2.

39 Continuous Operation
MTA Penia Kresnowati, Bandung Institute of Technology, Bandung, Indonesia and Monash University, Clayton, VIC, Australia
XD Chen, Xiamen University, Xiamen, China and Monash University, Melbourne, VIC, Australia
© 2011 Elsevier B.V. All rights reserved.

2.39.1 Introduction 527

2.39.2 Homogeneous System 529
2.39.3 Heterogeneous Systems 532
References 535

Glossary heterogeneous reaction reaction involves more than one

batch operation Operation is not continuous; it usually
starts with a mixture of all ingredients for the reaction;
harvesting is performed at the end of reaction duration.
continuous operation Steady continuous feed-ins and
outflows are maintained throughout, usually lasting for
hours and sometimes days.
fed-batch operation Operation is not continuous; it
usually involves feeding the batch reactor periodically or
intermittently with the substrates that are needed by the
culture, and which have been depleted somewhat to some
extent during the batch operation in order to improve
growth.
phase.
continuous brewery the process of beer making that is
performed in continuous operation.
CSTR continuous stirred tank (bio)reactor; a continuous
operation that is performed in a stirred tank (bio)reactor.
fuel ethanol fermentation a fermentation process involves
microorganism, such as yeast or bacteria, to yield ethanol.
biomass yield coefficient the ratio of the mass (or mole)
of biomass produced to the mass (or mole) of substrate
consumed.
plug flow Flow velocity remains uniform across a channel
or a reactor vessel (a one-dimensional operation).

Nomenclature V bioreactor volume [m3]

A surface area [m ] 2
v linear velocity [m.s−1]
Ci concentration of species i in the bioreactor [mol.m−3 YP/S product yield on substrate [(mol-P). (mol-S)−1]
or kg.m−3) YX/Sm maximum cell yield on substrate [(kg-X). (mol-S)−1]
D dilution rate [h−1]
De diffusion coefficient [m2.s−1] Greeks
Da Dahmkohler number [−] μ specific growth rate of the cell [h−1]
F volumetric flow rate [m−3.h−1]
J flux [m.h−1] subscripts
KS [mol.m−3], Monod parameter for cell growth i species i
Km [mol.m−3], Michaelis Menten parameter for in inlet
enzymatic reaction m maximum
mS maintenance [(mol-S).(kg-X)−1.h−1] P product
qi specific consumption or production rate of species i S substrate
[(mol-i).(kg-X)−1.h−1] X biomass
r reaction rate [(mol-S). h−1]

2.39.1 Introduction

A continuous bioreactor operation is defined as a bioreactor system that is operated continuously, with both the inlet and outlet
streams flowing simultaneously at approximately the same rate. The system within the bioreactor appears to be in a steady-state
operation.
Although bioprocess has been practiced to make useful products since the ancient Egyptian and Sumerian eras, the concept of
continuous culture only dates back from the nineteenth century, when the conversion process of waste beers and wines to vinegar
was developed [1]. The use of pure culture for the production of useful products via bioprocesses is very sensitive to contamination;
consequently, a continuous system was rarely applied until the development of the sterilization process. Instead, continuous

527
528 Bioreactions and Bioreactor Operation

process has been widely applied for waste treatments or other processes in which contamination is not an important issue.
Furthermore, sufficient knowledge about the process is also required to allow adequate process monitoring and control for
preventing process contamination or any other process failure.
In general, a process is operated as a continuous system when the process needs to be operated at a large scale, in order to ease
the operational difficulties and to increase the effective run time. Otherwise, a significant fraction of time in every batch cycle
needs to be spent for cleaning, sterilizing, emptying, and growing the inoculated microorganism to its maximum effective
number, which makes the actual fermentation run time very limited. From another perspective, continuous operation allows
continuous medium refreshment to the system and prevents an accumulation of toxic waste. In particular, for tissue culture
application, continuous culture also reduces manual handling of the culture, which is both labor intensive and likely to lead to
culture contamination. The comparison between different modes of operation – batch, fed-batch, and continuous – is summarized
in Table 1.
On the other hand, a small-scale continuous system, in particular a chemostat system, is also highly favorable for research
purposes. It offers a tool to maintain a constant growth rate condition by specifying a particular dilution rate of the chemostat. It
offers defined and constant microenvironmental conditions that are necessary to study the effect of a particular parameter on a
biological system. The development of chemostats by Novick and Szilard [2] marked the application of continuous operation in
bioprocesses. Nowadays, continuous bioreactor operations are performed in various bioreactor configurations, for example, a
homogeneous system such as a stirred tank bioreactor or a heterogeneous system such as a perfusion bioreactor, a membrane
bioreactor, a fluidized-bed bioreactor, and an airlift bioreactor.
The performance and analysis of continuous bioreactor operations is based on the mass and energy conservation balances, as
well as the underlying processes governing reaction kinetics and mass and energy transport. In some cases, the reactions involve cell
growth; in other cases, purified enzymes are used as the biocatalysts and hence no growth needs to be considered in the analysis. In
particular, in a heterogeneous system, the transport of material, such as substrates and products, from the bulk solution to the
biocatalysts (cell/enzymes) and vice versa will be an important aspect of the analysis. A step-by-step analysis of a continuous
bioreactor operation involves the following [3]:

1. Defining problem statements and goals.

2. Identifying the system; this includes defining the system boundaries and the interactions with its environments.
3. Identifying state variables that characterize the system.
4. Characterizing the state of the system using mass and energy balances. Separate mass balances are written for substrates,
products, and biocatalysts.
5. Performing balance calculations and rechecking the validity of the assumptions used.

Table 1 Comparison of various modes of operation

Batch Fed-batch Continuous

Rate and yield Low conversion rate, related to
time required to grow cells to a
sufficient cell number
Low product yield, related to a high
proportion of substrate needed to
grow cells to a sufficient number
Scale Low volumetric efficiency
Utilities Utilities’ consumption varies with
batch cycle; this implies high
utilities cost
Product quality Product quality varies per batch
Labor High labor requirement related to
the batch cycle (cleaning,
sterilizing, starting up, etc.)
Flexibility High flexibility to switch to other
production processes/qualities
Durability (infection) Less vulnerable to infection/
contamination
Design Simple process design
Control Can be operated with minimal
control system
Low conversion rate, related to
time required to grow cells to a
sufficient cell number
Low product yield, related to a high
proportion of substrate needed to
grow cells to a sufficient number
Low volumetric efficiency
Utilities’ consumption varies with
batch cycle; this implies high
utilities cost
Product quality varies per batch
High labor requirement related to
the batch cycle (cleaning,
sterilizing, starting up, etc.)
High flexibility to switch to other
production processes/qualities
Less vulnerable to infection/
contamination
More complex process design
More complex control system is
required, related to the dynamics
of the fed-batch process
High conversion rate, related to constant
biocatalyst (cell) concentration
High product yield, related to a small
proportion of substrate needed to grow cells
High volumetric efficiency, related to the use
of recycled or immobilized biocatalyst; this
also implies reduction in capital equipment
Constant demand of utilities; this implies low
utilities cost
Constant product quality
Low labor requirement
Fixed system, difficult to switch to other
process/quality specified
Highly vulnerable to infection/contamination,
in particular related to the large scale
involved
More complex process design
More complex control system is required,
related to the large scale involved
 Continuous Operation 529

2.39.2 Homogeneous System

A continuous operation in a homogeneous system can be well represented by a chemostat (Figure 1), a continuous stirred-tank
bioreactor (CSTR) in which substrates are continuously fed into the bioreactor to maintain a constant concentration of the nutrients
in the bioreactor. All but one nutrient required in the reaction is normally provided in excess. The one limiting nutrient is used to
control the performance of the chemostat.
A general mass balance over a chemostat system is stated in eqn [1]:

dCi X
V ¼ FCi ; in − FCi  qi CX V ½1
dt
|fflffl{zfflffl} |fflffl{zfflffl} |{z} |fflfflfflfflfflffl{zfflfflfflfflfflffl}
flow in flow out consumption=production chemostat
accumulation

Dividing both sides of eqn [1] by the chemostat volume gives eqn [2]:

dCi   X
¼ D Ci ; in − Ci  qi CX ½2
dt
Where D (h−1) is the dilution rate of the chemostat. Separate mass balances, that is, substrate, cell, and product balances, are needed
in the analysis.
The inlet stream is usually sterile (i.e., Cx,in = 0); hence the cell balance can be written as eqn [3]:

dCX
¼ −DCX þ μCX ½3
dt
Equation [3] states that in a steady-state condition (dCX/dt = 0), the dilution rate of the chemostat will fix the growth rate of the cell
(μ = D). However, when the dilution rate is set to exceed the maximum specific growth rate of the cell, cell washout will occur
(Figure 2). This unique property of the chemostat, which the growth rate can be manipulated by controlling the dilution rate,
provides a powerful experimental tool for the quantitative study of a biological system.
When the chemostat is intended for biomass production, most of the nutrients supplied will be used for cell growth. In this case,
the nutrients used for product formation or cell maintenance can be neglected. Subsequently, the limiting substrate and biomass
concentration in the chemostat can be calculated by assuming a cell growth model. If cell growth is assumed to follow Monod’s
relation [4], the limiting substrate concentration (CS) in the bioreactor can be calculated from the dilution rate using eqn [4]:

μm C S DKS
D¼μ¼ ⇒ CS ¼ ½4
CS þ KS μm − D
Cell concentration at steady-state condition can be calculated from the substrate balance as follows:

dCS
¼ DðCS; in − CS Þ − qS CX ½5
dt
At steady state, dCs/dt = 0, and substrate consumption is mainly for cell growth (qS ¼ μ=YXm=S ); hence, eqn [5] can be rewritten as:

μCX
DðCS; in − CS Þ ¼ ½6
YX=S
m

which gives

CX ¼ YXm=S ðCS;in − CS Þ ½7

F, C i,in

F, C i
V

Figure 1 Chemostat configuration. Feed and outlet streams are continuously flowing into and out from the, respectively bioreactor, respectively constant
chemostat volume can be achieved by either controlling the flow of the feed and outlet streams using pumps, a level control system, or a weight control
system.
530 Bioreactions and Bioreactor Operation

1.2 0.3

Cell or substrate concentration (C-mol l–1)

1 0.25

Biomass productivity (C-mol l–1 h–1)

0.8 0.2

0.6 0.15

0.4 0.1

0.2 0.05

0 0
0 0.1 0.2 0.3 0.4 0.5
Dilution rate (h–1)
Figure 2 Cell and substrate concentrations and biomass productivity profiles at various dilution rates (Ks = 5  10−3 (C-mol l−1); μm = 0.5 (h−1); Yx/s
m
= 0.57
−1
((C-mol X) (C-mol S) )). Dotted line represents biomass productivity, the black solid line represents cell concentration, and the gray solid line represents
substrate concentration.

Equation [7] indicates that the biomass concentration can be controlled by setting the concentration of a particular substrate in the
feed stream.
When the chemostat is intended for product formation, substrate conversion to product needs to be accounted in the
calculation, as stated in eqn [8]:

μCX qP CX
qS ¼ þ mS CX þ ½8
YX=S
m |fflffl{zfflffl} YP=S
|ffl{zffl} maintenance |fflffl{zfflffl}
cell growth product formation

where product formation may be growth-associated (primary metabolites), non-growth-associated (secondary metabolites), or a
combination of both, as is indicated in eqn [9]:

qP ¼ αμ þ  ½9
|{z} |{z}
growth-associated nongrowth-associated

Accordingly, the substrate balance can be rewritten as eqn [10] to solve for biomass concentration in the steady-state condition:

μCX qP CX
DðCS; in − CS Þ ¼ þ ½10
YXm=S YP = S

Product concentration in the steady-state condition can be calculated from the product balance in eqn [11]:

dCP ðαμ þ Þ
¼ DCP − qP CX ⇒ CP ¼ CX ½11
dt D
When an enzyme is used as a biocatalyst in the chemostat operation, the mass balance analysis only involves substrate and product
mass balance. Enzyme activity may be influenced by environment (e.g., temperature, and pH) and needs to be considered separately:

dCS
¼ DðCS; in − CS Þ − r ½12
dt
where r ((mol-S) h−1) is the rate of enzymatic reaction that may follow any of the common enzyme kinetics expressions, for
example, Michaelis–Menten, substrate inhibition, product inhibition, or Hill kinetics [5, 6].
Several variations in the chemostat configurations are sought to improve its performance. For instance, cells or enzymes can be
recycled (Figure 3(a)) to increase process productivity, to retain a higher concentration of biocatalyst, and to extend the use of the
active biocatalyst. In case of a chemostat operation that involves cell growth, cell recycling allows the chemostat to be operated at a
dilution rate higher than the maximum growth rate without the occurrence of washout (Figure 3(b)).
 Continuous Operation 531

(a)
αF, RCx1

F, Cs, in
Cx, in

V (1+α)F, Cx1 F, Cx

(b) 1 0.75

Biomass productivity (C-mol l−1 h−1)

Cell concentration (C-mol l−1)

0.8 0.6

0.6 0.45

0.4 0.3

0.2 0.15

0 0
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8
Dilution rate (h−1)
Figure 3 Chemostat with cell recycling configuration. (a) Setup of the chemostat with cell recycling system; (b) performance comparison between
chemostats with and without a recycling system simulated under model parameters (Ks = 5  10−3 (C-mol l−1); μm = 0.5 (h−1); Yx/s m
= 0.57 ((C-mol X)
−1
(C-mol S) ); α = 0.2; R = 3). The black line represents a system without recycling, while the gray line represents a system with recycling; the dotted line
represents biomass productivity, while the solid line represents cell concentration.

