CELLS GROWTH IN CONTINUOUS CULTURE

In batch culture - Growth, product formation, and substrate utilization terminate after a certain time interval,

We have already talked

about it in class, hope you
Everything fixed. So, bacteria keep consuming food, remember
but there is not constant supply of food.
Continuous In
Continuous Out

In continuous culture - fresh nutrient medium is

continually supplied to a well-stirred culture, and
products and cells are simultaneously withdrawn

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In continuous culture

• Fresh nutrient medium is continually supplied to a well-stirred culture, and products and cells are
simultaneously withdrawn

• Growth and product formation can be maintained for prolonged periods in continuous culture

• After a certain period of time, the system usually reaches a steady state where cell, product, and substrate
concentrations remain constant.

• Continuous culture provides constant environmental conditions for growth and product formation and
supplies uniform-quality product.

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The Ideal Chemostat - The primary types of continuous cultivation devices
• An ideal chemostat is the same as a perfectly mixed Continuous-Flow, Stirred-Tank Reactor (CFSTR).

• Most chemostats require some control elements, such as pH and dissolvedoxygen control units, to be useful.

• Fresh sterile medium is fed to the completely mixed and aerated reactor, and cell suspension is removed at the same rate.

• Liquid volume in the reactor is kept constant.

A material balance on the cell concentration around the chemostat yields:

F - the flow rate of nutrient solution (l/h),

VR - is the culture volume (l) (assumed constant),
X - the cell concentration (g/l),
µg and kd - growth and endogenous (or death) rate constants,
respectively (h-1).
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 under steady-state conditions, the cell
concentration remains constant or vice-versa,
when dX/dt = 0, then reactor reach steady state
and the change in biomass concentration (X)
So, now if you rearranged this to over time (t) is zero i.e. X remain constant

dX/dt = X - kd X
So, what is the unit of D here?
Then,

Usually, the feed media are sterile (we sterilize the media first by autoclaving), X0 = 0,

if the endogenous metabolism or death rate is negligible compared to the growth rate ( kd << mg or kd= 0) and if the system is
at steady state (dX/dt = 0), then

=D and, what is the unit of D here? Is it matching or not?

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 = D, so 2 importants can be derived from this eqn

• In a chemostat, cells are removed at a rate equal to their growth rate, and the growth rate of cells is equal to the
dilution rate.

• This property allows the investigator to manipulate growth rate as an independent parameter and makes the
chemostat a powerful experimental tool

Now recall your memory, what was ? Remember the Monod eqn?

So, what happens in reactor? – substrate/media is

where S is the steady-state limiting substrate concentration (g/l).
If D is set at a value greater than mm, the culture cannot reproduce
quickly enough to maintain itself and is washed out
continuously going in from one end and
product+biomass are continuously coming out from
another end. Now, we need to give time to bacteria
to stay inside the reactor isn’t it? So that they
consume substrate and keep growing and making
product. Otherwise, they will WASH OUT and
nothing will happen
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 Rate of change in Biomass concentration = Rate of Production – Rate of removal

dX/dt = mX - DX

Steady state Dilution rate decreased Dilution rate increased

D S D S
dX/dt = 0
m m
m = D
a plot of 1/mg versus 1/S can be used to estimate values for mm and Ks

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Using eq. 6.67, we can relate effluent substrate concentration to dilution rate for D < mm

S0 – initial substrate
S – some unutilized substrate
some substrate used some substrate
for biomass growth used for product
S0 and S - feed and effluent substrate concentrations (g/l), formation
qP - specific rate of extracellular product formation (g P/mg cells h),
YM X/S and YP/S - yield coefficients (g cell/g S and g P/g S).

The use of the superscript M on YX/S denotes a maximum value of the yield coefficient 8
I hope you do remember
what is D from previous
slides? So, can you derive
this easy eqn from 6.69?

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In eqs. 6.71 and 6.72, the yield coefficient (YX/S) is assumed to be constant, which is an approximation, since endogenous
metabolism has been neglected.

Usually, YX/S varies with the limiting nutrient and growth rate. Consider the effect of inclusion of endogenous
metabolism will have; eq. 6.66 becomes

Now substitute this into eqn 6.70

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where ms is the maintenance coefficient based on substrate S.
YAP X/S is the apparent yield = (S0-S)/X
When YX/S is written, it should be interpreted as YAPX/S.
While YM X/S is a constant, YAPX/S varies with growth conditions if kd > 0.
Remember why it is constant? – cause its bacteria’s
maximum efficiency which is constant. In real
experiments, we try to achieve the maximum
efficiency
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Values of YMX/S and ms can be obtained from chemostat experiments by plotting 1/YAPX/S against 1/D.

The slope is ms and the intercept is 1/YMX/S

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In the presence of endogenous metabolism, we can also show that

Can you solve this, how you are getting this eqn?

