions

. Bonded covalently to a cross linking agent and then to an insoluble

support such as clay

Gel entrapment - held inside a gel lattice such as silica gel, or within a network of cellulose or collagen matrix, or within a microcapsule of alginate

12

Advantages of using immobilised enzymes

The enzyme can be recovered easily and re-used many times – ideal for continuous culture

The product is not contaminated with the enzyme, reducing the cost of downstream processing and product purification

The enzyme is protected by the immobilising material and is therefore more stable and more tolerant to changes in temperature and pH

Suitable for continuous fermentation processes

Disadvantages of Using Immobilised enzymes

Requires specialised equipment that is initially more expensive and difficult to set up

Immobilised enzymes are less active because they do not mix freely with substrate – only the substrate molecules can move to bring about collisions with active sites

Some examples of the uses of immobilised enzymes

Production of amino penicillanic acid, a derivative of penicillin, that is used to produce a range of different penicillin based antibiotics. The enzyme

13

penicillin acylase is trapped in alginate beads in a reaction vessel, while penicillin passes through the vessel (see page 165 in textbooks)

In biosensors to monitor blood glucose concentrations – used by diabetics for a rapid and accurate assessment of blood glucose concentrations

Used for immobilisation of lactase in production of lactose free milk (cats and some humans are lactose intolerant)

14