cbe320b slides(part 1)

7/28/2019 CBE320b Slides(Part 1)

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CBE 320b BIOCHEMICAL

ENGINEERING III COURSE NOTES

Instructor: Dr. A. Margaritis, Ph.D., P.Eng., F.C.I.C.

Professor of Biochemical Engineering

http://www.eng.uwo.ca/people/amargaritis/

DEPARTMENT OF CHEMICAL AND BIOCHEMICAL

ENGINEERING

The University of Western Ontario

Faculty of Engineering

A. Margaritis 2006-2007


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TABLE OF CONTENTS

1. Introduction

Bioprocess Design Novel Bioreactor Types Design Criteria for Bioreactors2. Aeration and Oxygen Mass

Transfer in Bioreactor

Systems Oxygen Requirements by Microorganisms

The volumetric Mass Transfer Coefficient KLaand Methods of Measurements

Empirical Correlations of KLa


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3. Agitation of BioreactorSystems

4. Scale-up of BioreactorSystems

Scale-up Criteria Example of Geometric Scale-up

5. Sterilization of Liquid Media Kinetics of Thermal Death of Microorganisms

Batch Sterilization of Liquid Media

Continuous Sterilization of Liquid Media

Examples of Design for Continuous Liquid

Medium Sterilization in a Tubular Sterilizer


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6. Air Sterilization by FibrousBed Filters

Mechanisms of Air Filtration and Design ofFibrous Packed Beds

Example of Design of Fibrous Packed Bed forAir Sterilization


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1. Introduction


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GENERALIZED VIEW OF

BIOPROCESS

RAW MATERIALS

UPSTREAM PROCESSES

Inoculum

Preparation

Equipment

Sterilization

Media Formulationand

Sterilization

BIOREACTOR - FERMENTER

Reaction Kinetics

and Bioactivity

Transport Phenomena

and Fluid Properties

Instrumentation

and Control

DOWNSTREAM PROCESSES

SeparationRecovery and

Purification

Waste Recovery,

Reuse and Treatment

THE BOTTOM LINE

REGULATION ECONOMICS HEALTH AND SAFETY


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TYPICAL BIOPROCESS FLOW SHEET

RAW MATERIASNutrients and Reactants

in Aqueous Solution

(may contain insoluble

organic and/or inorganic

materials)

Air

CELL SEPARATION

1). CELL DISTRUPTION2). PRODUCT EXTRACTION

PRODUCT

CONCENTRATION

PROCESS

FINAL PRODUCT

DRYING

PURIFICATION

PRODUCT

SEPARATION

PREPARATION

OF BIOMASS

Innoculum Stages

FOAM CONTROL

Antifoam Addition

pH CONTROL

Acid-Alkali Addition

Extracellular

product

Intracellular

product

STERILIZATION

BIOREACTOR

Free Cells,

Immoblized Cells

or

Enzyme Bioreactor

PRODUCT RECOVERY


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TABLE 1. Basic Bioreactor Design Criteria___________________________________________________________________

Microbiological and Biochemical Characteristics ofthe Cell System (Microbial, Mammalian, Plant)

Hydrodynamic Characteristics of the bioreactor

Mass and Heat Transfer Characteristics of theBioreactor

Kinetics of the Cell Growth and Product Formation

Genetic Stability Characteristics of the Cell System

Aseptic Equipment Design

Control of Bioreactor Environment (both macro-and micro-environment)

Implications of Bioreactor Design on DownstreamProducts Separation

Capital and Operating Costs of the Bioreactor

Potential for Bioreactor Scale-up______________________________________________________________________


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TABLE 2. Summary of Bioreactor Systems__________________________________________________________

Bioreactor Cell Systems Products

Design used

__________________________________________________________ Air-Lift Bioreactor Bacteria, Yeast and SCP, Enzymes, Secondary

other fungi metabolites, Surfactants

Fluidized-Bed Immobilized bacteria, Ethanol, SecondaryBioreactor yeast and other fungi, metabolites, Wastewater

Activated sludge treatment

Microcarrier Immobilized (anchored) Interferons, Growth factors,

Bioreactor mammalian cells on Blood factors, Monoclonal

solid particles antibodies, Vaccines, Proteases,

Hormones

Surface Tissue mammalian, tissue Interferons, Growth factors,

Propagator growth on solid surface, Blood factors,

tissue engineering Monoclonal antibodies,Vaccines, Proteases, Hormones

__________________________________________________________


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TABLE 2. Summary of Bioreactor Systems(Contd)

