What do we use microbes for?

Biotechnology
The commercial use of living organisms or
biological processes to produce food, drugs,
enzymes or other useful products
Advantages of using microorganisms
• Rapid life cycles – some have a 20 minute
doubling time – so have large pop quickly
• Simple growth requirements – e.g. low temps
and pressure– so can be grown in fermenter in
any country /climate safely and cheaply
• Can be grown on unwanted industrial
waste/food
• Genetic engineering is simple as only a single
copy of gene
• Secrete products that are easily separated
• No ethical considerations
• Asexual reproduction – all offspring are clones
 Bioremediation
• Bioremediation is the use of
microorganisms to clean soil/oil spills
/ sewage
• The organisms convert the toxic
pollutants to non toxic substances.
• Pseudomonas bacterium was used to
break down crude oil and can be used
in treating oil spills.
• Similarly, solvents and pesticides can
also be treated using bioremediation.
• Bioremediation requires the right
growth conditions (water,
temperature, pH) for the organisms
that are using the contaminants as a
source of food.
• Because area of contamination may
be very large and it is too expensive
to remove contaminated material
• Microbe may be harvested and
contaminants retrieved
• Microbes/biogas can be incinerated
5
to release heat energy
 Yoghurt – indirect food production
• Milk is first pasteurized and homrogenised
and then cooled before bacteria is added
• Milk that has been fermented by Lactobacillus
bulgaricus and Streptococcus thermophilus in
a 1:1 ratio becomes the product that you
know as yoghurt. Both bacteria produce
extracellular polymers that give yoghurt a
smooth thick texture
• The lactose in the milk is converted to lactic
acid which gives it the typical flavour and the
acidity also denatures the milk protein making
it coagulate together
• Partial digestion of the milk by the bacteria
make the product easy for digestion
• Lactobacillus acidophilus, Lactobacillus casei
and Bifidobacterium are added as a probiotic
(live culture to benefit human health) to aid
gastrointestinal function
 Cheese – indirect food production
• Milk is first pasteurized and
homogenised
• Milk is treated with Lactobacillus
producing lactic acid from the lactose,
then mixed with rennet
• Rennet contains and enzyme rennin
(chymosin) obtained from the
stomachs of mammals which
coagulates the milk protein casein
• Kappa-casein is broken down turning
the soluble casein into an insoluble
form
• Calcium ions are used to bind the
casein together and precipitate it
from solution forming the curd
• The liquid portion (whey) is
removed from the curd by cutting,
stirring & heating
• The curd is then pressed into
moulds and the different
treatments while making the curd
determine the characteristic
flavour and texture of the cheese
• Final ripening and maturing will
further develop these
characteristics
• Some cheeses will be given an
inoculum of Penicillium to
produce ‘blue’ cheese
7
 Baking – indirect food production

• Bread is made by mixing flour,

water, salt and yeast
(Saccharomyces cerevisiae)
together to produce a dough
• The dough is left to prove
(ferment) in a warm place while
the yeast respire anaerobically to
produce carbon dioxide causing
the dough to rise
• Yeast cells are killed during
cooking
• Once baked, the alcohol
(produced by the anaerobic
fermentation in yeast) is
 Alcoholic drinks– indirect food
production
• Saccharomyces cerevisiae (yeast) is used
to produce alcohol by anaerobic
respiration
• Wine is produced using the natural
yeast present on the skin of the grapes
• The yeast uses the fructose and glucose
from the crushed grapes to respire and
release carbon dioxide and alcohol
• Beer is produced using germinating
barley grains in a process called malting
• As the grain germinates is converts
stored starch to maltose which is
anaerobically respired by yeast to
produce carbon dioxide and alcohol
• Hops is added to give it a bitter taste
Malting, mashing, fermentation,
maturation, finishing
 Single cell protein (e.g. Quorn) – Direct
food production
• The fungal protein or mycoprotein,
also called SCP (single-cell protein) is
produced from the fungus Fusarium
venenatum
• QuornTM is one of the best known
commercial products of SCP first
marketed in the early 1980s
• It is marketed as a meat substitute
for vegetarians and a healthy option
as it contains no animal fat or
cholesterol
• Other fungi, Kluyveromyces,
Scytalidium and Candida, are also
being used to produce protein and
can be grown on almost any organic
substrate (e.g. paper, whey)
• Bacteria multiply by simple cell division called binary fission. If they have
enough nutrients and a the optimum temperature for enzymes they double
every 20 minutes
 Microbial Growth and
metabolites

