Chapter 5: Operating Considerations for Bioreactors

for Suspension and Immobilized Cultures

Prof. Zakaria Al-Qodah

Department of Chemical Engineering

School of Engineering

2nd Semester (2019-2020)

Presentation Outline: Lectures 1-5
❖ Introduction: Choosing Cultivation Methods

❖ Modifying Batch and Continuous Reactors

❖ Immobilized Cell Systems

 Batch or Continuous Scheme?

❖ A simple batch and continuous-flow stirred-tank reactor

(CFSTR) represent extremes

❖ There are other reactors with intermediate characteristics

❖ The choice depends on:

▪ Productivity

▪ Genetic instability

▪ operability and reliability

.
 Productivity

In batch culture: four distinct phases are present:

▪Lag phase
▪Exponential growth phase
▪Harvesting
▪Preparation for a new batch (cleaning, sterilizing, and filling).

▪Assuming tl is the required time for lag phase, harvesting and

preparation 1 Xm
▪ Exponential time equal: c = ln + l
m X o
1 Xm
▪Cycle time c = ln + l (9.1)
m X o
 Productivity

where Xm is the maximal attainable cell concentration and

X0 is the cell concentration at inoculation.
X=Yx/s(S0-S)
The total amount of cell mass produced: X m − X o = YX / S So (9.2)

The rate of cell mass production in one batch cycle (rb) is :

(9.3)

As discussed in Chapter 6, the maximum productivity of a chemostat is

found by differentiating DX with respect to D and setting dDX/dD to zero.
 Productivity

We found that (6.83)

By the same procedure (9.4)

Thus, the best productivity that could be expected from a chemostat

where Monod kinetics apply is:

(9.5)
If So >> Ks Then equation 9.5 reduces to:
(9.6)
 Productivity

❖ The ratio of rate between continuous and batch is:

rc ,opt  mYX / S S o
=
rb YX / S S o = (9.7)
1 X
ln m + l
m X o
❖ Most commercial fermentations operate with:
rc ,opt
Xm/X0 = 10 to 20, tl = 5 and μm = 1 h-1, Accordingly: =8
rb
❖ Based on this productivity advantage we might be surprised to
learn that most commercial bioprocesses are batch systems.

❖ Why? There are several answers.

Batch or Continuous Culture? Productivity

❖ Eq. 9.7 applies only to growth-associated products.

❖ Many secondary products are not made by growing cells; growth

represses product formation.

❖ Under such circumstances, product is made only at very low

dilution rates, far below those values optimal for biomass
formation.

❖ For secondary products, the productivity in a batch reactor may

significantly exceed that in a simple chemostat.
Batch or Continuous Culture? Genetic instability
❖ Another primary reason for the choice of batch systems over a
chemostat is genetic instability.
❖ The biocatalyst in most bioprocesses has undergone extensive
selection.
❖ These highly “bred” organisms often grow less well than the parental
strain.
❖ A chemostat imposes strong selection pressure for the most rapidly
growing cell.
❖ Back-mutation from the productive specialized strain to one similar to
the less productive parental strain is always present.
❖ In the chemostat the less productive variant will become dominant,
decreasing productivity.
❖ In the batch culture the number of generations available (< 25 from slant
cultures to a commercial-scale fermenter) for the revertant cell to
outgrow the more productive strain is limited.
Batch or Continuous Culture? operability and reliability

❖ Another consideration is operability and reliability.

❖ Batch cultures can suffer great variability from one run to

another. Variations in product quality and concentration create
problems in downstream processing and are undesirable.

❖ However, long-term continuous culture can be problematic;

pumps may break, controllers may fail, and so on.

❖ Maintenance of sterility (absence of detectable foreign

organisms) can be very difficult to achieve for periods of
months, and the consequences of a loss of sterility are more
severe than with batch culture.
Batch or Continuous Culture? Market economics

❖ A continuous system forms the basis of a dedicated processing

system—dedicated to a single product.

❖ Many fermentation products are required in small amounts, and

demand is difficult to project.

❖ Batch systems provide much greater flexibility. The same

reactor can be used for two months to make product A and
then for the next three for product B and the rest of the year for
product C.
Modified Bioreactors: Chemostat with Recycle

❖ To keep the cell concentration higher than the normal steady-

state level, cells in the effluent can be recycled back to the
reactor.