____________________________________________________________________________________________________

Bioreactor Cell Systems used Products

Design

________________________________________________________________________________________

Membrane Bioreactors, Bacteria, Yeasts, Ethanol, Monoclonal anti-Hollow fibers and Mammalian cells, Plant bodies, Interferons, Growth

membranes used, cells factors, Medicinal products

Rotorfermentor

Modified Stirred Immobilized Bacteria, Ethanol, Monoclonal anti-Tank Bioreactor Yeast, Plant cells bodies, Interferons, Growth

factors

Modified Packed- Immobilized Bacteria, Ethanol, Enzymes, MedicinalBed Bioreactor Yeasts and other fungi products

Tower and Loop Bacteria, Yeasts Single Cell Protein (SCP)Bioreactors

________________________________________________________________________________________


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TABLE 2. Summary of Bioreactor Systems

(Contd)

_______________________________________

_____Bioreactor Cell System used Products

design

__________________________________________________________________________________________________________

___________

Vacuum Bioreactors Bacteria, Yeasts, Fungi Ethanol, Volatileproducts

Cyclone Bioreactors Bacteria, Yeasts, Fungi Commodity products,SCP

Photochemical Photosynthetic bacteria, SCP, Algae, Medicinal

Bioreactors Algae, Cyano bacteria, plant products,

Plant Cell culture, r-DNA Monoclonal antibodies,

plant cells Vaccines, Interferons

________________________________________________________________________________________


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Fig. 1.1. Schematic diagram of a tower bioreactor system with

perforated plates and co-current air liquid flow.

Medium

inlet

Air filter

OrificeCompressedair

Flow

meter

Peristaltic

pump

Medium

reservior

Constant temp.

water bath

Air exhoust

Pump

Jacket

Perforated

plate

Sparger

Broth

outlet

Sampling

nozzles


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Fig. 1.2. Schematic diagram of a tower bioreactor system

with multiple impellers and liquid down comer and

counter-current air liquid flow

Perforated

plate

Downcomer

Baffle

Impeller

FeedAir

Product

Air


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Fig. 1.3. ICI Deep Shaft Unit

AIR

PROCESS

AIR

OUTLET

RISER

DOWN-

COMER

SHAFT

LINING

INLET

SLUDGE

RECYCLE

START

-UP AIR

CONDENSATE


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FIG. 1.4. EMLICHHEIM FLOWSHEET

AIR

COMPRESSOR

DEEP

SHAFT

B

FLOATATION

LAGOON

BSAND

WASH

WATER

CLARIFIER

RECYCLE SLUDGE

RECYCLED

WATERSETTLEMENT

TANT

CONDENSATE,

MAE-UP WATER, AND

FLOCCULATING AGENT

DECANTER

CENTRIFUGE

SOIL ANDSLUDGE

Outer draft tube


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FIG. 1.5. Internal circulation patterns of fluidized Ca-alginate beads

containing immobilized cells ofZ. mobil is. All dimensions in cm.

0.10.953

6.895

21.30

28.40

2.876

26.43

1.176 2.620 4.530

Outer draft tube

Inner draft tube

4 Jets


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FIG. 1.6. Vacuum Fermenter

Dry ice

bath

Metering

pump

Receiving

tank

(bleed)Filter

Filter

Fermenter

Vacuum

controlReceiving

tank

(product)

Condenser

Level

control

Heating

water

Medium

reservoir

Rheostat

Vacuumpump

Air or O2

Chilled

water


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2. Aeration and Oxygen Mass

Transfer in Bioreactor

Systems


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Living Cells:Bacteria,

Yeasts,

Plant cells,

Fungi,Mammalian Cells

Require Molecular Oxygen O2 asfinal Electron Acceptor in Bioxidation

of Substrates (Sugars, Fats, Proteins,

etc.)


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Substrate O2

Electrons H2O

Products of

Oxidation

CO2

ProductsCell mass

FIG. 2.1. Bio-oxidation of Substrate with Molecular

Oxygen as the Final Electron Acceptor

OXIDATION REDUCTION REACTION


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OXIDATION-REDUCTION REACTION

Glucose is oxidized to make CO2

Oxygen is reduced to make H2O

Fig. 2.1. Shows the biochemical pathway foraerobic oxidation of carbohydrates, fattyacids, and amino acids (AA) via the Tri-carboxylic acid cycle (T.A.C.) and electron

Transport System.