• Bacteria multiply by simple cell division called binary fission. If they have
enough nutrients and a the optimum temperature for enzymes they double
every 20 minutes
 Standard growth curve in a
closed culture

Population
Death (Decline) phase
Lag phase:
Log Phase (exponential phase)
1. Death rate greater than cell
division rate
2. Organism doubles its
numbers after each generation –
E.coli doubles every 20 minutes
in optimum conditions.
3. Nutrients run out
4. Space and nutrients do not
limit growth
5. Toxins build up too high
6. Times vary depending on species of
organism/type of media
Stationary
phase
7. Bacteria adjust to new conditions
e.g. Take in water
8. Cell division rate equal to death
rate
9. Activating genes
10. Nutrient levels have decreased
Waste products (e.g. CO2 /ethanol)
build up
11. Synthesising enzymes to metabolise
molecules in the media
12. A change in pH / temp. Enzymes
start to denature
13. Cells active but do not reproduce
14. carrying capacity
• Lag phase:
Bacteria adjust to new conditions
e.g. Take in water
Activating genes
Synthesising enzymes to metabolise molecules
in the media

Cells active but do not reproduce

Times vary depending on species of

organism/type of media
Log Phase (exponential phase)
Organism doubles its numbers after each
generation – E.coli doubles every 20 minutes
in optimum conditions.
Space and nutrients do not limit growth
Stationary phase
Cell division rate equal to death rate
Nutrient levels have decreased
Waste products (e.g. CO2 /ethanol) build up
Change in pH / temp

TIP in an open system this would be called the

carrying capacity
Death (Decline) phase
Death rate greater than cell division rate
Nutrients run out
Toxins build up to high

Shows a sigmoid growth curve during the first

three phases
 Stretch and challenge:
Suggest a reason for the shape of
this growth curve
Diauxic growth – has 2
lag phases because
media contains 2
substrates e.g. Glucose
and sucrose
B
C
D
A
n = 55 G= t/n = 1440 / 55 = 26.18
t = 24 hours = 24x60 = 1440 minutes
26.2
Bacteria calculations
Calculate number of bacteria
in 1cm3 of the original
sample
1. Original unknown pop 2. 0.1 of A 3. 0.01 of A

0.1cm3 0.1cm3 0.1cm3

4. 0.001 of A 5. 0.0001 of A 6. 0.00001 of A

0.1cm3 0.1cm3 0.1cm3
 Serial Dilutions
1. Original unknown pop 2. 0.1 of A 3. 0.01 of A
Using plate 5
0.1cm3 0.1cm3 0.1cm3
• Number of colonies = 23
• So pop in 0.1cm3 of original is
23/0.0001 = 230000

• Pop in 1cm3 of original is 230000/0.1=

2,300,000 = 2.3x106

4. 0.001 of A 5. 0.0001 of A 6. 0.00001 of A

0.1cm3 0.1cm3 0.1cm3
Metabolites 2ometabolite
(e.g. Antibiotic)

1o metabolite
e.g. CO2
 Primary metabolite
A substance produced as a consequence of
normal growth (energy metabolism)
Production matches growth in pop of organism
Produced by all organisms
Examples
CO2, Ethanol, lactate, enzymes, Amino acids
Q:What would be the effect of:
a) CO2 on a bacteria colony?
b) Ethanol on yeast?
 Secondary metabolite
Produced in response to an environmental change
(especially now that organism are competing for
nutrients/space because they’re declining)
Not needed for growth
Produced at start of log phase and will continue
during stationary phase
Examples
Antibiotics – Kills competing organisms (esp
bacteria)
Secondary metabolite
2ometabolite
(e.g. Antibiotic)