❖ Advantages of Cell Recycle

1. Increase productivity for biomass production

2. Increase stability by dampening perturbations of input stream
properties
Modified Bioreactors: Chemostat with Recycle
Chemostat with Recycle: Biomass Balance

Input – Output + Generation = Rate of Change

(9.8)

where α is the recycle ratio based on volumetric flow rates, C is the conc. ratio
of cell concentration in the cell recycle stream to the cell concentration in the
reactor effluent,

A chemostat can be operated at dilution rates higher than the

Chemostat with Recycle: Biomass Balance

The net result is that the substrate concentration decreases

Chemostat with Recycle: Substrate Balance

(9.10)

(9.11)

(9.12)

(9.13)

The net result is that the Biomass concentration increases

Example 9.1
 Multiple Chemostat Systems

❖ In some fermentations processes, particularly for secondary

metabolite production, the growth and product-formation steps need
to be separated, since optimal conditions for each step are different.

❖ Conditions such as temperature, pH, and limiting nutrients may be

varied in each stage, resulting in different cell physiology and cellular
products in multistage systems.

Growth stage Product formation stage

 Multiple Chemostat Systems (cont.)

❖ Biomass and substrate balances on the first stage yield the following
equations (ignoring endogenous metabolism

The biomass balance for the second stage yields

At steady state, eq. 9.17 becomes

 Multiple Chemostat Systems (cont.)

❖ The substrate balance for the limiting substrate in the second stage
is:

At steady state, eq. 9.19 becomes:

Where

Equations 9.18 and 9.20 can be solved simultaneously for X2 and S2 by

substituting in both equations or any other
functional form that describes 2.
Two
, Chemostat Systems with a feed stream to the second stage
Fed-batch Operation
,

❖ In fed-batch culture, nutrients are continuously or semi-continuously

fed, while effluent is removed discontinuously

❖ Such a system is called a repeated fed-batch culture, semi

continuous system or variable-volume continuous culture.

❖ Used to overcome substrate inhibition or catabolite repression by

intermittent feeding of the substrate.

❖ If the substrate is inhibitory, intermittent addition of the substrate

Fed-batch Operation
,

❖ In batch culture the biomass concentration at a certain time is given by:

❖ When X reaches its maximum value, Xm, the substrate concentration is very
low, S  S0, and also X0  X. Then we can write:

❖ Suppose at this point 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. X=Xt/V dX/dt=
❖ The rate of increase in culture volume is:

❖ Integration gives:
Fed-batch
, Operation

❖ The rate of change in biomass concentration is:

❖ Since dXt/dt = netXt, dV/dt = F, and F/V = D, eq. 9.31 becomes

dX V net X t − X t F  net X t − X t F / V (  net − D) X t

= 2
= =
dt V V V

❖ Nearly all the substrate in a unit volume is consumed, then dX/dt = 0.

❖ net = D (9.33)
,
Fed-batch Operation: Substrate balance
❖ If maintenance energy can be neglected:

❖ then

❖ The balance on the rate-limiting substrate without maintenance energy is

❖ At quasi-steady state, Xt = VXm and essentially S = 0

dX VdX / dt − X t dV / dt
=0= 2
→ VdX / dt − X t
F = 0 → dX / dt = X m F
dt V
❖ dX / dt = X m F = FY m
X /S So (9.37)
Fed-batch
,
Operation: Substrate balance

❖ Integration of eq. 9.37 from t = 0 to t with the initial amount of biomass in

the reactor being Xt0 yields

❖ That is, the total amount of cell in the culture increases linearly with time in
a fed-batch culture.

❖ Dilution rate and therefore μnet decrease with time in a fed-batch culture.

❖ Since net = D at quasi-steady state, the growth rate is controlled by the

dilution rate.

❖ The use of unstructured models is an approximation, since μnet is a function

of time.
Fed-batch
, Operation: Product balance

❖ Product profiles in a fed-batch culture can be obtained by using the

definitions of YP/S or qP.

❖ P = YP / S So 9.39

❖ or the potential product output is FP = FYP / S So 9.40

dPt
=q p X t
❖ When qP is constant, dt 9.41

❖ Substituting Xt = V Xm =( V + Ft) Xm into eq. 9.41 yields

O

❖ 9.42
Fed-batch
, Operation: Product balance

❖ Integration of eq. 9.42 yields

❖ In terms of product concentration, eq. 9.43 can be written as:

❖ If the cycle time tw is constant and the system is always at quasi-steady

state, then P at the end of each cycle is given by:
Fed-batch
, Operation: Product balance

❖ where Dw = F/Vw, Vw is the culture volume at the end of each cycle

❖ V0 is the initial volume,

❖  is the fraction of culture volume remaining at each cycle (= V0/Vw),

❖ and tw is the cycle time. The cycle time is defined as

❖ Substitution of eq. 9.46 into eq. 9.45 yields:

❖ fed-batch culture is its use in some antibiotic fermentations

Figure
, 9.9.
Perfusion
, Systems

❖ An alternative to fed-batch culture is a perfusion system, which are used

most often with animal cell cultures.

