Bioreactor Monitoring & Control BioreactorMonitoring&Control

Bioreactor Monitoring & Control BioreactorMonitoring&Control

Basic principles of process control Basicprinciplesofprocesscontrol
Fermentationmonitoring
Di l d Dissolvedoxygen
pH
Temperature
Offgasmonitoring
Substrate(glucose)
Useful references Usefulreferences
Ogunnaike,B.A.,andRay,W.H.,ProcessDynamics, g y y
Modeling,andControl,OxfordUniversityPress,
1994.
Vogel H C Todaro C L eds Fermentation & Vogel,H.C.,Todaro,C.L.,eds.,Fermentation&
BiochemicalEngineeringHandbook2
nd
Ed.,Elsevier
1996.
Sh l M L d K i F Bi E i i Shuler,M.L.andKargi,F.,BioprocessEngineering:
BasicConcepts2
nd
Ed.,PrenticeHall,2001.
VanImpe,J.F.M.,VanrolleghamP.A.,andIserentant, p , , g , ,
D.I.,eds.,AdvancedInstrumentation,Data
Interpretation,andControlofBiotechnological
Processes, Kluwer Academic Publishers, 1998. Processes,KluwerAcademicPublishers,1998.
ProcessControlBasics:Theconceptof
l processcontrol
F
offgas
Feed in
F
i
F
gas
Presentation of problem:
1. Control level of liquid
F
o
2. Control concentration of
O
2
in fermentor
Level probe

Broth out
Definitions:
DO probe
Air in
Chemostat
F
air
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
ProcessControlBasics:Theconceptof
l processcontrol
F
offgas
Feed in
F
i
F
gas
Two types of input variables
Those we can manipulate are
control variables
F
o
control variables
Those we cannot control are
disturbance variables
W t l id tif
Level probe

Broth out
We can separately identify
variables that we can measure.
DO probe
Air in
Chemostat
F
air
FeedbackControlConfiguration
DISTURBANCE
Input
Output
Final Control
El t
Process
Sensor
Element
Transmitter
DECISION
Transmitter
INFORMATION
DECISION
Controller
Control action is taken based on information that there is a process upset 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
FeedbackControl:RegulatoryControl
3.5
4
4.5
underdamped
critically damped
o
n
s
e
2.5
3
3 5
overdamped
c
e
s
s

r
e
s
p
o
1.5
2
P
r
o
c
0
0.5
1
SP
disturbance
0 5 10 15 20 25
time
Feedback Control: Regulatory Control
6
FeedbackControl:RegulatoryControl
4
5
n
s
e
3
4
s
s

r
e
s
p
o
n
1
2
underdamped
critically damped
Change SP
P
r
o
c
e
s
0
0 2 4 6 8 10 12 14 16
time
Standard Feedback Controllers StandardFeedbackControllers
ProportionalController(P)
Proportional + Integral Controller (PI) Proportional+IntegralController(PI)
Proportional+Integral+DerivativeController(PID)
offgas
Feed in
Bioreactor systems generally
come set up so that you dont
need to know which type of need to know which type of
control strategy has been
employed they just do it

samples
Standard:
temperature
Air in
pH
dissolved oxygen
Process Monitoring: Hardware Sensors ProcessMonitoring:HardwareSensors
Hardware sensors Hardwaresensors
Dissolvedoxygenprobe
pH probe pHprobe
Temperatureprobe
Offgas monitoring (IR + flow sensor; mass spec) Offgasmonitoring(IR+flowsensor;massspec)
DissolvedCO
2
probe
Substrate (glucose) flow sensor Substrate(glucose)flowsensor
Fermentorvolume(vialoadcell)
Bioreactor Control: Nonlinear Process BioreactorControl:NonlinearProcess
PI or PID control strategies can be used for PIorPIDcontrolstrategiescanbeusedfor
simplymanagingprocessvariablessuchas
temperatureandpressure.
pHissomewhatmoresophisticatedtoavoid
oscillationandoffset
DOismanagedbycascadecontrol
Critical variables, such as nutrient feed rate(s) Criticalvariables,suchasnutrientfeedrate(s)
maynothavesimplehardwaresensors
available(glucosemonitoring)
Wewillbeginwithoxygen g yg
Oxygenisneededforrespirationforaerobiccultures.
StrictaerobeswilldiewithoutsufficientO
2
FacultativeaerobeswillnotdieintheabsenceofO
2
butwill
producebyproductsduetotheneedtotransferelectronsto
metaboliteswhenO
2
isnotpresenttodothejob.
d b d l d b Oxygenismonitored byadissolvedoxygenprobe
Oxygenisfedasagasbutcanonlybeutilizedintheliquid
phase 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 O
2
, best with oxygen but can grow also without it. In the absence of O
2
,
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 ChallengeswithDOcontrol
Oxygenwilldissolveinwaterupto yg p
[DO]
sat'd
~7ppm(=7mg/L)at30C.
Thisconcentrationissufficientfor
bugstogrow,whatislimitingisthe g g g
rate oftransferfromtheairbubbleto
theliquid.
T
0
C x (mol O
2
/mol H
2
O) [O
2
] mg O
2
/l H
2
O
10 0 0000064 11 377 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 Oxygentransferengineeringstyle
Ifwetakeasnapshotofa
bubble interface during a bubbleinterfaceduringa
fermentationrun,hereis
whatwesee
Distance
Air
bubble
liquid
bubble
Bubbleinterface
Assumptions:airbubbleandliquidaretwowellmixedsystems
Oxygentransfer engineeringstyle
Theoxygenconcentrationsvarybothwithdistanceandwithtime.
Theactualmechanismofoxygentransferiscomplex,soengineersuseanempirical
methodtodescribetheprocess.
O f (OTR) k (C* C ) Oxygentransferrate(OTR)=k
L
a(C*C
L
)
C* is the saturation concentration of O
2
in the liquid
C is the actual liquid concentration (measured by DO probe) C
L
is the actual liquid concentration (measured by DO probe)
k
L
a is the Liquid side mass transfer coefficient.
TransferringtheO
2
fromthebubbleinterfaceintotheliquidistheratelimiting
steptogettheairintotheliquid.
Oxygensupplyvs.demand
Oxygen transfer rate (OTR) = k a(C*C )
Oxygensupply iswhatistransferredfromthebubbleintotheliquid
|
|