1o metabolite
e.g. CO2
 PAG 7.1
Investigating the effect of
antiseptics on bacterial growth
Evidence:
Drawing of the agar plate with its clear zones
around the antibiotic and filter paper discs,
measurements of these clear zones and have
drawn conclusions about the anti-bacterial
properties of each antibiotic. All work should be
clearly dated.
Zone of Inhibition (Clear Zones)
Extension questions
1. What was the purpose of the lit Bunsen burner while you were
working?.
2. Why did you only lift the lid slightly from the agar plate when
placing the discs on the agar?
3. Why did you not seal the plate completely?
4. Why is 20-25C a more suitable incubation temperature than
37C?
5. What was the purpose of using a sterile filter paper disc
soaked in sterile distilled water in area D?
Answers to extension questions
What was the purpose of the lit Bunsen burner while you were
working? To create an up-draught to limit aerial contamination.
Why did you only lift the lid slightly from the agar plate when
placing the discs on the agar?
To avoid contamination from microbes in the air.
ASEPTIC TECHNIQUE
Why did you not seal the plate completely?
To avoid the growth of anaerobic microbes which can be
dangerous to human health, O2 needed for aerobic resp of E.coli
Why is 20-25C a more suitable incubation temperature than
37C? To avoid growing microbes likely to inhabit the human body
as this is human body temperature.
What was the purpose of using a sterile filter paper disc soaked in
sterile distilled water in area D?
It acts as a negative control.
Write what the parts A-K are on your
mini whiteboards
 gases out
sterile air in
product out
acid/base in nutrients in

pH probe temperature probe

cooling water out

stirrer
Antifoam

stirrer paddle

air outlets

cooling water in
Fermenters
• How is each of these conditions
controlled
• Why do we need to control
them?

1. Temperature
2. pH
3. Concentration of nutrients
4. Oxygen Concentration
5. Source of nitrogen
Fermenters
1. Temperature:
a)Probe/thermocouple measures temp –
controls rate of cold water into jacket
– cold water removes excess heat
energy
b) Metabolic reactions are exothermic-
so rise in temp – if too high enzymes
are denatured (organism dies)
Need optimum temp to maximise
growth/product
Fermenters
2. pH:
• Buffer keeps pH constant
• 1o and 2o products alter pH – enzymes
work best at an optimum pH – denature
at other pHs – cell would die – less
product
Fermenters
3. Conc of nutrients
• Sterile nutrients added through tubes
• Paddle distributes evenly
• This will affect rate of product
formation. High conc will increase 1o
metabolite formation (maintain log)
• Low conc will increase penicillin (2o
metabolite) formation (for stationary)
Fermenters
4. Oxygen conc
• Filtered air bubbled in using a sparger
• Allows aerobic respiration. (but some
require anaerobic condition e.g ethanol
production in yeast)
Fermenters
5. Nitrogen
• Add ammonium phosphates, ammonia,
amino acids
• Needed for microorganism to
synthesise proteins (enzymes), amino
acids, nucleic acids, for growth/cell
division
 Asepsis – the a______ of unwanted m_________
Why do we want asepsis?
1. Avoid entry of u________ m________.
2. No c______ with the culture for n______

3. No reduction in the y______ of useful products

4. No c____________ of p_______
5. Prevent your microbe escaping!
 Batch Vs Continuous culture
Batch Culture:
• Mix microorganism starter population and fixed quantity of nutrient
solution at beginning.
• Grow for a fixed period.
• Empty fermentation tank and harvest product periodically. Start
again.
• Makes Penicillin (2o Metabolite) as stationary growth phase (see fed
batch)

Continuous Culture:
• Nutrients added and products removed from fermentation tank at
regular intervals (or continuously). Growth maintained in log phase
• Used to harvest GM Insulin and mycoprotein (Quorn) (1o
metabolite)
How are continuous and batch fermentation
different?
 Compare and contrast batch and continuous culture
Batch Continuous

