respirometria (2)
TRANSCRIPT
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Bioreactores y BioreaccinAnlisis de reactores biolgicos de aplicacin
novedosa
6.1. Caractersticas y aplicaciones de reactores con
microorganismos inmovilizados.
6.2. Caractersticas y aplicaciones de los reactores biolgicos de
membrana.6.3. Consideraciones generales de diseo para el uso de foto-
bioreactores.
6.4. Consideraciones de diseo para el uso de bioreactores
para el cultivo de clulas de mamfero y de
plantas.6.5 Tendencias en el desarrollo de bioreactores biolgicos.
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Kinetic and Stoichiometric parametersTraditional methods for parameters estimation are based on substrate measurements during
batch and continuous lab experiments.
X -Difficult to measure low substrate concentrations
X -Time consuming
Relevancia: The development and optimization ofindustrial fermentation processes ask for a clear
picture of the relevant microbial kinetics.
Pulse respirometry for characterization
of aerobic biodegradation process
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Indirect measurement ofsubstrate
Drawbacks of traditional methods may be overcome
by other techniques such as:
! dissolved organic carbon
! chemical oxygen demand (COD)
! biological oxygen demand (BOD)
!
oxygen uptake rate (OUR
)! CO2 production.
Kovarova-Kovar and Egli, 1998
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Allows the indirect measurement of substrate
consumption rates by monitoring the biological
Oxygen Uptake Rate (OUR), under well defined
conditions
RESPIROMETRY TECHNIQUE
Respirometry
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Oxygen uptake rate (OUR)
OURmeasuring
allows the retrieval of kineticparameters within a good confident interval
Spanjers et al., 1995; Vanrolleghem et al., 1995; Ellis et al., 1996
"YO2/S
"YX/S
" KS" Rmax
"!max
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Substrate vs. Oxygen measurements
Substrate OxygenSophisticated
techniques
Probe
normally expensive easily available at lowcost
Specific No specificOff-line In situ
Sensitivity ( mg L-1) Sensitivity ( 10 !g L-1)
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Respirometry techniques
Static
IntermitentAir supply
Dynamic
ContinuousAir supply
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Respirometry in Biofilm reactors
Static
IntermitentAir supply
Lack ofhomogeneity
Dynamic
ContinuousAir supply
KLaO2determination
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Respirometry in Biofilm reactors
Static Dynamic
DO
SP
Time
DO
Time
SP
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Respirometry in Biofilm reactors
Dynamic
ContinuousAir supply
KLaO2determination
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DO ProbepH Probe
Dissolved oxygen bench meter
4.8
5
5.2
5.4
5.6
5.8
6
6.2
0 10 20 30 40 50 60 70
t (d)
O2
(mg/l
)
t (min)
4.8
5
5.2
5.4
5.6
5.8
6
6.2
0 10 20 30 40 50 60 70
t (d)
O2
(mg/l
)
t (min)
4.8
5
5.2
5.4
5.6
5.8
6
6.2
0 10 20 30 40 50 60 70
t (d)
O2
(mg/l
)
t (min)
4.8
5
5.2
5.4
5.6
5.8
6
6.2
0 10 20 30 40 50 60 70
t (d)
O2
(mg/l
)
t (min)
Respirogram
Data acquisition system
Air Supply
Simple and low cost equipment
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4.8
5
5.2
5.4
5.6
5.8
6
6.2
0 10 20 30 40 50 60 70
t (d)
O
2
(mg/l)
t (min)
4.8
5
5.2
5.4
5.6
5.8
6
6.2
0 10 20 30 40 50 60 70
t (d)
O
2
(mg/l)
t (min)
4.8
5
5.2
5.4
5.6
5.8
6
6.2
0 10 20 30 40 50 60 70
t (d)
O
2
(mg/l)
t (min)
4.8
5
5.2
5.4
5.6
5.8
6
6.2
0 10 20 30 40 50 60 70
t (d)
O
2
(mg/l)
t (min)
In siturespirometry involves:
1. Feeding suspension .
1
Dynamic Respirometry protocol
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4.8
5
5.2
5.4
5.6
5.8
6
6.2
0 10 20 30 40 50 60 70
t (d)
O
2
(mg/l)
t (min)
4.8
5
5.2
5.4
5.6
5.8
6
6.2
0 10 20 30 40 50 60 70
t (d)
O
2
(mg/l)
t (min)
4.8
5
5.2
5.4
5.6
5.8
6
6.2
0 10 20 30 40 50 60 70
t (d)
O
2
(mg/l)
t (min)
4.8
5
5.2
5.4
5.6
5.8
6
6.2
0 10 20 30 40 50 60 70
t (d)
O
2
(mg/l)
t (min)
In siturespirometry involves:
