Growth Requirements

Microbial growth an increase in a population of

microbes rather than an increase in size of an individual

Result of microbial growth is discrete colony an
aggregation of cells arising from single parent cell
Reproduction results in growth

Organisms use a variety of nutrients for their energy
needs and to build organic molecules and cellular
structures

Most common nutrients those containing necessary
elements such as carbon, oxygen, nitrogen, and hydrogen

Microbes obtain nutrients from variety of sources

Growth Requirements
Growth Requirements

Nutrients: Chemical and Energy Requirements
Sources of carbon, energy, and electrons
Two groups of organisms based on source of carbon
Autotrophs use an inorganic source of carbon (carbon dioxide)
Heterotrophs catabolize reduced organic molecules (proteins,
carbohydrates, amino acids, and fatty acids)

Two groups of organisms based on use of chemicals or light as
source of energy
Chemotrophs acquire energy from redox reactions involving inorganic
and organic chemicals
Phototrophs use light as their energy source

Nutrient Requirements
Energy Source
Phototroph
Uses light as an energy source
Chemotroph
Uses energy from the oxidation of reduced chemical
compounds
Nutrient Requirements
Electron (Reduction potential) Source
Organotroph
Uses reduced organic compounds as a source for reduction
potential
Lithotroph
Uses reduced inorganic compounds as a source for
reduction potential
Nutrient Requirements
Carbon source
Autotroph
Can use CO
2
as a sole carbon source
(Carbon fixation)
Heterotroph
Requires an organic carbon source; cannot use CO
2
as a
carbon source
Nutrient Requirements
Nitrogen source
Organic nitrogen
Primarily from the catabolism of amino acids
Oxidized forms of inorganic nitrogen
Nitrate (NO
3
2-
) and nitrite (NO
2
-
)
Reduced inorganic nitrogen
Ammonium (NH
4
+
)
Dissolved nitrogen gas (N
2
) (Nitrogen fixation)
Nutrient Requirements
Phosphate source
Organic phosphate
Inorganic phosphate (H
2
PO
4
-
and HPO
4
2-
)
Nutrient Requirements
Sulfur source
Organic sulfur
Oxidized inorganic sulfur
Sulfate (SO
4
2-
)
Reduced inorganic sulfur
Sulfide (S
2-
or H
2
S)
Elemental sulfur (S
o
)
Nutrient Requirements
Special requirements
Amino acids
Nucleotide bases
Enzymatic cofactors or vitamins
Nutrient Requirements
Prototrophs vs. Auxotrophs
Prototroph
A species or genetic strain of microbe capable of growing
on a minimal medium consisting a simple carbohydrate
or CO
2
carbon source, with inorganic sources of all other
nutrient requirements
Auxotroph
A species or genetic strain requiring one or more complex
organic nutrients (such as amino acids, nucleotide bases,
or enzymatic cofactors) for growth
Nutrient Transport Processes
Simple Diffusion
Movement of substances directly across a
phospholipid bilayer, with no need for a transport
protein
Movement from high low concentration
No energy expenditure (e.g. ATP) from cell
Small uncharged molecules may be transported via
this process, e.g. H
2
O, O
2
, CO
2
Nutrient Transport Processes
Facilitated Diffusion
Movement of substances across a membrane with the
assistance of a transport protein
Movement from high low concentration
No energy expenditure (e.g. ATP) from cell
Two mechanisms: Channel & Carrier Proteins
Nutrient Transport Processes
Active Transport
Movement of substances across a membrane with the
assistance of a transport protein
Movement from low high concentration
Energy expenditure (e.g. ATP or ion gradients) from
cell
Active transport pumps are usually carrier proteins
Nutrient Transport Processes
Active Transport (cont.)
Active transport systems in bacteria
ATP-binding cassette transporters (ABC transporters):
The target binds to a soluble cassette protein (in periplasm of
gram-negative bacterium, or located bound to outer leaflet of
plasma membrane in gram-positive bacterium). The target-
cassette complex then binds to an integral membrane ATPase
pump that transports the target across the plasma membrane.
Nutrient Transport Processes
Active Transport (cont.)
Active transport systems in bacteria
Cotransport systems: Transport of one substance from a low
high concentration as another substance is simultaneously
transported from high low.