|
O mg
2
|
|

|
mM
or
Oxygentransferrate(OTR)=k
L
a(C C
L
)
Ifwehaveabacteriaculturethatconsumesalotofoxygen,thenthemeasured
concentrationofO
2
intheliquidcandroptozero.
|
.

\
- h l
|
.

\
h
or
2
Howcanweincrease the
oxygensupply??
DO
Oxygensupplyvs.demand
Whatcellsconsume orthedemand
TherateofO
2
consumptionistheoxygenuptakerate.
2
p yg p
TheunitsarethesameasforOTR=>
|
.
|

\
|
-

h l
O mg
2
|
.
|

\
|
h
mM
or
dO
OURisarate OUR=
dt
dO
2
molsO
2
consumed/time
ifO
2
isthelimitingsubstrate,andtheO
2
limitationcomesintoplay,then
2
g ,
2
p y,
OTR=OUR
orthedemand=supply ==>canthedemandbegreaterthanthesupply?
Theamountofoxygenrequiredbycellsdependsontheirgrowthrateand
onthetotalnumberofcells.
Even cells that are not growing need oxygen, and this is called the Evencellsthatarenotgrowingneedoxygen,andthisiscalledthe
maintenancerequirement.
Oxygensupplyvs.demand
Howcanweincrease supply??
Oxygen transfer rate (OTR) = k a(C*C )
Wecaneitherincreasek
L
aorwecanincreaseC*
Oxygentransferrate(OTR)=k
L
a(C C
L
)
Howcanwegetoxygentomoveacrossthebubbleinterfacefaster?
4 0
) (
| |
79%N
2
21%O
2
6 . 0
4 . 0
) (
1 . 0
gs L
M
w P
a k u
|
.
|

\
|
=
Airbubble
Misthemassofthefluid.
0.1 isexperimentallydetermined(shouldbe
determinedforeachculturebroth).
gs
u
issuperficialgasvelocity.
Increase O
2
Supply to Bioreactor
Strategy 1: Increase flow of gas into bioreactor
IncreaseO
2
SupplytoBioreactor
4 0
| | 6 . 0
4 . 0
) (
1 . 0
gs L
M
w P
a k u
|
.
|

\
|
=
Oxygen controller
Flowmeter
Dissolved
oxygen (DO)
Air compressor
oxygen (DO)
probe
Sparger
Increase O
2
Supply to Bioreactor
Mixing
Strategy 2: Increase mixer
d
IncreaseO
2
SupplytoBioreactor
Motor
controller
speed.
4 . 0
) ( P
| | 6 . 0
) (
1 . 0
gs L
M
w P
a k u
|
.
|

