Bioreactor Monitoring & Control

CHE 194
Spring 2015
Presented by Dr. Claire Komives
(See the accompanying video on Canvas)

Bioreactor Monitoring & Control

Basic principles of process control
Fermentation monitoring
Dissolved oxygen
pH
Temperature
Offgas monitoring
Substrate (glucose)

Useful references
Ogunnaike, B.A., and Ray, W.H., Process Dynamics,
Modeling, and Control, Oxford University Press,
1994.
Vogel, H.C., Todaro, C.L., eds., Fermentation &
Biochemical Engineering Handbook 2nd Ed., Elsevier
1996.
Shuler, M.L. and Kargi, F., Bioprocess Engineering:
Basic Concepts 2nd Ed., Prentice Hall, 2001.
Van Impe, J.F.M., Vanrollegham P.A.,and Iserentant,
D.I., eds., Advanced Instrumentation, Data
Interpretation, and Control of Biotechnological
Processes, Kluwer Academic Publishers, 1998.

Process Control Basics: The concept of

process control
Fgas

Feed in

Presentation of problem:

offgas
Fi

Level probe

DO probe
Air in
Fair

Fo

Chemostat

1.

Control level of liquid

2.

Control concentration of
O2 in fermentor

Broth out
Definitions:
Input Variables are those that independently
stimulate the system and can thereby induce
change in the internal conditions of the process
Output Variables are those by which one obtains
information about the internal state of the system

Process Control Basics: The concept of

process control
Fgas

Feed in

Two types of input variables

offgas

Those we can manipulate are

control variables

Fi

Level probe

DO probe
Air in
Fair

Fo

Chemostat

Broth out

Those we cannot control are

disturbance variables

We can separately identify

variables that we can measure.

Feedback Control Configuration

DISTURBANCE

Input

Output
Process
Sensor

Final Control
Element

Transmitter
Transmitter

DECISION
Controller

INFORMATION

Control action is taken based on information that there is a process upset

Upset occurs when the process output variable deviates from the setpoint.

Feedback Control: Regulatory Control

5

underdamped

Process response

4.5
4

critically damped

3.5

overdamped

3
2.5
2
1.5
1

SP

disturbance

0.5
0
0

10

15

time

20

25

Feedback Control: Regulatory Control

6

Process response

underdamped
critically damped

Change SP

0
0

10

time

12

14

16

Standard Feedback Controllers

Proportional Controller (P)
Proportional + Integral Controller (PI)
Proportional + Integral + Derivative Controller (PID)

offgas

Feed in

Air in

Bioreactor systems generally

come set up so that you dont
need to know which type of
control strategy has been
employed they just do it

samples

Standard:
temperature
pH
dissolved oxygen

Process Monitoring: Hardware Sensors

Hardware sensors
Dissolved oxygen probe
pH probe
Temperature probe
Offgas monitoring (IR + flow sensor; mass spec)
Dissolved CO2 probe
Substrate (glucose) flow sensor
Fermentor volume (via load cell)

Bioreactor Control: Nonlinear Process

PI or PID control strategies can be used for
simply managing process variables such as
temperature and pressure.
pH is somewhat more sophisticated to avoid
oscillation and offset
DO is managed by cascade control
Critical variables, such as nutrient feed rate(s)
may not have simple hardware sensors
available (glucose monitoring)

We will begin with oxygen

Oxygen is needed for respiration for aerobic cultures.

Strict aerobes will die without sufficient O2

Facultative aerobes will not die in the absence of O2 but will
produce byproducts due to the need to transfer electrons to
metabolites when O2 is not present to do the job.
Oxygen is monitored by a dissolved oxygen probe
Oxygen is fed as a gas but can only be utilized in the liquid
phase
The E. coli we use in the 194 lab is a facultative aerobe and therefore grows
best with oxygen but can grow also without it. In the absence of O2,
however, it will produce byproducts that inhibit the growth. Thus, we aim
to avoid an oxygen-limited situation in the process.

Challenges with DO control

Oxygen will dissolve in water up to
[DO]sat'd ~ 7 ppm (= 7 mg/L) at 30C.
This concentration is sufficient for
bugs to grow, what is limiting is the
rate of transfer from the air bubble to
the liquid.