Molecular oxygen O2 accepts all theelectrons released from the substrates duringaerobic metabolism.

Pyruvate


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FIG. 2.2. Aerobic oxidation of carbohydrates, fatty acids, and amino acids via the TCA

cycle and the Electron Transport System (ETS) through which electrons are transported

and accepted by molecular oxygen (O2).

ATP is produced from the phosphorylation of ADP. The ETS iscomposed of the following: FP1 = NADH; FP2 = succinatedehydrogenase; Q = Co-enzyme Q; Cytochrome b, c, a, and a3.The final electron acceptor O2 is reduced to water. Oxygen comes

from the liquid phase and diffuses through the cell.

Pyruvate

Acetyl CoA

alpha-

Ketoglutarate

Marate

Isocitrate

Fumarate

Succinate

2H2H

2H

2H

2H

2H

Citrate

CO2

CO2 NADFPi

FPiADP+Pi

Q b

ADP+Pi

ATP ATP

c a a3

O2

H2O

ADP+Pi

CO2

Oxaloacetate

Amino acids

Fatty acids

Respiratory chain phosphorylation

--Electron transport along the respiratory chain--

OXIDATION REDUCTION REACTION


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OXIDATION-REDUCTION REACTION

(CONTD)

Question: How do we ensure that weprovide enough O2 so that the cell

growth in a bioreactor is not limiting?

Answer: Must ensure that O2 istransferred fast enough from the airbubbles (gas phase) to the liquid phase(usually water) where all cells are

present and growing.


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LIQUID PHASE

O2

O2

O2

O2

Dissolved O2

in liquid phase,

nutrients

(medium mostly

water)

AIR BUBBLE

LIQUID FILM

CELLO2

INTERNAL

CELL

RESISTANCE

LIQUID FILM

CELL-LIQUD

INTERFACE

Electron

TransportSystem +

TCA cycle

enzymes

GAS FILM

GAS-LIQUD

INTERFACE

FIG. 2.3. The oxygen transport path to the microorganism. Generalized path of oxygen

from the gas bubble to the microorganism suspended in a liquid is shown. The variousregions where a transport resistance may be encountered are as indicated


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LIQUID PHASE (CONTD)

At Steady-state with no O2accumulation in the liquid phase:

What are the O2 requirements ofmicroorganisms?

Rate of O2 Transfer (OTR) = Rate of O2 Uptake (OUR)

(Air bubbles Liquid) by Growing Cells

2 1 OXYGEN REQUIREMENTS OF


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2.1 OXYGEN REQUIREMENTS OF

MICROORGANISMS

We define: QO2 = Respiration rate coefficient fora given microorganism.

Units of QO2:

(mass of O2

consumed) (unit wt. of dry biomass) .(time)

Biomass means the mass of cells in abioreactor vessel.

Some units of QO2:mM O2/(g dry wt. of biomass) (hr.)

gO2/(g dry wt.) (hr.)

LO2/(mg dry wt.) (hr.)

CONVERSION FACTORS


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CONVERSION FACTORS:

1 M O2 = 32 x 10-6 g O21 L = 1 x 10-6 L at S.T.P.1 mole O2 = 22.4 L O2 at S.T.P.

In general:QO2 = f(microbial species and type of cell, age of

cell, nutrient conc. in liquid medium, dissolved O2

conc., temperature, pH, etc.)

For a given: 1) type of species of cell2) age of cell

3) nutrient concentration

4) temperature

5 H

d if O t ti C i th li iti f t i ll


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and if O2 concentration, CL, is the limiting factor in cell

growth, then QO2 is a strong function of dissolved O2

concentration CL (= mg O2/L). The relationship between QO2

and CL is of the Monod type.

OxygenCONC. (CL)

O2

0

2

4

6

8

10

12

0 2 4 6 8 10 12 14 16 18 20

QO2ma

KO

QO2max

/

QO

CLCRI.

FIG. 2.4. Respiration coefficient QO2 as a function of the dissolved oxygen concentrationCL.

where: KO2 = O2 conc. at QO2 /2


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where: KO2 O2 conc. at QO2 max/2

CL CRIT. = Critical O2 conc. beyond which O2 isnot limiting

QO2 = QO2max = constant

At CLCRIT. respiration enzymes of Electron Transport System are saturated

with O2.