❖ The basic characteristic is constant medium flow, cell retention, and in some
cases selective removal of dead cells.

❖ High cell density can be achieved.

❖ Cell retention is usually achieved by membranes or screens or by a

centrifuge capable of selective cell removal.

❖ When a membrane is used, the system has characteristics of an immobilized

cell system except the cells are usually maintained in suspension and mixed.

❖ With a selective removal/recycle the system approaches the cell recycle

Perfusion
,
Systems
 Immobilized Cell Systems

Restriction of cell mobility within a confined space

 Potential Advantages:

❖ Provides high cell concentrations per unit of reactor volume.

❖ Eliminates the need for costly cell recovery and recycle.

❖ May allow very high volumetric productivities.

❖ May provide higher product yields, genetic stability, and shear

damage protection.

❖ May provide favorable microenvironments sresulting in uch as cell-

cell contact, nutrient-product gradients, and pH gradients higher
yields.
Potential Disadvantages

❖ If cells are growing (as opposed to being in stationary

phase) and/or evolve gas (CO2, physical disruption of
immobilization matrix could result.

❖ Products must be excreted from the cell to be recovered

easily.

❖ Mass transfer limitations may occur as in immobilized

enzyme systems.
Methods of Immobilization
Methods of Immobilization (cont.)
Methods of Immobilization (cont.)
Methods of Immobilization (cont.)
Methods of Immobilization (cont.)
Methods of Immobilization (cont.)
Methods of Immobilization (cont.)
Methods of Immobilization (cont.)
Analysis of Biofilm Mass Transfer
Analysis of Biofilm Mass Transfer (cont.)
Analysis of Biofilm Mass Transfer (cont.)
Analysis of Biofilm Mass Transfer (cont.)
Analysis of Biofilm Mass Transfer (cont.)
Effectiveness Factor
Effectiveness Factor
Spherical Particle of Immobilized Cells, Fig. 9.14
Diffusion Effects in Cells Immobilized in a Porous
Matrix

At steady state, the intraparticle diffusion rate of

substrate equals to the reaction rate in a
spherical shell:
d[S ] 2 d[S ]
De 4r − De 4r 2 = v4r 2 r
dr r + r dr r
R

v is the reaction rate

per unit volume of support (mg/cm3-s).
De is the effective diffusivity (cm2/s). r r+Δr
Diffusion Effects in Cells Immobilized in a
Porous Matrix
Dividing the two sides of the equation by 4r
yields
d[S ] 2 d[S ] 2
De r − De r
r + r r
dr dr = vr 2
r
d d[S ] 2
When r →0
dr
( De
dr
r ) = vr 2

Re-arrange this equation

d 2[S ] 2 d[S ] 2
De( r + 2r ) = vr
dr 2 dr
 Diffusion Effects in Enzymes
Immobilized in a Porous Matrix
Dividing the two sides of the equation by r2, yields,

d 2[S ] 2 d[S ] Vm" [S ]

De( + )=v v=
dr 2 r dr K m + [S ]

Then
d 2[S ] 2 d[S ] Vm" [S ]
De( + )=
dr 2 r dr K m + [S ]
"
Vm is the maximum reaction rate per unit volume of support
(mg/cm3-s).
De is the effective diffusivity (cm2/s).
Analysis of Mass Transfer in Spherical Particle
Particle Effectiveness
Particle Effectiveness (cont.)
Particle Effectiveness (cont.)
Particle Effectiveness (cont.)
Particle Effectiveness (cont.)
Bioreactors Using Immobilized Cells, Fig.
Bioreactors Using Immobilized Cells, Fig.
Bioreactors Using Immobilized Cells, Fig.
Bioreactors Using Immobilized Cells, Fig.
 Immobilized Enzyme Reactors

Recycle packed column reactor:

- allow the reactor to operate at high fluid velocities.
- a substrate that cannot be completely processed on a single pass
Fluidized Bed Reactor:
- a high viscosity substrate solution
- a gaseous substrate or product in a continuous reaction system
- care must be taken to avoid the destruction and
decomposition of immobilized enzymes
- An immobilized enzyme tends to decompose
upon physical stirring.
- The batch system is generally suitable for the production
of rather small amounts of chemicals.