respirometria (2)

8/10/2019 respirometria (2)

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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


KLaO2determination


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Static Dynamic

DO

SP

Time

DO

Time

SP


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Dynamic


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)


1. Feeding suspension.

2. A new pseudo steady-state (absence of soluble substrate).

1

2



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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)


1. Feeding suspension .

2. A new pseudo steady-state, (absence of soluble substrate).

3. Injection of substrate pulses.

1

2 3



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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)

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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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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Injection of pulse of

increasing concentration


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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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Dynamic


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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HeterogeneousKLaO2

Rashig

Rings

Fixed Biomass


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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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Kinetic and stochiometric characterization before and after

biofilm disruption

l


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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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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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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.

respirometria (2)

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