For example: lactose permease in E. coli:
As hydrogen ions are moved from a high concentration
outside low concentration inside, lactose is moved from a
low concentration outside high concentration inside
Nutrient Transport Processes
Active Transport (cont.)
Active transport systems in bacteria
Group translocation system: A molecule is transported while
being chemically modified.

For example:
phosphoenolpyruvate: sugar phosphotransferase systems (PTS)

PEP + sugar (outside) pyruvate + sugar-phosphate (inside)




Nutrient Transport Processes
Active Transport (cont.)
Active transport systems in bacteria
Iron uptake by siderophores:

Low molecular weight organic molecules that are secreted by
bacteria to bind to ferric iron (Fe
3+
); necessary due to low
solubility of iron; Fe
3+
- siderophore complex is then transported
via ABC transporter




Microbiological Media
Liquid (broth) vs. semisolid media
Liquid medium
Components are dissolved in water and sterilized
Semisolid medium
A medium to which has been added a gelling agent
Agar (most commonly used)
Gelatin
Silica gel (used when a non-organic gelling agent is required)
Microbiological Media
Chemically defined vs. complex media
Chemically defined media
The exact chemical composition is known
e.g. minimal media used in bacterial genetics experiments
Complex media
Exact chemical composition is not known
Often consist of plant or animal extracts, such as soybean
meal, milk protein, etc.
Include most routine laboratory media,
e.g., tryptic soy broth

Microbiological Media
Selective media
Contain agents that inhibit the growth of certain bacteria
while permitting the growth of others
Frequently used to isolate specific organisms from a
large population of contaminants
Differential media
Contain indicators that react differently with different
organisms (for example, producing colonies with
different colors)
Used in identifying specific organisms
Measurement of
Microbial Growth
Serial dilution and colony counting
Also know as viable cell counts
Concentrated samples are diluted by serial dilution
The diluted samples can be either plated by spread
plating or by pour plating
- Quantifying cell concentration:

- direct: no suspended solid and interference
compounds.

Cell mass concentration preferred
dry weight, optical density (turbidity) (600-
700nm Wave Length)

cell number density:
Petroff-Hausser slide (Hemocytometer), plate
counts, etc.
Measurement of
Microbial Growth
Serial dilution (cont.)
Diluted samples are spread onto media in petri dishes
and incubated
Colonies are counted. The concentration of bacteria
in the original sample is calculated (from plates with
25 250 colonies, from the FDA Bacteriological
Analytical Manual).
A simple calculation, with a single plate falling into
the statistically valid range, is given below:
ml) in plated, lume factor)(vo (dilution
counted colonies #
sample original in
ml
CFU
=
Measurement of
Microbial Growth
Serial dilution (cont.)
If there is more than one plate in the statistically
valid range of 25 250 colonies, the viable cell count
is determined by the following formula:
Measurement of
Microbial Growth
Where:
C = Sum of all colonies on all plates between 25 - 250
n
1
= number of plates counted at dilution 1
(least diluted plate counted)
n
2
= number of plates counted at dilution 2
(dilution 2 = 0.1 of dilution 1)
d
1
= dilution factor of dilution 1
V= Volume plated per plate
V * * ...] ) * 1 . 0 ( ) * 1 [(
C
ml
CFU
d n n
1 2 1
+ +
=

Membrane filtration
Used for samples with low microbial concentration
A measured volume (usually 1 to 100 ml) of sample
is filtered through a membrane filter (typically with a
0.45 m pore size)
The filter is placed on a nutrient agar medium and
incubated
Colonies grow on the filter and can be counted
Measurement of
Microbial Growth
Measurement of
Microbial Growth
Turbidity
Based on the diffraction or scattering of light by
bacteria in a broth culture
Light scattering is measured as optical absorbance in
a spectrophotometer
Optical absorbance is directly proportional to the
concentration of bacteria in the suspension
Measurement of
Microbial Growth
Mass determination
Cells are removed from a broth culture by
centrifugation and weighed to determine the wet
mass.
The cells can be dried out and weighed to determine
the dry mass.
Measurement of enzymatic activity or other cell
components
Measurement of
Microbial Growth
Microscopic cell counts
Calibrated Petroff-Hausser counting chamber, similar
to hemocytometer, can be used
Generally very difficult for bacteria since cells tend to
move in and out of counting field
Can be useful for organisms that cant be cultured
Special stains (e.g. serological stains or stains for viable
cells) can be used for specific purposes
Serial dilution and colony counting
Also know as viable cell counts
Concentrated samples are diluted by serial dilution
KINETICS AND TECHNOLOGY
OF NUTRIENT LIMITATION
Type of culture;
- Batch; varies during culture cycle
- Fed-batch; is controlled or regulated after a certain time
- Continuous; is controlled