T 0C

x (mol O2/mol H2O)

[O2] mg O2/l H2O

10

0.0000064

11.377

20

0.0000052

9.277

30

0.0000044

7.832

40

0.0000039

6.954

50

0.0000036

6.327

Oxygen transfer engineering style

If we take a snapshot of a
bubble interface during a
fermentation run, here is
what we see

Air
bubble

liquid

Distance

Bubble interface

Assumptions: air bubble and liquid are two well mixed systems

Oxygen transfer - engineering style

The oxygen concentrations vary both with distance and with time.
The actual mechanism of oxygen transfer is complex, so engineers use an empirical
method to describe the process.

Oxygen transfer rate (OTR) = kLa(C*-CL)

C* is the saturation concentration of O2 in the liquid
CL is the actual liquid concentration (measured by DO probe)

kLa is the Liquid side mass transfer coefficient.

Transferring the O2 from the bubble interface into the liquid is the rate-limiting
step to get the air into the liquid.

Oxygen supply vs. demand

Oxygen supply is what is transferred from the bubble into the liquid
Oxygen transfer rate (OTR) = kLa(C*-CL)

mM
mg O2

or

h
h

If we have a bacteria culture that consumes a lot of oxygen, then the measured
concentration of O2 in the liquid can drop to zero.

DO

How can we increase the

oxygen supply??

Oxygen supply vs. demand

What cells consume - or the demand
The rate of O2 consumption is the oxygen uptake rate.
mM
The units are the same as for OTR =>

or
h
OUR is a rate

OUR =

dO2
dt

mg O2

mols O2 consumed/time

if O2 is the limiting substrate, and the O2 limitation comes into play, then
OTR = OUR

or the demand = supply ==> can the demand be greater than the supply?
The amount of oxygen required by cells depends on their growth rate and
on the total number of cells.
Even cells that are not growing need oxygen, and this is called the
maintenance requirement.

Oxygen supply vs. demand

How can we increase supply??
Oxygen transfer rate (OTR) = kLa(C*-CL)

We can either increase kLa or we can increase C*

How can we get oxygen to move across the bubble interface faster?
0.4

79% N2
21% O2

P( w)
0.6
k L a 0.1
gs
M
M is the mass of the fluid.

Air bubble

0.1 is experimentally determined (should be

determined for each culture broth).

gs

is superficial gas velocity.

Increase O2 Supply to Bioreactor

Strategy 1: Increase flow of gas into bioreactor
0.4

P( w)
0.6
k L a 0.1

gs
M
Oxygen controller

Flowmeter

Air compressor

Sparger

Dissolved
oxygen (DO)
probe

Increase O2 Supply to Bioreactor

Mixing
Motor
controller

Strategy 2: Increase mixer

speed.

0.4

P( w)
0.6
k L a 0.1
gs
M

Increase O2 Supply to Bioreactor

How can we increase C*?
Oxygen transfer rate (OTR) = kLa(C*-CL)
C* is the saturation concentration of O2 in the liquid phase
We can increase it by increasing the concentration of O2 in the GAS
phase

Feed pure O2 or a mixture of O2 + air

If this strategy can be avoided, it should be

added cost to purchase purified O2

safety issues associated with handling pure O2

Increase O2 Supply to Bioreactor

DO Control involves a combination of both -- called Cascade Control
increasing stir speed

increasing air flow rate

There is a maximum possible stir rate
Air flow rate cannot be increased indefinitely
Flooding is the phenomena where there is so much air blown
into the reactor that you create a pocket of air in the middle of
the reactor around the impellers just beating the air and not
the liquid
When flooding occurs, there is a notable drop in the power supply to the
mixer.

pH

What is pH
Pure water dissociates to yield 10-7 moles/L of H+ at 250C:

H2O <----> H+ + OHSince water dissociates to produce one OH- ion for each H+ ion, it is
obvious that

10-7 OH- ions are produced simultaneously.

The product of [H+] and [OH-] always remains constant. When the value for
one of the species changes the other also changes accordingly.

