HOW CELLS GROW

IN CONTINUOUS
CULTURE
INTRODUCTION
Continuous Culture

 To keep a culture growing indefinitely. This can be done if:

fresh nutrient medium are continually supplied
products and cells are simultaneously withdrawn
 Provides constant environmental conditions for growth product formation and
supplies uniform-quality product.
 Determine the response of microorganisms to their environment and to produce
the desired products under optimal environmental conditions.
SOME SPECIFIC
DEVICES FOR
CONTINUOUS CULTURE
The primary types of continuous cultivation devices are the:
Some Specific
●

●
Chemostat
Turbidostat
Devices for
●
Plug flow reactors (PFR) Continuous
Culture
These units are modified by recycle of cells.
Chemostat
A schematic of a continuous-culture device laboratory setup (chemostat)
 The cell concentration in the culture vessel is maintained constant by
monitoring the optical density of the culture and controlling the feed
flow rate.

When the turbidity of the medium exceeds the set point, a pump is
Turbidostat activated and fresh medium is added.

The culture volume is kept constant by removing an equal amount of

culture fluid.
 The turbidostat is less used than the chemostat.

Turbidostats can be very useful in selecting subpopulations able to

Turbidostat withstand a desired environmental stress.

The selection of variants or mutants with desirable properties is very

important.
Typical laboratory setup for a turbidostat
 Since there is no back-mixing in an ideal PFR, fluid elements
containing active cells cannot inoculate other fluid elements at
different axial positions.
 Liquid recycle is required for continuous inoculation of nutrient
media.
 In a PFR, substrate and cell concentrations vary with axial
position in the vessel.

Plug Flow
Reactor
THE IDEAL CHEMOSTAT
  Perfectly Mixed Continuous-flow, Stirred-tank
Reactor (CFSTR)
 Control elements: pH, dissolved oxygen, temperature
Ideal  Fresh sterile medium is fed to the completely mixed
Chemostat and aerated (if required) reactor.
 Suspension is removed at the same rate.
 Liquid volume in the reactor is kept constant.
Simplified
Chemostat
  F is the volumetric flowrate of
nutrient solution (l/h)
 VR is the volume (l) (constant)
 X is the cell concentration (g/l)
 P is the extracellular product (g/l)
Cell Growth in  µg and kd are growth and
endogenous rates, respectively (h -1)
Ideal Chemostat  qp is the specific rate of extracellular
product formation (g P/ g cells-h)
 Yp/s is the product yield coefficient (g
P/g S)
 Subscript 0 denotes the parameters
at the feed medium
 x0 = x0 = x0=
0 0 0
k d≈ k d> kd>
0 0 0
qp= qp= qp>
1 0 0 0

Cell Growth in Ideal Chemostat

Washed
Out
THE CHEMOSTAT
AS A TOOL
Chemostat
 Chemostat is a culture vessel designed for
continuous culture.
 It has fixed volume and flow rate, and thus a
fixed dilution rate.
 Used as a tool to study mutation and
selection of cultures.
 Natural or induced mutation
 Errors in DNA replication – 10-6 to 10-8
gene per generation.
 Used for the selection of special organisms.
DEVIATIONS FROM
IDEALITY
In fermentations such as the utilization or production of polysaccharides or mycelial
fermentations, the fermentation broth may be highly viscous and it may be difficult to maintain
complete mixing.

Incomplete mixing is the rule in industrial-scale fermenters. The presence of incompletely mixed
regions in a “chemostat” culture is common. The term “chemostat” is actually applicable only to
perfectly mixed vessels.
 A simple, two-compartmental model of a “chemostat”
culture

 The reactor is divided into two regions: a well-mixed

region and a stagnant region. Feed and effluent streams
pass through the well-mixed region 1, and mass exchange
takes place between the two regions.
At steady state,