reflects the physiological state or intracellular environment
control control intracellular environment

Growth in Batch Culture
Growth is generally used to refer to the
acquisition of biomass leading to cell division, or
reproduction

A batch culture is a closed system in broth
medium in which no additional nutrient is added
after inoculation of the broth.
Growth in Batch Culture
Typically, a batch culture passes through four
distinct stages:
Lag stage
Logarithmic (exponential) growth
Stationary stage
Death stage
Growth in Batch Culture
Mean Generation Time
and Growth Rate
The mean generation time (doubling time) is the
amount of time required for the concentration of
cells to double during the log stage. It is
expressed in units of minutes.

Growth rate (min
-1
) =


Mean generation time can be determined directly
from a semilog plot of bacterial concentration vs
time after inoculation
time generation mean
1
Mean Generation Time
and Growth Rate
Growth of E. coli 23716,
9-20-01 batch culture
y = 0.0187e
0.0069x
R
2
= 0.9928
0.01
0.1
1
10
0 200 400 600 800 1000 1200 1400 1600
time, min
A
4
2
5
Mean Generation Time
and Growth Rate
Lag phase
A period of adaptation for the cells to their new
environment

New enzymes are synthesized.

A slight increase in cell mass and volume, but no increase in cell
number

Prolonged by low inoculum volume, poor inoculum condition (high
% of dead cells), age of inoculum, nutrient-poor medium

Multiple lag phases: (diauxic growth) medium contains more than
one carbon source
Batch Growth Kinetics
Batch Growth Kinetics
Exponential growth phase

In this phase, the cells have adjusted to their new
environment and multiply rapidly (exponentially)

Balanced growth all components of a cell grow at the
same rate.

Growth rate is independent of nutrient concentration,
as nutrients are in excess.
Batch Growth Kinetics
Deceleration growth phase


Very short phase, during which growth decelerates due
to either:

Depletion of one or more essential nutrients

The accumulation of toxic by-products of growth (e.g.
Ethanol in yeast fermentations)

Period of unbalanced growth: Cells undergo internal
restructuring to increase their chances of survival
Batch Growth Kinetics
Stationary Phase:

With the exhaustion of nutrients (S0) and build-up of
waste and secondary metabolic products

- The growth rate equals the death rate.

- There is no net growth in the organism population.

- Cells may have active metabolism to produce secondary
metabolites.
Primary metabolites are growth-related: ethanol by S. cerevisae.
Secondary metabolites are non-growth-related: antibiotics,
pigments.
Batch Growth Kinetics
Death Phase:
The living organism population decreases with time, due to
a lack of nutrients and toxic metabolic by-products.

The rate of death usually follows:
constant. rate death order - first the is
'
'
d
d
k
N k
dt
dN
=
Growth Kinetics
Introduction
- Autocatalytic reaction: The rate of growth is directly related to cell
concentration

substrates + cells extracellular products + more cells
S + X P + nX

S: substrate concentration (g/L); X: cell mass concentration (g/L);
P: product concentration (g/L); n: increased number of biomass.

