Topic 02:
Microbial Oxygen Uptake Kinetics
• After having investigated principles and methods of
quantifying the oxygen transfer OTR (from gas phase
to solution):
• Now we will investigate the oxygen uptake rate OUR
behaviour of bacteria
• Leading to combining both kinetics in a typical reactor
where often OUR = OTR. This allows easy online
interpretation of the whole bioprocess
1
OUR – Variation during batch culture
In batch culture OUR changes strongly over time due to
increase in biomass (X)
depletion of substrate (S).
X
OUR
[Substrate]
Time
However OUR can be considered constant:
• over short time intervals (min)
• in continuous culture
2
 Very useful tool to study microbes and reactor behaviour
OUR – Significance
1. Critical indicator of culture status (respiration rate).
2. Indicator or growth (relationship X* / OUR).
3. Indicator of health, inhibition etc ( if X = constant).
4. Essential for culture optimisation.
5. Should be ideally monitored online.
*) X= biomass concentration (e.g. g Dry Weight/L)
3
OUR – Determination
1. Aerate to maximum
2. Stop aeration
cL
3. Monitor cL
Time (sec)
Conclusion:
1. OUR is linear over most cL values
2. A critical D.O. exists
4
OUR – Dependency on DO
Maximum rate
D.O.
(mg/L)
about half maximum
rate
Time (sec)
The next slide shows the green and blue part of this curve
but as the rate as a function of D.O.
5
OUR – Dependency on DO
OUR
(mg/L/h)
The OUR is mostly
independent of D.O.
(zero order kinetics)
At very low D.O. the OU
R is strongly dependent on
D.O.
(close to 1st order kinetics)
D.O. (mg/L)
6
OUR – Dependency on DO
Examinable concepts:
OUR
(mg/L/h)
D.O. saturation
D.O. limitation
First order reaction
Zero order reaction
Michaelis Menten kinetics
Driving Force
Equilibrium
D.O. (mg/L)
7
Critical DO
OUR (mg/L/h)
Dependence of OUR on the dissolved
oxygen concentration (DO or cL )
0.5
1
2
DO (mg/L)
Conclusions:
1. Typical Michaelis Menten relationship
2. ks at about 0.1 ppm (critical D.O.: 0.2 mg/L)
3. Over most DO concentations
8
Simultaneous OTR and OUR makes the
bioreactor more complex
• The previous slides have shown OUR
without new air input
• The next slides consider oxygen
transfer rate (OTR) at the same time
as oxygen uptake rate (OUR)
9
Simultaneous OUR – OTR
 Oxygen Steady State
Klein T, Schneider K, Heinzle (2012) Biotechnology&Bioengineering 110: 535-542
10
OUR – Indirect online monitoring
Steady state:
1. OUR constant
2. OTR constant
Air Off
3. DO constant
4. OUR = OTR
A dcL/dt = 0
DO (ppm)
Air On
B dcL/dt = OUR
= - QO2.X
A
B
C
Time (sec)
When dcL/dt = 0
C dcL/dt = OTR - OUR
= kLa (cs - cL) - QO2.X
→ OUR = OTR
→ OUR = kLa(cs – cL)
Conclusion: When kLa is known, steady state OUR can be
calculated from the dissolved [oxygen] (D.O.) (cL)
12
OUR – Dependency on DO
Feed
On
DO (ppm)
Feed
On
Feed
Off
Feed
Off
Feed
Off
Time (min)
The addition of feed to a starving culture of microbes
results in an instantaneous increase of OUR, which
Causes a drop in the D.O.
14
OUR – calculation from in situ DO monitoring
1. Calculation of OUR from kLa and cL
Given:
• Reactor with airflow that gives a known kLa of . kLa = 20 h-1
• Due to bacterial OUR a steady state DO establishes at 2 mg/L
• OUR = kLa * (cS-cL)
kLa = 20 h-1, cL = 2 mg/L, cS= 8 mg/L OUR = ?