When O2conc. is the limiting substrate then

analogous to the Monod equation:

max.S

= ________ (S = substrate conc. (g/L)KS + S

= 1 dX (h-1) [Ks = S (g/L), at max/2]

X dt

1.2.

2

22

L

LMAX

C

CQQ


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Table 1 shows typical values of QO2 measured byWarburg respirometer.

Table 2 shows typical data for critical oxygenconcentration CL,CRIT. (mmol O2/L).

FIG. 2 shows the variation of QO2 withfermentation time for the microorganismBacil lus subtil is, where QO2 reaches a maximum

value during the exponential growth phase.

FIG.3 shows the effect of agitation rate (revolutionsper minute) on the value of QO2 for the bacteriumNocardia erythropolis, growing on hexadecane to

produce biosurfactants.

TABLE 1 Cell suspensions in glucose Oxygen uptake determined in


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_______________________________________________________________________

Microbial Species Temp. Culture Resp. Rate Coeff.

(o

C) age (hr.) QO2 (L O2)/(mg dry wt.) (hr.)

____________________________________________________________B.aerogenes 36; 30 17; 48 47; 50Azotobacter choococcum 22 36 2,000-10,000

A.subti l is (cel ls) 37 6-8 170C.subti l is (spores) 32 98-147 10

Corynebacteria species 30 48-96 67

E. col i 40; 32 20 200; 272

L . bulgari cus 45; 37 8 55; 34

M icrococcus lu teus 35 30-34 15

M icrobacter ium avium 37 84 1Mycobacter ium tuberculosis 38 252 4

Pseudomonas fluorescens 26 30 58

_______________________________________________________________________

TABLE 1. Cell suspensions in glucose. Oxygen uptake determined in

constant volume Warburg respirometer

TABLE 2 Typical values of C in the Presence of Substrate


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_______________________________________________________________________

Microorganism Temp. (oC) CL CRIT.

(mmol O2)/L

____________________________________________________________

Azotobacter vinelandii 30 0.018-0.049

E. coli 37.8 0.0082

E.coli 15 0.0031Serratia marcescens 31 0.015

Pseudomonas deni tr i f icans 30 0.009

Yeast 34.8 0.0046

Yeast 20 0.0037

Penicil l ium chrysogenum 24 0.022

Penicil l ium chrysogenum 30 0.009

Aspergil lus oryzae 30 0.020_______________________________________________________________________

Adopted from R. K. Finn, P.81 in: N. Blakebrough (ed),Biochemical Engineer ing Science. Vol. 1, Academic Press, Inc., New

York, 1967

TABLE 2. Typical values of CL CRIT in the Presence of Substrate


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FIG. 2. 5a: Oxygen uptake rate, QO2X () and broth viscosity ()during batch aerobic fermentation ofBacill us subtil is. b:Respirationrate coefficient,QO2 () and volumetric mass transfer coefficient, KLa ().Taken from A.Richard and A. Margaritis, Rheology, Oxygen Transfer, and Molecular Weight Characteristics of Poly(glutamic acid)

Fermentation byB. subtilis, Biotechnology and Bioengineering, Vol. 82 No. 3, p. 299-305, (2003)

FIG 2 6 Eff f i i h i i ffi i (Q ) i 20 L b h


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FIG. 2.6. Effect of agitation on the respiration coefficient (QO2) in a 20 L batch

fermentation ofNocardia erythropolis. () 250 r.p.m, () 375 r.p.m, () 500 r.p.m.(Adopted from Kennedy et al. In Dev. Ind. Microbiol., 20 (1978) 623-630)


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2.2 THE VOLUMETRIC MASS

TRANSFER COEFFICIENTkLa AND METHODS OF

MEASUREMENT

Mass Balance of Oxygen in Unit Liquid


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Volume

AIR BUBBLE

LIQUID FILM

GAS FILM

GAS-LIQUDINTERFACE

Lk

a

C L*

UNIT LIQUID

VOLUME

CELLS

(CONC. X)

O2 C L

OXYGEN

(CONC. C )LBULK

LIQUID

PHASE

O2 TRANSFER

FIG. 2.7 Schematic diagram of the mass balance of oxygen transfer in unit liquid volume


37/96


Volume (Contd)

Rate of = net rate of O2

Accumulation supply from air

of O2 bubblesrate ofO2 consumption by

cells

dCLdt

= kLa(C*L - CL) - QO2X......(2.2)


38/96


Volume (Contd)

where: dCL/dt in (mmol O2/L.h)

kLa in (h-1

)C

*L, CL in (mmol O2/L)

QO2 in (mmol O2/(g dry

wt. cell)(h)X in (g dry wt. Cell/L)


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Volume (Contd)

At steady state:

dCL

dt

kLa(C*

L- C

L) = Q

O2X.........(2.3)

= 0

At all times CL = constant


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Volume (Contd)

Oxygen transfer rate from air

bubbles to liquid = OTR

OTR = kLa (C*L CL)

OTRkLa =(C*L - CL)

......(2.4)



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Volume (Contd)For a given OTR and CL

*(= PyO2/H), please note that as

kLa increases, then CL also increases.