Net specific growth rate (1/time):

t: the time
dt
dX
X
1
net

Growth Kinetics
Introduction
Net specific replication rate (1/time):


dt
dN
N
1
R

: '
R

Gross specific replication rate (1/time)

:
d
k
The rate of cell death (1/time)
R

d
k = '
R
R

N : Cell number concentration (cell number /L)
Growth in Continuous Culture
A continuous culture is an open system in which
fresh media is continuously added to the culture at a
constant rate, and old broth is removed at the same rate.
This method is accomplished in a device called a
chemostat.
Typically, the concentration of cells will reach an
equilibrium level that remains constant as long as the
nutrient feed is maintained.
0.05% glucose run assuming delay of 20 min and 28% yield
0.000
0.050
0.100
0.150
0.200
0.250
0 500 1000 1500 2000 2500
time (min)
b
a
c
t
e
r
i
a

(
g
/
l
)
observed
initial data
predicted
0.05% glucose run assuming delay of 20 min and 28% yield
0.000
0.050
0.100
0.150
0.200
0.250
0 500 1000 1500 2000 2500
time (min)
b
a
c
t
e
r
i
a

(
g
/
l
)
observed
initial data
predicted
4.1.1. TYPES OF CONTINUOUS CULTURE
Method of control;
- Chemostat - regulated by control of concentration of
limiting nutrient

- Turbidostat - regulated by biomass using optical density
(photoelectric cell)

- Biostat - regulated by systems monitoring biomass other
than optical density (e.g CO
2
production)

Cell
Number
Time in Hours
Steady State
The development of growth in a chemostat
Inoculation

max

Population density increases
Nutrient limitation causes decrease in
Growth rate equals loss of cell biomass
Mathematical relationships of
growth in chemostats
Relationship between growth rate or specific
growth rate and medium flow can be described
mathematically
The medium flow through the system is
represented by the term dilution rate

D =
D = dilution rate (h
-1
)
V = culture volume (l)
F = flow rate (l h
-1
)
F
V
4.1.2. KINETICS OF CONTINUOUS CULTURE
Thus:
Mass balance or the rate of change of cells in reactor = RATE of
ACCUMULATION minus RATE of LOSS

dX /dt = .X - D.x

- Mass balance of the substrate = INPUT minus LOSS DUE TO OUTFLOW
minus SUBSTRATE USED BY BIOMASS

dS / dt = D. Sr - D. S - . X / Y
X = Total biomass
D = Dilution rate
x = Biomass concentration
= Specific growth rate
Y = Yield
S = Substrate conc in fermenter
S
r
= Substrate conc in reservoir
The empirically derived equation for the relationship between
specific growth rate and [S] is Monod equation

D =
max
. S / (Ks + S)

This is the most basic model for continuous culture


NOTE; When dX / dt = 0 (at steady state) then D =

This equation demonstrates how the steady state substrate
concentration in the chemostat is determined by the dilution rate
INCORPORATE MONOD MODEL
Exponential phase Chemostat
of batch culture operating in
steady-state

Growth rate of culture

Specific growth rate of culture

Biomass

Available nutrients

Culture volume

Toxic metabolites

Constant, Variable, Increasing, Decreasing
Increasing
Constant
Increasing
Decreasing
Constant
Increasing
Constant
Constant
Constant
Constant
Constant
Constant
Batch versus Chemostat
4.1.5. APPLICATION OF
CONTINUOUS CULTURE
INDUSTRY;
Waste-treatment
Single-cell protein
Continuous beer production
Continuous amino acids, organic acids production
Continuous ethanol
Continuous bakers yeast

4.1.6 ADVANTAGES / DISADVANTAGES OF CC
Advantages
Uniformity of operation
Process demands are constant
i.e. continuous cycle of sterilisation, fermentation, harvesting, extraction
Once in steady-state demands re process control are constant
i.e. oxygen demand

Disadvantages
Susceptibility to contamination
Duration of run is longer increased chance of contamination
Strain degeneration arising from large number of generations
Contamination with "fitter" competitor e.g. lower Ks

4.1.7. MODIFICATIONS OF BASIC
CHEMOSTAT
MULTI-STAGE
Different environments or growth rates in the various reactors (e.g. 1st
biomass, 2nd product)

SINGLE STAGE WITH CELL RECYCLE
Application in activated sludge waste-treatment
Relationship between D and different when recycle used.

EFFECT OF FEEDBACK;
1. Increase biomass conc. in fermenter - lower in effluent
2. Decrease residual substrate
3. Maximise rate of product formation
4. Dcrit is increased - useful when substrate is dilute

F
1

S
R

X
1

S
1

V
1

F
O2

S
R2

X
2

S
2

V
2

F
2

Chemostats in series
CONTINUOUS CULTURE PRINCIPLES

Also applied in;

UP-FLOW REACTORS (often with immobilised cells)

PLUG-FLOW SYSTEMS

4.2. NUTRIENT LIMITATION ALSO
APPLIED IN FED-BATCH
4.2.1 Fed-Batch

Takes advantage of fact that residual substrate concentration
may be maintained at very low levels

Type of continuous culture but volume is not constant.
Quasi-steady state achieved.