[H+] x [OH-] = 10-14

The concentration of H+ ions can be increased when compounds are added
which release H+ ions such as H2SO4:

H2SO4

<---->

2H+ + SO42-

Control of pH in Bioreactor
Calculate the concentration of hydrogen ions
from the pH
[H+] (molar concentration) = 10-pH
eg for pH = 7.0

and 7.2

[H+] = 10-7 molar or 0.0000001

[H+] = 10-7.2 molar or 0.0000000631

notice that increasing the pH by 0.2 decreases the [H+] by 37%

Control of pH in Bioreactor
Most bacteria can grow over a wide range of pH, although many enzymes upon
which microbial growth depends function only within a narrower range of pH.
The bacteria then must maintain their internal pH near a fixed optimal value.

Bacteria (E. coli) that grow at neutral pH (6.0-8.0) are called neutrophiles.
Regardless of the external pH, the internal pH is maintained at ~7.6.
pH is maintained by ion pumps on the membrane of the bacteria.
Operation of the pumps requires energy input
Effort put into maintaining the pH will be at the expense of other cellular
functions
Bugs tend to grow more slowly when the pH is not at the optimum.
Any processes that involve interaction with the external
medium, such as uptake of nutrients, secretion of proteins, etc.
will be directly affected by the external pH.

pH control

pH controller

Acid pump

pH probe
Base pump

What causes the pH to change?

Overfeeding substrate can cause the cells to produce
organic acids, such as acetate pH drops
Lack of carbohydrate substrate causes the cells to
consume protein in the media producing NH3
NH4OH pH rises
When producing a protein product, cells consume
ammonia from the media from the cellular demand
for more nitrogen causes the release of a proton
pH drops.

pH control in CHE 194 process

We use NH4OH for base to control the pH.
We dont need any acid because the process
control is designed to avoid overshooting.
We wait for a pH spike (production of base)
and then we turn on the glucose feed pump.
A very slow glucose feed is used to enable
growth of bacteria and avoid oxygen
limitation. (explained later in this
presentation)

Temperature Control

Heat management is another engineering task

The amount of heat (Q) to be added or removed depends on the

density of the culture, the volume of the culture, and the growth rate
of the culture.

QGR VL XYH

Heat management in a laboratory bioreactor is

analogous to that at a large scale

Flowmeter

Cooling water
Temperature
probe
Heating jacket
At large scale, heating jacket is replaced with a steam jacket

Temperature
controller

Heating Jacket on Fermentor

TOUT
H
TIN

Our (lab 109) heating jacket is electrical, not using hot water or
steam. We use it to heat the bioreactor and to help maintain
temperature. We use cooling water in a little heat exchanger to cool
the system. The combination of heating and cooling inputs carefully
maintains the temperature.

Metabolic Heat Generation

40-50% of energy produced by substrate catabolism is converted to ATP, the rest is
released as heat.
Heat of
Combustion
Glucose + NH3 + O2 -------> CO2 + H2O + cells
(HC) of cells 20
- 25 kJ/kg

YH is the metabolic heat evolved per gram of cell produced

YH depends on the degree of oxidation of the substrate

YH ~ 2.4 kcal/g on glucose

YH ~ 5.6 kcal/g on ethanol
YH ~ 8.3 kcal/g on methanol

Total heat evolved (QGR) depends on

QGR VL XYH

Oxygen uptake
rate

YH ~ 16.4 kcal/g on CH4

For aerobic fermentations,

Units kcal/hr

QGR 0.12QO2

Units kcal/lithr

Where is the heat production the greatest?

250.000

200.000

150.000

100.000

Dissolved Oxygen
Optical Density

50.000

0.000
0.000

QGR VL XYH

20.000

40.000

60.000

80.000

100.000

Time from inoculation (hrs)

120.000

Cooling water addition

Q QGR QS
Total heat to be removed by cooling water is due to metabolic
heat generation plus heat added from the stirring

Q UAC T
Heat is removed by running cooling water through the cooling
coils in the reactor
U is the overall heat transfer coefficient of the coils
AC is the total area of the coils in contact with the broth
T is the log-mean temperature difference between the broth
and the cooling water

T Tin T Tout
T
lnT Tin / T Tout

Offgas Analysis

Offgas composition is an indicator of culture activity

Bug: glucose + NH3 + 2O2

C4H7O2N (bug) + 4H2O + 2CO2

Respiration:
glucose + 36 Pi + 36 ADP + 6 O2

6 CO2 + 6 H2O + 36 ATP

Based on this simple description of the chemistry, we can

see that production of CO2 is an indicator of living cells. A
high rate of CO2 production coupled to O2 consumption is
an indicator of growth.