Mass balance analysis of a chemostat system with recycling is expressed in eqns [13–16]. This system involves a sterile feed
stream; α, fraction of outlet stream recycled back to the chemostat; and R, the concentrating factor of the biomass separator. At
steady state, the cell balance is expressed as:
0 ¼ FCX; in þ αFRCX1 − ð1 þ αÞFCX1 þ μCX1 V ½13
which gives

μ ¼ ð1 þ α − αRÞD ½14
Accordingly, if cell growth is assumed to follow Monod’s equation [4], the substrate concentration in eqn [4] can be rewritten as:

μ ð1 þ α − αRÞD
CS ¼ K S ¼ KS ½15
μm − μ μm − ð1 þ α − αRÞD
whereas the calculation of cell concentration from the substrate balance in eqn [6] can be rewritten as:
 
YX=S
m
ð1 þ α − αRÞD
CX ¼ CS ; in − KS ½16
ð1 þ α − αRÞ μm −ð1 þ α − αRÞ
A serial chemostat configuration can be applied in order to facilitate different microenvironmental requirements of the process. An
additional feed stream can be introduced to the second/succeeding bioreactor to provide different microenvironmental conditions
in that particular bioreactor (Figure 4). For example, a series of aerobic and anaerobic chemostats can be applied to successively
facilitate cell growth and ethanol formation in a continuous ethanol production process such as in a continuous brewery [7].
A real example of a continuous homogeneous bioreactor system is the New Zealand continuous brewery process [7–11]. This
system comprises three cascading stirred bioreactors (Figure 5), involving both the serial and recycle systems. The first bioreactor
mainly functions as the holding tank for worth, the substrate for beer fermentation. This bioreactor is aerated to provide an aerobic
environment that facilitates cell growth. A recycle stream from the second bioreactor, containing active yeasts and partially
532 Bioreactions and Bioreactor Operation

F, C i, in
Fadd2, C i, in 1

F1, C i, 1

V1 Fadd n, C i, in n

F2, C i, 2
V2 Fn–1,
C i,n–1

Vn
Fn, C i,n

Figure 4 Serial chemostat configuration.

CO2

Bioreactor 1
Worth

Bioreactor 2

Bioreactor 3

Separator Beer maturation

Yeast recycle

Figure 5 Schematic process diagram of the New Zealand continuous brewery process. Adapted from Mousdale DM (2008) Biofuels: Biotechnology,
Chemistry, and Sustainable Development. New York: CRC Press.

fermented beer, is also fed to this bioreactor as the seeding culture. The second and third bioreactors are the main fermentation
vessels. They are not aerated, and operate continuously to produce ethanol. The second bioreactor produces partially fermented
beer, which is fine-tuned in the third bioreactor. The system was in operation from 1957 to 1985.

2.39.3 Heterogeneous Systems

In a heterogeneous system, reactions occur at a phase or condition different from that of the bulk solution. This is the case for
bioreactors involving anchorage-dependent cells that form a biofilm or the wall of the bioreactor or on any other surface and
bioreactors involving immobilized biocatalysts that stabilize the activity of the biocatalyst or to extend the use of the biocatalyst.
These typical applications are usually performed in a perfusion bioreactor, a packed-bed bioreactor, a fluidized-bed bioreactor, an
airlift bioreactor, a membrane bioreactor, and also in a stirred tank bioreactor (Figure 6).
 Continuous Operation 533

(a) (d)
Gas
outlet Media
Media Cell
inlet layer
outlet
Media
Carriers for outlet
cell growth

(e)
Media
(b) inlet
Gas Media
outlet outlet
Media
outlet

Carriers for
cell growth Gas
Media inlet
outlet
(f)
Media
(c) inlet
Gas
outlet Media
outlet

Carriers for
cell growth
Media Media
Gas inlet inlet
inlet
Figure 6 Heterogeneous bioreactor system: (a) Packed-bed bioreactor; (b) fluidized-bed bioreactor; (c) airlift bioreactor; (d) perfusion bioreactor;
(e) membrane bioreactor; and (f) stirred tank bioreactor.