CLASSIFICATION OF FED-BATCH
OPERATION
Without feed-back control - programmed feed-rate
1. Intermittent addition
2. Constant rate
3. Exponentially increased rate

With feed-back control
1. Indirect feed-back
e.g. respiration rate, dissolved oxygen, pH
2. Direct feed-back
concentration of substrate in culture exerts control
4.2.2 INDUSTRIAL APPLICATION OF FED-BATCH
Penicillin
Glucose, phenyl acetic acid, ammonia source
Cephalosporin
Glucose, methionine
Streptomycin
Glucose, ammonia source
Glutamic acid
Urea, ethanol, (acetic acid)
Amylase
Carbon source
Bakers Yeast
Glucose
Citric acid
Glucose, ammonia

Factors that Influence Growth
Growth vs. Tolerance
Growth is generally used to refer to the acquisition of
biomass leading to cell division, or reproduction
Many microbes can survive under conditions in which
they cannot grow
The suffix -phile is often used to describe conditions
permitting growth, whereas the term tolerant describes
conditions in which the organisms survive, but dont
necessarily grow
For example, a thermophilic bacterium grows under
conditions of elevated temperature, while a
thermotolerant bacterium survives elevated
temperature, but grows at a lower temperature
Factors that Influence Growth
Obligate (strict) vs. facultative
Obligate (or strict) means that a given condition is
required for growth
Facultative means that the organism can grow under
the condition, but doesnt require it
The term facultative is often applied to sub-optimal
conditions
For example, an obligate thermophile requires elevated
temperatures for growth, while a facultative thermophile
may grow in either elevated temperatures or lower
temperatures
Factors that Influence Growth
Temperature
Most bacteria grow throughout a range of approximately
20 Celsius degrees, with the maximum growth rate at a
certain optimum temperature
Psychrophiles: Grows well at 0C; optimally between
0C 15C
Psychrotrophs: Can grow at 0 10C; optimum between
20 30C and maximum around 35C
Mesophiles: Optimum around 20 45C
Moderate thermophiles: Optimum around 55 65 C
Extreme thermophiles (Hyperthermophiles):
Optimum around 80 113 C
Factors that Influence Growth
pH
Acidophiles:
Grow optimally between ~pH 0 and 5.5
Neutrophiles
Growoptimally between pH 5.5 and 8
Alkalophiles
Grow optimally between pH 8 11.5
Factors that Influence Growth
Salt concentration
Halophiles require elevated salt concentrations to
grow; often require 0.2 M ionic strength or greater
and may some may grow at 1 M or greater; example,
Halobacterium
Osmotolerant (halotolerant) organisms grow over a
wide range of salt concentrations or ionic strengths;
for example, Staphylococcus aureus
Factors that Influence Growth
Oxygen concentration
Strict aerobes: Require oxygen for growth (~20%)
Strict anaerobes: Grow in the absence of oxygen; cannot
grow in the presence of oxygen
Facultative anaerobes: Grow best in the presence of
oxygen, but are able to grow (at reduced rates) in the
absence of oxygen
Aerotolerant anaerobes: Can grow equally well in the
presence or absence of oxygen
Microaerophiles: Require reduced concentrations of
oxygen (~2 10%) for growth
Quorum Sensing
A mechanism by which members of a bacterial
population can behave cooperatively, altering
their patterns of gene expression (transcription) in
response to the density of the population
In this way, the entire population can respond in a
manner most strategically practical depending on
how sparse or dense the population is.
Quorum Sensing
Mechanism:
As the bacteria in the population grow, they secrete a
quorum signaling molecule into the environment (for
example, in many gram-negative bacteria the signal is
an acyl homoserine lactone, HSL)
When the quorum signal reaches a high enough
concentration, it triggers specific receptor proteins
that usually act as transcriptional inducers, turning on
quorum-sensitive genes