In an imperfectly mixed chemostat with a stagnant biotic phase, the dilution rate can
exceed mm without washout. The washout dilution rate is obtained by setting S0 = S1 =
S2
Effect of Environmental
Conditions on Microbial
Growth
Major Physical
Factors TEMPERATURE
pH
Affecting
Microbial
Growth SOLUTES AND
WATER
OXYGEN
ACTIVITY LEVEL
  Changes in osmotic concentration of the surroundings can affect
microbial growth as a selectively permeable plasma membrane
separates the microorganisms from their surroundings.
 In a hypertonic environment, the prokaryotes increase their
SOLUTES internal osmotic concentration through the synthesis or uptake of
choline, proline, glutamic acid and other amino acids.
AND WATER  A few prokaryotes like Halobacterium salinarium raise their
ACTIVITY osmotic concentration with potassium ions.
 Halophiles grow optimally in the presence of NaCl or other salts at
a concentration above about 0.2M.
 The plasma membrane of halophiles is also stabilized by high
concentration of sodium ions.
  Water activity (aw) is the amount of water available
to microorganisms and this can be reduced by
interaction with solute molecules (osmotic effect).
 Water activity is inversely related to osmotic
pressure.
SOLUTES  Microorganisms differ greatly in their ability to
AND WATER adapt to habitats with low water activity.
ACTIVITY  In a low aw habitat, the microorganisms must
expend extra effort to grow as it should maintain a
high solute concentration to retain water.
 Most of the microorganisms grow at aw =0.98 or
higher.
  It refers to the acidity or alkalinity of a solution. It is a measure of
the hydrogen ion activity of a solution and is defined as the
negative logarithm of the hydrogen ion concentration.
 pH = -log [H+] = log (1/H+)
 The pH scale ranges from 1.0 to 14.0 and most microorganisms
grow vary widely from pH 0 to 2.0 at the acid end to alkaline lakes
and soil that may have pH values between 9.0 and 10. The pH can
affect the growth of microorganisms and each species has a
definite pH growth range and pH growth optimum.
pH  Acidophiles
 Neutrophiles
 Alkalophiles
 Cyanidium caldarium (algae) and archaeon Sulfolobus
acidocaldarium are inhabitants of acidic hot springs; both grow
well around pH 1 to 3 and at high temperature.
 Drastic changes/variations in cytoplasmic pH can harm
microorganisms by disrupting the plasma membrane or inhibiting
the activity of enzymes and membrane transport proteins.
  Prokaryotes die if the internal pH drops much below 5.0 to 5.5.
External pH alterations also might alter the ionization of nutrient
molecules and thus reduce their availability to the organism. The
microorganism needs to maintain a neutral cytoplasmic pH and
for this the plasma membrane may be relatively impermeable to
protons.
 Neutrophiles
 Extreme alkalophiles
pH  The antiport systems probably correct small variations in pH. In
case of too much acidity (below 5.5 to 6.0) S.
typhimurium and E.coli synthesize an array of new proteins as part
of what has been called as their acidic tolerance response.
 If the external pH decreases to 4.5 or lower, chaperones such as
acid shock proteins and heat shock proteins are synthesized.
Microorganisms can change the pH of their own habitat by
producing acidic or basic metabolic waste products.
  Beyond a certain point of higher temperature,
slow growth takes place and damages the
microorganisms by denaturing enzymes,
transport carriers and other proteins.
 At very low temperature, membranes solidify
and enzymes don't work rapidly.
TEMPERATURE  In summary, when organisms are above their
optimum temperature, both the function and
cell structure is affected at low temperature,
function is affected.
 The cardinal temperatures vary greatly
between microorganisms.
  Microorganisms are classified into five classes
based on their temperature ranges for growth.
 1.  Psychrophiles
 2.  Psychrotrophs or Facultative
TEMPERATURE Psychrophiles
 3.  Mesophiles
 4.  Thermophiles
 5.  Hyperthermophiles
  Most microorganisms always are subjected to pressure of 10
atmospheres (atm).
 The hydrostatic pressure can reach to 600 to 1100 atm in the deep
PRESSURE sea with temperature about 2°C to 3°C.
 Organisms can survive and adapt at these extreme conditions and
many are barotolerant, increased pressure does adversely affect
them but not as much as it does to nontolerant bacteria.
  Types of Microorganism Based on their
Oxygen Capacity
Aerobe
Anaerobe
OXYGEN Obligate aerobes
LEVEL Facultative anaerobes
Aerotelerant anaerobes
Obligate anaerobes
Microaerophiles 
OXYGEN
LEVEL
RADIATION

 Electromagnetic Spectrum
HOW CELLS GROW IN
CONTINUOUS CULTURE
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