1. Feeding suspension.
2. A new pseudo steady-state (absence of soluble substrate).
1
2
Dynamic Respirometry protocol
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4.8
5
5.2
5.4
5.6
5.8
6
6.2
0 10 20 30 40 50 60 70
t (d)
O
2
(mg/l)
t (min)
4.8
5
5.2
5.4
5.6
5.8
6
6.2
0 10 20 30 40 50 60 70
t (d)
O
2
(mg/l)
t (min)
4.8
5
5.2
5.4
5.6
5.8
6
6.2
0 10 20 30 40 50 60 70
t (d)
O
2
(mg/l)
t (min)
4.8
5
5.2
5.4
5.6
5.8
6
6.2
0 10 20 30 40 50 60 70
t (d)
O
2
(mg/l)
t (min)
In siturespirometry involves:
1. Feeding suspension .
2. A new pseudo steady-state, (absence of soluble substrate).
3. Injection of substrate pulses.
1
2 3
Dynamic Respirometry protocol
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Dynamic Respirometry protocol
4.8
5
5.2
5.4
5.6
5.8
6
6.2
0 10 20 30 40 50 60 70
t (d)
O
2
(mg/l)
t (min)
4.8
5
5.2
5.4
5.6
5.8
6
6.2
0 10 20 30 40 50 60 70
t (d)
O
2
(mg/l)
t (min)
4.8
5
5.2
5.4
5.6
5.8
6
6.2
0 10 20 30 40 50 60 70
t (d)
O
2
(mg/l)
t (min)
4.8
5
5.2
5.4
5.6
5.8
6
6.2
0 10 20 30 40 50 60 70
t (d)
O
2
(mg/l)
t (min)
2 2
0 0
2/
t t
ex L bl
O S
P P
OUR dt k a (O O )dt Y
S S
!= =
" "
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Dynamic Respirometry protocol
4.8
5
5.2
5.4
5.6
5.8
6
6.2
0 10 20 30 40 50 60 70
t (d)
O
2
(mg/l)
t (min)
4.8
5
5.2
5.4
5.6
5.8
6
6.2
0 10 20 30 40 50 60 70
t (d)
O
2
(mg/l)
t (min)
4.8
5
5.2
5.4
5.6
5.8
6
6.2
0 10 20 30 40 50 60 70
t (d)
O
2
(mg/l)
t (min)
4.8
5
5.2
5.4
5.6
5.8
6
6.2
0 10 20 30 40 50 60 70
t (d)
O
2
(mg/l)
t (min)
XOS !+!=!2
COD/L Units
S
XO
!
!+!=
21
SXSO YY
//21 +=
/ 2/1
X S O SY Y= !
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Dynamic Respirometry protocol
Injection of pulse of
increasing concentration
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Dynamic Respirometry protocol
max
max
2/
ex
O S
OURR
Y
=
max /
max
2/
ex X S
O S
OUR Y
Y X =
!
!
Injection of pulse of
increasing concentration
i i d i hi i
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Kinetic and stoichiometriccharacterization
!
A total of 6 parameters can be obtained by in situ pulserespirometry:
! YO2/S
!
YX/S
! OURexmax
! Rmax
!
KS! !max
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Conclusions
!
Currently in situ pulse respirometry has been applied in:! Bubble colum reactors
! Airlift reactors
! Stirred tank reactors
!
Partitionary reactors! Biofilm reactors
! To charaterize,
!
Mixed cultures (activated sludge, nitrification)! Pure cultures (pseudomonas sp., comamonas sp., rhodococcus sp.)
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Introduction
in
out
YO2/S
YX/S
KSRmax
!max
In situ
Respirometry
Biofilm
reactor
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Introduction
Stoichiometric and kinetic analyses are
critical tools for developing efficient
processes for the optimum cell growth,nutrient utilization and production of high-
value cell culture-based products such as
therapeutic proteins, vaccines orenvironmental applications.
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Biofilm reactors
! In a Biofilm reactor, microorganisms are attached to asolid substratum which can be gravels, stones, plastic,sand, or activated carbon particles.