Analysis of these bioreactor operations needs more complex calculations. The multiphase condition requires a transport analysis
of material from and to the biocatalyst to account for the difference in the microenvironment of the biocatalyst and the bulk
solution. From the perspective of the degree of mixing, some types of bioreactors should be analyzed as a plug flow system or a
combination of series of stirred tanks and plug flow reactors. Because of cell immobilization inside the bioreactor, continuous
media inflow may result in cell accumulation in the bioreactor. Biocatalyst distribution in the bioreactor may need to be considered
in the analysis. Flow distribution inside the bioreactor may also need to be considered in the calculation.
To what extent transport from the bulk solution to the biocatalyst may affect the overall reaction rate of the system can be
checked by analyzing the Damköhler number (Da), which is defined as:

max: rate of bioconversion

Da ¼ ½17
max: rate of diffusion

Da ≈ 1 shows that the resistance of transport and reaction are comparable, Da << 1 shows that the reaction progresses slower than the
transport of material from or to the bulk solution, whereas Da >> 1 shows that the system is limited by transport diffusion from bulk
solution to the biocatalyst (transport-limited condition). At a steady-state condition, the overall reaction rate is further governed by
the limiting process, as is shown in eqn [18] for enzymatic reaction and eqn [19] for cellular reaction in which JS is the flux of
substrate from the bulk solution to the catalyst:
 
∂CS CS
JS A ¼ −De ; S A ¼ rm ½18
∂x Km ; S þ CS
 
∂CS
JS A ¼ −De ; S A ¼ qS CX ½19
∂x

A further implication of this is the limited penetration of substrate into the immobilizing agent. In particular, for immobilized cell
systems, this results in limited thickness of the cell layer (Figure 7).
534 Bioreactions and Bioreactor Operation

(a) (b)

∂2Cs 2 ∂Cs
De = qsCx
∂r 2 r ∂r

∂Cs
r=0 =0
∂r

r=R Cs = Cs,bulk

r=0 r=R

(c)
1
0.9
0.8
0.7
0.6
Cs/Cs0

0.5
0.4 R = 5 µm
R = 10 µm
0.3
R = 25 µm
0.2 R = 50 µm
R = 75 µm
0.1 R = 100 µm
0
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
r/R
Figure 7 Substrate penetration in an immobilized cell biocatalyst. (a) Immobilized cell biocatalyst; (b) model on substrate diffusion in cell biocatalyst;
and (c) effects of biocatalyst size on substrate distribution.

The analysis of a unidirectional flow bioreactor system, for example, a fixed-bed bioreactor, a fluidized-bed bioreactor, or a perfusion
bioreactor, can be approached as a plug flow system. Fluid flows through a series of infinitely thin homogeneously mixed cells and there
is no mixing in the axial direction. The balance is developed for each individual cell and integrated over the length of the bioreactor:

∂Ci X
¼ − ð∇Ci vÞ − ð∇Ji Þ þ R ½20
∂t |fflfflffl{zfflfflffl} |ffl{zffl} |fflfflffl{zfflfflffl}ij
convection diffusion reaction

When flow distribution inside the bioreactor is between the two extremes of homogeneously mixed (Continuous Stirred Tank
Bioreactor or CSTR) and plug flow, the system can be analyzed as a combination of CSTR compartments (Figure 8).

Figure 8 Various flow configurations inside the bioreactor. The far left describes the homogeneously mixed Continuous stirred-tank bioreactor (CSTR),
the far right describes the plug flow system, and in between are some possible flow configurations that may be approached as sets of CSTR.
 Continuous Operation 535

CO2

Substrate

Bioreactor 1

Bioreactor 2

Bioreactor 3

Bioreactor 4
Product

Bioreactor 5

Bioreactor 6
Product
Separator

Yeast slurry bleeding system

Yeast paste
Figure 9 Schematic process diagram of a fuel ethanol fermentation plant with self-flocculating yeast. Adapted from Zhao XQ and Bai FW (2009) Yeast
flocculation: New story in fuel ethanol production. Biotechnology Advances 27: 849–856. Copyright 2009, with permission from Elseveir.

A real example of a heterogeneous bioreactor system is that a fuel ethanol fermentation plant of BBCA (China BBCA Group
Corp.), with self-flocculating yeast in China [12, 13]. This plant comprises a cascade of six suspended-bed bioreactors (Figure 9) and
uses corn meal hydrolysate as the main substrate. Yeasts are self-immobilized by flocculation and are retained within the bioreactor
by baffles. A yeast slurry bleeding system is used to control the fermentation temperature, by passing some part of the slurry through
a heat exchanger system, and to control the biomass concentration within the bioreactor.

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[11] Davies AW (1988) Continuous fermentation – 30 years on. 20th Convention of the Institute of Brewing, Australia and New Zealand Section. Brisbane, QLD, Australia.
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