Title: The growth curveHomework: complete learning package 12

3 May 2023

Learning Outcomes

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

Standard Growth curve

Growth curve in a closed culture

• Lag phase– Bacteria adjusting to new

conditions– Takes a while for enzyme

production• Log phase

– Number of bacteria increase rapidly

• Stationary Phase– Rate of growth is equal

to rate of death• Decline Phase

– Death rate is greater than “birth rate”

• The first three stages represent a sigmoid growth curve

Primary and secondary metabolites

• Primary metabolites– Substances produced by on

organism as part of its growth– Amino acids, proteins,

enzymes, nucleic acids, ethanol and lactate

– Production matches growth of population of organism

• Secondary metabolites– Substances produced not as

part of normal growth– Antibiotic chemicals are

mostly secondary metabolites– Begins after main growth

period and does match population growth.

Learning Outcomes

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

Large-Scale production

• Microorganisms are cultured in large containers called fermenters

• The growing conditions within the fermenter are manipulated and controlled– Precise growing conditions• Temperature• Type and time of the addition of the nutrient• Oxygen concentration• pH

A batch fermenter

Large scale production

• Three examples are– The production of penicillin– The production of protease enzymes– The production of mycoprotein

Learning Outcomes

• Compare and contrast the processes of continuous culture and batch culture.

• Describe the differences between primary and secondary metabolites.

Metabolism and metabolites

• Metabolism (process)– Sum total of all the chemical reactions– Processes produce• New cell and cell components• Chemicals• Waste products

• Metabolites (products)– A substance produced during cell processes

Primary and secondary metabolites

• Primary metabolite– Substance produced by

organism as part of it’s normal growth

– E.g. amino acids, proteins, enzymes

– Production of primary metabolites matches the growth in population

• Secondary metabolite– A substance only produced

at a particular growth phase– No direct involvement in

fundamental metabolite processes

– Production usually begins after the main growth phase of the micro organisms

Batch culture

• Starter population is mixed with a specific quantity of nutrient solution

• Allowed to grow for a fixed period• Products removed• Fermentation tank emptied• Examples

• Penicillin production• Enzyme production

Continuous Culture

• Nutrients are added and products are removed from the fermentation tank at regular intervals

• Examples• Insulin production from genetically modified E.Coli• Production of mycoprotein

Learning outcomes

• Explain the importance of asepsis in the manipulation of microorganisms

Asepsis

• Asepsis – absence of unwanted microorganisms

• Aseptic techniques– Any measure taken during a biotechnological

process to prevent contamination by unwanted microorganisms

The importance of asepsis

• Unwanted microorganisms– Compete with the culture microorganisms– Reduce the yield of useful products– Cause spoilage of the product– Produce toxic chemicals– Destroy the culture microorganism or its products.

Methods to maintain asepsis• Ensure all fermenters and attachments are sterile

– Cleaning with pasteurised steam– Chemical sterilisation

• Sterilise all liquids, solids and gases that enter the reaction vessel

• Maintain a pressure difference between the air in the room where fermentation is taking place and outside– Maintains a steady airflow out of the room

• Ensure culture of microorganisms is pure• Ensure the workers do not introduce unwanted

microorganisms from their skin.

Learning Outcomes

• Describe how enzymes can be immobilised.• Explain why immobilised enzymes are used in

large-scale production.

Immobilising enzymes

• Enzymes act as catalysts in metabolic reactions• Enzymes are useful in industrial processes– Specificity– Temperature of enzyme action

• Enzymes in solution need to be separated from the products.

• Immobilised enzymes can be re-used many times and leaves the product enzyme free.

Methods for immobilising enzymes

• Gel entrapment– Example – immobilising lactase in alginate– Stages• Enzyme solution is mixed with sodium alginate solution• Droplets of this solution are added to a solution of

calcium chloride• The droplet turns into a bead which contains the

enzyme

Immobilising lactase in alginate

Immobilising lactase in alginate

• The beads can be tightly packed into a column

• The liquid substrate can be trickled over the beads

• The product trickles out of the bottom of the column

• The product is collected and purified.

Methods of immobilising enzymes

• Adsorption / carrier bound– Enzyme molecules are

mixed with immobilising support e.g. glass beads or clay

• Covalent Bonding / cross-linked– Enzyme molecules

covalently bonded to a support

Methods of immobilising enzymes

• Entrapment / inclusion– Enzymes trapped in their natural

state in a gel bead– Reaction rate can be reduced as

substrate needs to get through the trapping barrier

• Membrane separation– Substrate separated from the

mixture by a partially permeable membrane.

Advantages of immobilised enzymes

• The advantages of using immobilised enzymes over enzymes in solution are– Immobilised enzymes can be reused– Product is enzyme free– Immobilised enzymes are more tolerant to pH and

temperature changes