\
|
=
Increase O
2
Supply to Bioreactor IncreaseO
2
SupplytoBioreactor
How can we increase C*?
Oxygentransferrate(OTR)=k
L
a(C*C
L
)
C* is the saturation concentration of O
2
in the liquid phase
We can increase it by increasing the concentration of O
2
in the GAS
phase p ase
Feed pure O
2
or a mixture of O
2
+ air
If this strategy can be avoided, it should be
dd d t t h ifi d O added cost to purchase purified O
2
safety issues associated with handling pure O
2
Increase O Supply to Bioreactor IncreaseO
2
SupplytoBioreactor
DO Control involves a combination of both -- called Cascade Control 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 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 y p
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 When flooding occurs, there is a notable drop in the power supply to the
mixer.
pH pH
What is pH WhatispH
Pure water dissociates to yield 10
-7
moles/L of H
+
at 25
0
C:
H
2
O <----> H
+
+ OH
-
Since water dissociates to produce one OH
-
ion for each H
+
ion, it is
obvious that 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
[H ] x [OH ] 10
The concentration of H
+
ions can be increased when compounds are added
which release H+ ions such as H
2
SO
4
:
H
2
SO
4
<----> 2H
+
+ SO
4
2-
Control of pH in Bioreactor ControlofpHinBioreactor
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 molar or 0.0000001
[H+] = 10-
7.2
molar or 0.0000000631
ti th t i i th H b 0 2 d th [H ] b 37% 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.
ControlofpHinBioreactor
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. 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.
H i i t i d b i th b f th b t i 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
di h t k f t i t ti f t i t medium, such as uptake of nutrients, secretion of proteins, etc.
will be directly affected by the external pH.
H t l pH control
pH controller
Acid pump
p co t o e
pH probe
Acid pump
BBase pump
What causes the pH to change? WhatcausesthepHtochange?
Overfeeding substrate can cause the cells to produce Overfeedingsubstratecancausethecellstoproduce
organicacids,suchasacetate pHdrops
Lackofcarbohydratesubstratecausesthecellsto y
consumeproteininthemedia producingNH
3

NH
4
OH pHrises
Whenproducingaproteinproduct,cellsconsume
ammoniafromthemediafromthecellulardemand
f h l f formorenitrogen causesthereleaseofaproton
pHdrops.
pH control in CHE 194 process pHcontrolinCHE194process
We use NH
4
OH for base to control the pH WeuseNH
4
OHforbasetocontrolthepH.
Wedontneedanyacidbecausetheprocess
control is designed to avoid overshooting controlisdesignedtoavoidovershooting.
WewaitforapHspike(productionofbase)
d h h l f d andthenweturnontheglucosefeedpump.
Averyslowglucosefeedisusedtoenable
h f b i d id growthofbacteriaandavoidoxygen
limitation.(explainedlaterinthis
i ) presentation)
Temperature Control TemperatureControl
Heatmanagementisanotherengineeringtask
Q
Q
Q
Q
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.
H L GR
XY V Q =
Heatmanagementinalaboratorybioreactoris
analogoustothatatalargescale
Flowmeter
T t Temperature
controller
Coolingwater
Temperature
probe
Heatingjacket
At large scale, heating jacket is replaced with a steam jacket
Heating Jacket on Fermentor
Our (lab 109) heating jacket is electrical, not using hot water or 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 temperat re maintains the temperature.
MetabolicHeatGeneration
4050%ofenergyproducedbysubstratecatabolismisconvertedtoATP,therestis
releasedasheat.
Heat of
Glucose+NH
3
+O
2
>CO
2
+H
2
O+cells
Heatof
Combustion
(H
C
)ofcells20
25kJ/kg
Y
H
isthemetabolicheatevolvedpergramofcellproduced
Y
H
dependsonthedegreeofoxidationofthesubstrate
Y
H
~2.4kcal/gonglucose
Y
H
~5.6kcal/gonethanol
H
p g
Totalheatevolved(Q
GR
)dependson
XY V Q
k l/h
Y
H
~8.3kcal/gonmethanol
Y
H
~16.4kcal/gonCH
4
Oxygenuptake
rate
H L GR
XY V Q =
Unitskcal/hr
Foraerobic fermentations,
2
12 . 0
O GR
Q Q ~
Unitskcal/lithr
Whereistheheatproductionthegreatest?
250.000
150.000
200.000
100.000
Dissolved Oxygen
0 000
50.000
Dissolved Oxygen
Optical Density
0.000
0.000 20.000 40.000 60.000 80.000 100.000 120.000
H L GR
XY V Q =
Time from inoculation (hrs)
Cooling water addition Coolingwateraddition
S GR
Q Q Q + =
Total heat to be removed by cooling water is due to metabolic
heat generation plus heat added from the stirring
T UA Q AT UA Q
C
A =
Heat is removed by running cooling water through the cooling
coils in the reactor coils in the reactor
U is the overall heat transfer coefficient of the coils
A
C
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
( ) ( )
i
T T T T ( ) ( )
( ) ( ) | |
out in
out in
T T T T
T T T T
T