Lets recall old eqns

So, from these eqns, we can have,

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Lets solve a problem, and all will be clear, when, where and how to use all those eqns you just read

Hint = you need to draw 2 different graphs, you

can use excel

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Chemostat with Recycle

Microbial conversions are autocatalytic, and the rate of conversion increases with cell concentration. To keep the cell
concentration higher than the normal steady-state level in a chemostat, cells in the effluent can be recycled back to the
reactor.

Cell recycle increases the rate of conversion (or productivity) and also increases the stability of some systems (e.g.,
wastewater treatment) by minimizing the effects of process perturbation.

Cells in the effluent stream are either centrifuged, filtered, or settled in a conical tank for recycling.

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A material balance on cell (biomass) concentration around the fermenter yields the following equation:

Where,
a is the recycle ratio based on volumetric flow rates,
C is the concentration factor or ratio of cell concentration in the cell recycle stream to the cell concentration in the reactor
effluent,
F is nutrient flow rate,
V is culture volume,
X0 and X1 are cell concentrations in feed and recycle streams,
X2 is cell concentration in effluent from the cell separator

At steady state, and if dX1/dt = 0 and X0 = 0 (that is, sterile feed); then eq. 9.8 becomes

I am sure, you can derive it from 9.8 easily…will take 2/3 steps maxm, F/V = D, if not forgotten
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Since C > 1 and a(1 - C) < 0, then mnet < D.

That is, a chemostat can be operated at dilution rates higher than the specific growth rate when cell recycle is used.

A material balance for growth-limiting substrate around the fermenter yields

Have you all got it? a(1 - C) < 0,

so if you divide any number by <1,
it will always increased, isn’t it?

Therefore, the steady-state cell concentration in a chemostat is increased by a factor of 1/(1 + a - aC) by cell
recycle.
If kd= 0, then, µnet = µg , and
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The substrate concentration in the effluent is determined from eq. 9.9 and the Monod eq. 6.30, where endogenous
metabolism is neglected, and is

Can you solve it by yourself? Simple steps,

hints: follow this 2 eqns

If kd= 0, then, µnet = µg , and

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Problem

In a chemostat with cell recycle, as shown in Fig. 9.1, the feed flow rate and culture volumes
are F = 100 ml/h and V = 1000 ml, respectively. The system is operated under glucose limitation, and the
yield coefficient, YMX/S is 0.5 gdw cells/g substrate. Glucose concentration in the feed is

S0 = 10 g glucose/l. The kinetic constants of the organisms are µm = 0.2 h-1,

Ks = 1 g glucose/l. The value of C is 1.5, and the recycle ratio is a = 0.7

The system is at steady state.
a. Find the substrate concentration in the recycle stream (S).
b. Find the specific growth rate (µnet)of the organisms.
c. Find the cell (biomass) concentration in the recycle stream

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 Fed-batch Operation

• In fed-batch culture, nutrients are continuously or semicontinuously fed, while

effluent is removed discontinuously (Fig. 9.8).
• Fed-batch culture is usually used to overcome substrate inhibition or catabolite
repression by intermittent feeding of the substrate. If the substrate is inhibitory,
intermittent addition of the substrate improves the productivity of the
fermentation by maintaining the substrate concentration low.
• Fed-batch operation is also called the semicontinuous system or variable-volume
continuous culture.

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Consider a batch culture where the concentration of biomass at a certain time is given by

When biomass concentration reaches its maximum value (Xm), the substrate concentration is very low (cause majority of

substrate has already been consumed, isn’t it?), So, S << S0, and also X0 << X.

That is, Xm =YMX/SS0.

Suppose that at Xm @ YMX/SS0, a nutrient feed is started at a flow rate F, with the substrate concentration S0.
The total amount of biomass in the vessel is Xt = VX,
where V is the culture volume at time t. The rate of increase in culture volume is

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 According to derivative rule,

All substrate consumed,

so growth rate is zero
When the substrate is totally consumed, S = 0 and X = Xm = YMx/sS0.
Furthermore, since nearly all the substrate in a unit volume is consumed, then dX/dt = 0. This is an example of a
quasi-steady state.
A fed-batch system operates at quasi-steady state when nutrient consumption rate is nearly equal to nutrient feed rate.
Since dX/dt = 0 at quasi-steady state, then

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 There are all same.

where St is the total amount of the rate-limiting substrate in the culture and S0 is the concentration of substrate in the
feed stream.
At quasi-steady state, Xt = VXm and essentially all the substrate is consumed, so no significant level of substrate can
accumulate (means St = 0). Therefore,

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 Equation 9.31 at quasi-steady state with S = 0 yields

Xm =YMX/SS0 slide no 20

At quasi-steady state, dX/dt = 0 , Xt = VXm

Using these 2 eqns, you can easily derive 9.37 from 9.31

Integration of eq. 9.37 from t = 0 to t with the initial amount of biomass in the reactor being Xt0 yields

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Problem

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