Offgas
analyzer

Offgas analysis

Condenser

Air out is <79% N2 and

<21% O2 and 0.033% <
CO2< 6%

Flowmeter

Air in is 79% N2
and 21% O2 and
0.033% CO2

Air compressor

Sparger

Offgas composition is an indicator of culture activity

Carbon dioxide Evolution Rate (CER) is the rate of production of
CO2.
CO2,out CO2,in
Time interval

Is the CER

Since we know the concentration of CO2 in the air is 0.033%, by

measuring the CO2,out we can determine CER.
Note that this is measured for the whole culture volume, so
we can divide out the culture volume for a more meaningful
value.

Offgas composition is an indicator of culture activity

Likewise,

O2,out O2,in

Time interval

Is the OUR

We can measure the CER and OUR on-line using a mass

spectrometer.
The mass spectrometer measures concentrations, and we
need to know total amounts.

By comparing the O2 and CO2 concentrations with the N2 we

can determine what the flow rate out is, since N2 is neither
produced or consumed by the bugs.

Calculation of OUR & CER

Handout example problem

Best practice: design your feeding strategy such

that O2 is not the limiting nutrient
Depending on your product, you may choose Carbon or Nitrogen to be
your limiting nutrient, and you can choose different limiting nutrients
during different phases of the run.
When the cells are growing, the feed rate can be determined from:
FS =

YX S

X (t )

FS is the substrate feed rate,

is the specific growth rate of the organisms

YX/S is the yield coefficient (rate of cell production/rate of substrate uptake)

X(t) is the time dependent concentration of organisms in the bioreactor.
Can we estimate the cell growth rate and cell concentration from
on-line measurements?

Best practice: design your feeding strategy such

that O2 is not the limiting nutrient, contd
Since we know (we can measure) the yield of cells on oxygen, we can estimate the
cell concentration by

dX (t )
YX O OUR (t )
dt

OUR can be
measured on-line

Together with an estimate of X(t) we can estimate from

1 dX

X (t ) dt

(t )

OUR(t )
X (t 0 ) t
OUR(t )dt
0
YX O

IOUR - can be
measured on-line

Best practice: design your feeding strategy such

that O2 is not the limiting nutrient, contd
Putting everything back into our Feed rate calculation, we have
FS =

YX S

X (t )

1
OUR (t )
FS

YX O IOUR X (t0 )
YX S X (t0 ) IOUR
YX O

If the cells have everything they need (balanced growth), the you are
estimating may be close to max. If your feed rate should exceed the
amount needed for the growth of the cells, you will accumulate
substrate and it will result in the formation of byproducts.
It solves the problem to choose some fraction of max, such as 0.7max

Off-line analysis methods used

for fermentation

Off-line analysis
Advantages:
No interfacing required
Flexibility
Low cost
Small sample volumes

Disadvantages:
Requires removing samples
Requires tracking the sampling time
Requires operator interaction, potential for bias
error from operator

Offline Analysis Methods

Glucose
Glucose concentration is measured by a glucose analyzer. For E. coli
fermentations, glucose should be kept above about 5 g/liter and below
about 20 g/liter for optimum growth.
The Yellow Springs Instruments (YSI) Bioanalyzer is a standard
instrument for fermentation analysis. It can measure other sugars, ethanol
and methanol, as well.

A small sample from the fermentor is centrifuged to settle the bacteria

and the supernatant is fed through a sipper tube into the analyzer. It is
automatically calibrated.

High Performance Liquid Chromatography (HPLC)

HPLC is useful for measuring fermentation by-products, such as ethanol,
organic acids acetic acid, succinic acid, lactate, etc., and specific amino
acids and virtually all small molecules and proteins.
Each molecule should have a specific method to be analyzed.

Offline Analysis Methods

Determining cell mass concentration
Direct methods:

Dry Weight
Solids-free medium.
Packed Cell Volume
Optical Density Method
Spectrophotometer.
550 or 600 nm wavelengths.
light absorbance cell mass/volume

OD 0.3 need to measure dry-weight of the cells.

Typical Off-line analyses

Optical density (OD) analysis of cell
concentration
Glucose can be measured off-line
NH3 can be measured by chemical analysis
Phosphate and other macro-nutrients
Protein activity assays
HPLC everything else