! Most of Biofilm reactor applications are in wastewatertreatment and bioremediation area (trickling filter,rotating disk, submerged filters or fluidized filter).
! Biofilm reactor applications to produce value-addedproducts are still remained in bench-scale or pilot scale
Kuan-Chen et al., 2010
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Biofilm reactors
!
Traditional biofilm reactors characterization is achievedfollowing two principal routes
Off situ analysis
Difficult to obtainrepresentative samplesSampling the
filter media
Analysis of several pseudosteady-states
Time-consuming task
Not take into account potentialbiofilm accumulation
Inffluent andeffluent mass
balance
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Respirometry in Biofilm reactors
Dynamic
ContinuousAir supply
KLaO2determination
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Dynamic Respirometry pathway
KLaO2
YO2/S
YX/SKS
Rmax
!max
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Estimation of KLaO2!
Dynamic gassing-out method! Sulfite method
HomogeneousKLaO2
Suspended Biomass
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Estimation of KLaO2!
Dynamic gassing-out method! Sulfite method
HeterogeneousKLaO2
Rashig
Rings
Fixed Biomass
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Estimation of KLaO2!
Dynamic gassing-out method! Sulfite method
HeterogeneousKLaO2
Rashig
Rings
#Higher UGin the packed section
#KLaO2is correlated with UG
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Estimation of KLaO2
The internal volume of the reactor is composed of two sections:(i)the filter-bed and (ii)the top section with no filter-bed.
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Estimation of KLaO2
Julio Prez et al., (2006) reported that KLaO2
in the filter-bed was 3 to 7 times higher than
in the top section!!
$ Dynamic gassing out method
$Sulfite method
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Effect of heterogeneous KLaO2 on kineticparameters (Ordaz et al., 2011)
Maximum Error of 55%!!
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Effect of mixing timeon kinetic parameters(Ordaz et al., 2011)
Mixing time:
Time require to achieve a predefined level
of homogeneity
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Effect of mixing timeon kinetic parameters(Ordaz et al., 2011)
Maximum Error of 150 % !!
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Erratic values of YO2/Sand YX/S
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Basic approach to test respirometry inbiofilm reactors
DO Probe pH Probe
InfluentEffluent
Air Supply
$
Avoid Heterogeneity in KLaO2$Reduce mixing time
Model support
media
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Steady values of YO2/Sand YX/S
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Apparent parameters vs. Real parametersin biofilm reactors
! Only apparent parameters has been estimated.
! Diffusion effects has not taken into consideration.
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Apparent parameters vs. Real parametersin biofilm reactors
Kinetic and stochiometric characterization before and after
biofilm disruption
l
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Apparent parameters vs. Real parametersin biofilm reactors
Kinetic and stochiometric characterization before and after
biofilm disruption
Biomass UGR(cm s-1)
YX/S
(mg COD mg-1
COD)
Fixed 0.13 0.496 0.004 (A)
0.25 0.484 0.017 (A)
0.50 0.489 0.016 (A)
1.00 0.458 0.038 (A)Suspended 0.13 ND
0.25 0.393 0.042 (B)
0.50 0.502 0.007 (A)
1.00 0.533 0.006 (A)
l
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Apparent parameters vs. Real parametersin biofilm reactors
Kinetic and stochiometric characterization before and after
biofilm disruption
BiomassUGR
(cm s-1)
OURexmax(mg O2L
-1h-1)
Fixed 0.13 41.32 2.78 (G)
0.25 43.23 3.90 (G)
0.50 48.71 3.37 (G)
1.00 53.85 3.40 (G)
Suspended 0.13 ND
0.25 113.21 9.47 (H)
0.50 131.97 1.17 (H)
1.00 139.07 23.76 (H)
A R l
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Apparent parameters vs. Real parametersin biofilm reactors
Kinetic and stochiometric characterization before and after
biofilm disruption
Diffusion effects may hide someparameters
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Conclusions
!
Respirometry is a technique that allows a rapid kinetic andstoichiometric characterization of several kinetic andstoichiometric parameters with relative low experimentaleffort with low cost equipment.
!
It has been succesfully applied in suspended cultures, howeverin biofilm reactors respirometry technique has been poorlystudied.
! The group has focus in the evaluation of Dynamicrespirometry for biofilm characterization and it has beenfound that several factors such as Kla, mixing time and diffusion may influence drastically the results obtained.