OUR = 20 h-1 x 6 mg/L = 120 mg/L/h
Conclusion:
OUR can be determined immediately
Online OUR monitoring is possible (online respirometry)
Useful for Degradability tests, toxicity tests, process optimisation
15
OUR – calculation from in situ DO monitoring
2. Determination of kLa in situ (dynamic method)
Since under steady state: OTR = OUR
kLa = OUR
(cs – cL)
16
OUR – applications of online DO monitoring
3. Calculation of new OUR from old OUR and cL
Original OUR = 120 mg/L/h at cL of 4 mg/L
After further growth DO lowered to 2 mg/L
What is the new OUR?
kLa =
OUR
(cS – cL)
=
120 mg/L/h
4 mg/L
= 30 h-1
OUR = kLa * (cS – cL) =
30 h-1 * (8 mg/L – 2 mg/L) = 180 mg/L
17
OUR – applications of online DO monitoring
4. Calculation of new kLa from old kLa and cL
Original kLa = 30 h-1 at cL of 2 mg/L
After increasing airflow the new cL was 5 mg/L
What is the new kLa?
OUR = kLa * (cS – cL) = 30 h-1 * (8 mg/L – 2 mg/L) = 180 mg/L/h
OUR
new kLa =
(cS – cL)
=
180 mg/L/h
3 mg/L
= 60 h-1
18
OUR- Comparison of Methods for kLa determination
1. Static Gassing Out Method (N2)
De-oxygenate solution, monitor DO increase over time.
Determine kLa (a) graphically or (b) mathematically
2. Sulfite Method
Sulfite reactos spontaneously with D.O (in the presence of
a cobalt catalyst – as also used in our lab session)
Titration of sulfite consumption during oxygenation trial
- indirect measurement
+ no oxygen probe required
+ allows direct monitoring of standard OTR
standard OTR = kLa . cs
3. Dynamic Method (in situ kLa)
+ measured real in situ value considering changes of
medium such as viscosity, particles, surface tension ...
- depends upon known OUR
+ only slight process interuption necessary
+ only works when DO >> critical DO
19
OUR- Comparison of Methods for kLa determination
4. Oxygen Balance Method = Direct Monitoring
OTR = specific air flow . ([O2]in - [O2]out )
mg/L.h
L(g)/L(l).h
mg/L(g)
Measures the oxygen concentration in the exit air of the reactor
+ integrates over the whole reactor volume
+ not affected by fine air bubbles (which still transfer
some oxygen for a while even after stopping of air flow)
+ no response lag by oxygen probe
- Longer response time to step changes
- Lower precision of oxygen readings in air than in solution
20
OUR – indirect respiration activity monitoring
There is very useful information in the OUR response of microbial
cultures to the addition of substrates or inhibitors
D.O.
(mg/L)
Add
feed
Time
Effect of minute feed addition on D.O. profile of
aerated starving microbial culture
21
OTR
OUR
D.O
D.O.
(mg/L)
cS
D.O
cS
Add
feed
Time
Effect of minute feed addition on D.O. profile of
aerated starving microbial culture
22
OUR – indirect respiration activity monitoring
OUR
(mg/L/h)
D.O.
(mg/L)
Add
feed
Time
Effect of minute feed addition on D.O. profile of
aerated starving microbial culture
23
OUR – indirect respiration activity monitoring
40
OUR
(mg/L/h)
20
20 mg/L/h
* 0.1h =
2 mg/L
6
12
Time (min)
24
OUR response to feed spike by starving microbial culture.
Numerical integration (counting squares) allows to determine
the amount of oxygen used due to the feed spike addition.
24
Lecture overview L 4-6
Lab1 (computerlab): Intro to CBLA use, oxygen solubility,
show bioprosim, download material, use memory sticks,
Henry’s law, temperature effect on oxygen solubility. Use
of spreadsheets for data processing
Lecture 4: In situ method of determining kLa
sulfite method of determining kLa
Lecture 5: Online OUR monitoring as a key bioprocess
monitoring tool. Saturation behaviour of OUR. Critical DO.
Respirometric testing of substrates and inhibitors.
Numeric integration of rate data