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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
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OUR
[Substrate]
Time
X
In batch culture OUR changes strongly over time due toincrease in biomass (X)depletion of substrate (S).
However OUR can be considered constant:• over short time intervals (min)• in continuous culture Very useful tool to study microbes and reactor behaviour
OUR – Variation during batch culture
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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)
OUR – Significance
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1. Aerate to maximum
2. Stop aeration
3. Monitor cL
c L
Time (sec)
Conclusion:
1. OUR is linear over most cL values2. A critical D.O. exists
OUR – Determination
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D.O.(mg/L)
Time (sec)
Maximum rate
about half maximum rate
OUR – Dependency on DO
The next slide shows the green and blue part of this curvebut as the rate as a function of D.O.
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OUR(mg/L/h)
D.O. (mg/L)
The OUR is mostly independent of D.O.(zero order kinetics)
At very low D.O. the OUR is strongly dependent on D.O. (close to 1st order kinetics)
OUR – Dependency on DO
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OUR(mg/L/h)
D.O. (mg/L)
Examinable concepts:
D.O. saturationD.O. limitationFirst order reactionZero order reactionMichaelis Menten kineticsDriving ForceEquilibrium
OUR – Dependency on DO
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0.5 1 2
OU
R (
mg
/L/h
)
Cri
tica
l D
O
DO (mg/L)
Dependence of OUR on the dissolved oxygen concentration (DO or cL )
Conclusions:
1. Typical Michaelis Menten relationship2. ks at about 0.1 ppm (critical D.O.: 0.2 mg/L)3. Over most DO concentations
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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)
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Simultaneous OUR – OTR Oxygen Steady State
10Klein T, Schneider K, Heinzle (2012) Biotechnology&Bioengineering 110: 535-542
dcL/dt = 0
dcL/dt = OUR
DO
(p
pm
)
Air OffAir On
Time (sec)
A B C
A
B
C
= - QO2.X
dcL/dt = OTR - OUR
= kLa (cs - cL) - QO2.X
Steady state: 1. OUR constant
2. OTR constant
3. DO constant
4. OUR = OTR
When dcL/dt = 0 → OUR = OTR→ OUR = kLa(cs – cL)
Conclusion: When kLa is known, steady state OUR can becalculated from the dissolved [oxygen] (D.O.) (cL)
OUR – Indirect online monitoring
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FeedOn
FeedOn
FeedOff
FeedOff
FeedOff
DO
(p
pm
)
Time (min)
The addition of feed to a starving culture of microbesresults in an instantaneous increase of OUR, which Causes a drop in the D.O.
OUR – Dependency on DO
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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 immediatelyOnline OUR monitoring is possible (online respirometry)Useful for Degradability tests, toxicity tests, process optimisation
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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)
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3. Calculation of new OUR from old OUR and cL
Original OUR = 120 mg/L/h at cL of 4 mg/LAfter further growth DO lowered to 2 mg/LWhat is the new OUR?
kLa = = = 30 h-1 OUR 120 mg/L/h(cS – cL) 4 mg/L
OUR = kLa * (cS – cL) = 30 h-1 * (8 mg/L – 2 mg/L) = 180 mg/L
OUR – applications of online DO monitoring
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4. Calculation of new kLa from old kLa and cL
Original kLa = 30 h-1 at cL of 2 mg/LAfter increasing airflow the new cL was 5 mg/LWhat is the new kLa?
new kLa = = = 60 h-1 OUR 180 mg/L/h(cS – cL) 3 mg/L
OUR = kLa * (cS – cL) = 30 h-1 * (8 mg/L – 2 mg/L) = 180 mg/L/h
OUR – applications of online DO monitoring
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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 ofa 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
OUR- Comparison of Methods for kLa determination
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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
OUR- Comparison of Methods for kLa determination
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D.O.(mg/L)
Time
Effect of minute feed addition on D.O. profile of aerated starving microbial culture
Add feed
There is very useful information in the OUR response of microbial cultures to the addition of substrates or inhibitors
OUR – indirect respiration activity monitoring
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D.O.(mg/L)
Time
Effect of minute feed addition on D.O. profile of aerated starving microbial culture
Add feed
OTR
D.O cS
OUR
D.O cS
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D.O.(mg/L)
Time
Effect of minute feed addition on D.O. profile of aerated starving microbial culture
Add feed
OUR(mg/L/h)
OUR – indirect respiration activity monitoring
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OUR – indirect respiration activity monitoring
Time (min)
OUR response to feed spike by starving microbial culture.
Numerical integration (counting squares) allows to determinethe amount of oxygen used due to the feed spike addition.
OUR(mg/L/h)
6 12 24
20
40
20 mg/L/h * 0.1h =
2 mg/L
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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 kLasulfite 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
Lecture overview L 4-6