= A
/ ln
Offgas Analysis OffgasAnalysis
Offgas composition is an indicator of culture activity Offgascompositionisanindicatorofcultureactivity
Bug: glucose+NH
3
+2O
2
C
4
H
7
O
2
N(bug)+4H
2
O +2CO
2
Respiration:
glucose+36Pi+36ADP+6O
2
6CO
2
+6H
2
O+36ATP
B d thi i l d i ti f th h i t Basedonthissimpledescriptionofthechemistry,wecan
seethatproductionofCO
2
isanindicatoroflivingcells.A
highrateofCO
2
productioncoupledtoO
2
consumptionis
2 2
anindicatorofgrowth.
Offgasanalysis
Offgas
analyzer analyzer
Condenser
Flowmeter
Airoutis<79%N
2
and
<21%O
2
and0.033%<
CO < 6% CO
2
<6%
Aircompressor
Airinis79%N
2
and21%O
2
and
0.033%CO
2
Sparger
2
Offgas composition is an indicator of culture activity Offgascompositionisanindicatorofcultureactivity
CarbondioxideEvolutionRate(CER)istherateofproductionof
CO
2
.
CO
2
,
out
CO
2
,
in
Is the CER
Timeinterval
IstheCER
Since we know the concentration of CO
2
in the air is 0.033%, by SinceweknowtheconcentrationofCO
2
intheairis0.033%,by
measuringtheCO
2
,
out
wecandetermineCER.
Note that this is measured for the whole culture volume so Notethatthisismeasuredforthewholeculturevolume,so
wecandivideouttheculturevolumeforamoremeaningful
value.
Offgas composition is an indicator of culture activity Offgascompositionisanindicatorofcultureactivity
O O
Timeinterval
O
2
,
out
O
2
,
in
IstheOUR
Likewise,
WecanmeasuretheCERandOURonlineusingamass
spectrometer.
Themassspectrometermeasuresconcentrations,andwe
needtoknowtotalamounts.
BycomparingtheO
2
andCO
2
concentrationswiththeN
2
we
candeterminewhattheflowrateoutis,sinceN
2
isneither
producedorconsumedbythebugs.
Calculation of OUR & CER CalculationofOUR&CER
Handout example problem Handout exampleproblem
Best practice: design your feeding strategy such
that O
2
is not the limiting nutrient that O
2
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. during different phases of the run.
When the cells are growing, the feed rate can be determined from:
) (t X
Y
S
X

F
S
=
F
S
is the substrate feed rate,
is the specific growth rate of the organisms
Y
X/S
is the yield coefficient (rate of cell production/rate of substrate uptake) Y
X/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 O
2
is not the limiting nutrient contd
Since we know (we can measure) the yield of cells on oxygen, we can estimate the
ll t ti b
that O
2
is not the limiting nutrient, cont d
cell concentration by
) (
) (
t OUR Y
dt
t dX
O
X
=
OUR can be
measured on-line
dt
O
Together with an estimate of X(t) we can estimate from
dt
dX
t X ) (
1
=
}
=
t
d OUR
t X
t OUR
t
0
) (
) (
) (
) (
IOUR b
}
+ dt t OUR
Y
t
O
X
0
0
) (
) (
IOUR - can be
measured on-line
Best practice: design your feeding strategy such
that O
2
is not the limiting nutrient contd that O
2
is not the limiting nutrient, cont d
Putting everything back into our Feed rate calculation, we have
) (t X
Y
S
X

F
S
=
| | ) (
) (
) ( 1
0
t X IOUR Y
X
t OUR
F
X
S
+ = | | ) (
) (
0
0
IOUR
Y
t X
Y
O
X
O
X
S
X
S
+
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 substrate and it will result in the formation of byproducts.
It solves the problem to choose some fraction of
max
, such as 0.7
max
Off-line analysis methods used
for fermentation
Offline analysis Off lineanalysis
Advantages:
Nointerfacingrequired
Flexibility
Lowcost
Smallsamplevolumes
Disadvantages:
Requiresremovingsamples q g p
Requirestrackingthesamplingtime
Requires operator interaction, potential for bias Requiresoperatorinteraction,potentialforbias
errorfromoperator
Offline Analysis Methods Offline Analysis Methods
Glucose
Glucose concentration is measured by a glucose analyzer. For E. coli
f t ti l h ld b k t b b t 5 /lit d b l 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 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 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 Offline Analysis Methods
Determining cell mass concentration
Di t th d Direct methods:
Dry Weight
Solids-free medium.
Packed Cell Volume
Optical Density Method
Spectrophotometer.
550 or 600 nm wavelengths.
light absorbance cell mass/volume light absorbance cell mass/volume
OD 0.3 need to measure dry-weight of the cells.
Typical Offline analyses TypicalOff lineanalyses
Optical density (OD) analysis of cell Opticaldensity(OD)analysisofcell
concentration
Glucose can be measured off line Glucosecanbemeasuredoffline
NH
3
canbemeasuredbychemicalanalysis
Phosphateandothermacronutrients
Proteinactivityassays y y
HPLC everythingelse