Advantages

Disadvantages
Draw a continuous fermenter to
show how it works and keeps
aseptic conditions
 Fed Batch
• For 2o Metabolite
• Glucose added at intervals during stationary
for respiration
• This maintains culture and prevents death
phase;
• This maintains in stationary phase and
prevents rapid growth ;
 Why it is used for this purpose?
What reaction the enzyme speeds up? Name the enzyme.
What are the advantages of using it for this job?
 Enzymes
• We can use enzyme rather than whole
microorganism (which may produce waste
products)
• We need to purify enzymes using ‘downstream
processing’ – grow microbe then extract, purify and
concentrate enzyme
• Enzymes are specific to a reaction due to active
sites so no other reactions/by products
• Work at relatively low temperatures
• If high temp needed use thermophilic bacteria :
Bacillus stearothermophilus – these have
thermostable enzymes
What reaction the enzyme speeds up?

Name the enzyme.

Why it is used for this purpose?

What are the advantages of using it for this job?

What reaction the enzyme speeds up?

Name the enzyme.

Why it is used for this purpose?

What are the advantages of using it for this job?

What reaction the enzyme speeds up?

Name the enzyme.

Why it is used for this purpose?

What are the advantages of using it for this job?

What reaction the enzyme speeds up?

Name the enzymes.

Why it is used for this purpose?

What are the advantages of using it for this job?

 Enzymes
• We can use enzyme rather than whole
microorganism (which may produce waste
products)
• We need to purify enzymes using ‘downstream
processing’ – grow microbe then extract, purify and
concentrate enzyme
• Enzymes are specific to a reaction due to active
sites so no other reactions/by products
• Work at relatively low temperatures
• If high temp needed use thermophilic bacteria :
Bacillus stearothermophilus – these have
thermostable enzymes
Immobilised enzymes

• Carrier Bound:
Enzyme is attached to a
support unit by:
a) Adsorption –involves hydrophobic or ionic
bonds to an insoluble substance such as
porous carbon, glass beads, clay, resin, gold
b) Covalent bonds – attaches enzyme to
support and also has covalent bonds
between enzyme proteins. Cross-links are
made using glutaraldehyde
Immobilised enzymes

• Entrapment:
Enzyme trapped in semi permeable
protein alginate gel or network of
cellulose.
Enzyme in not bound to anything
OR
Enzyme trapped inside a micro
capsule
 Immobilised enzymes
• Membrane separation
Enzymes separated from substrate by partially
permeable membrane. Substrate and product
can pass through however enzyme cannot
What is Cat Milk?
 Immobolised enzymes
Lactose Galactose + Glucose

Carry out the practical that demonstrates the

use of immobolised enzymes
Answer the questions
• 10cm3 enzyme
• 10cm3 alginate
• Mix in beaker
• 20cm3 calcium chloride in different beaker
• Drip the enzyme/alginate mixture into CaCl2
• Wait to make beads
• Strain
• Put into empty syringe
• Place beaker underneath
• Add substrate and collect product underneath
• Re pour product into beads for 5 mins
• Do Benedict’s test on product
Immobilised Enzymes
Advantages
Purification/down stream
costs are low as product not
contaminated
Disadvantages
Enzyme less active as do not
mix freely with substrate

Enzymes immediately More expensive to set up:

available for reuse – useful in needs additional
continuous processes and time/equipment/materials
reduces cost
Immobilised enzymes more Contamination costly : whole
stable at higher temp/pH as system need to be stopped
matrix protects the enzyme
molecule – higher temps
increase rate of reaction
•• glucose isomerase for the conversion of glucose
to fructose
•• penicillin acylase alters penicillin structure for the
formation of semisynthetic penicillins
•• lactase for the hydrolysis of lactose to glucose and
galactose
•• amino acylase for production of pure samples of
L-amino acids
•• glucoamylase for the conversion of
dextrins/starches to glucose
L- and D-Amino Acids. Amino acids can occur in L- and D-
forms, but only L-forms are used by cells. Every amino
acid (except glycine) can occur in two isomeric forms, because of
the possibility of forming two different enantiomers (stereoisomers)
around the central carbon atom.
Suggest the role of the
zinc ion?