Where:

CL*

= saturated oxygen conc. (mole O2/Lit)

P = total pressure inside air bubble (atm)

yO2 = mole fraction of oxygen in air (0.21)H = Henrys constant (atm.Lit/mole O2)

This is an important way of controlling the dissolved

oxygen concentration CL which also affects the metabolic

activity of aerobic cells their rate of growth and the rate

of production of different metabolic products.

For pure oxygen, yO2 = 1.00

Methods of Measurement of K a


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Methods of Measurement of KLa

in a Bioreactor

Two basic methods for Measuring

KLa

Chemical methods (no cells present)

Physical Methods (with/without

cells)

Chemical Methods of K a


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Chemical Methods of KLa

MeasurementThe Sulphite Batch Oxidation Method.

SO3

2-F,

Water out

Water in

rpm

Motor

Influent

Air flow, rate

Air outlet

FIG. 2.8. Schematicdiagram of a stirred tank batch reactor


44/96

Chemical Methods of KLa

Measurement (Contd)

Liquid Solution = 0.5 M Na2SO3 (Sodium

sulphite), with Cu++ as catalyst.

Sparge air through the bioreactor vessel at a

given volumetric flow rate Q and impellerspeed (R.P.M.)

Make sure that [SO3-2

] is in excess (i.e. 0.5 M

Na2SO3

Chemical Methods of KLa Measurement


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(Contd)

Oxygen oxidizes the sulphite ion tosulphate.

SO3-2 +12

O2Cu++

SO4-2 .......(2.5)

(SULPHITE) (SULPHATE)

The rate of chemical reaction is extremelyfast.

The controlling step is diffusion of O2molecules through the liquid film

surrounding the air bubbles.



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(Contd)

Rate of reaction = R = k2[O2][SO3-2]

~ k1[O2] =

= -

i.e. k1 ~ k2[SO3-2]= constant

2d[SO3-2]1

dt



47/96


(Contd)

i.e. R is zero order to sulphite concentration

[SO3-2

] because it is in excess.? From stoichiometry shown in Eq. (2.5)

dt1 d[SO3-2]2

R = (- ) = (KLa)(CL* - CL)...(2.6)



48/96


(Contd)

The reaction with [SO3-2

] is extremely

fast.

As a result, the O2 gas molecules are

consumed as soon as they diffuse into

the liquid phase. Therefore, the D.O. concentration in

the liquid phase, CL 0.



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(Contd)

Equation (2.6) becomes:

R = (KLa)(CL*) = (KLa)( )PyO2

H = (-12

)d[SO3-2]

dt......(2.7)

Assuming a perfeftly mixed vessel,


50/96


(Contd)

Use iodometric titration to measure

[SO3-2

] as a function of time, t, as theair bubbles pass through the

bioreactor vessel at a given R.P.M.



51/96


(Contd)

SLOPE = - ~ -d[SO3-2]

dt t

[SO3-2]

TIME, t, (min)

[SO

3-]

0

10

20

30

40

50

60

7080

0 2 4 6 8

LOPE = -~ -d[SO3-2]

dt t

[SO3-2]

FIG. 2.9. Concentration of SO3-2

as a function of oxidation time

Chemical Methods of K a Measurement


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(Contd) For a given:

Aeration rate Q

Agitation Speed R.P.M.

Total air pressure P

Volumetric mass transfer coefficientK

La can be calculated from Equation

(2.7) as:

KLa =

)(H)(- )(2 t

[SO3-2]1

PyO2

......(2.8)

-

In Situ Measurement of K a Q and


53/96

In Situ Measurement of KLa, QO2, and

CL* During Cell Growth in a Bioreactor

Consider a Stirred Tank Bioreactor System,

Where Cell Growth takes Place at a Given

Set of Conditions:Aeration

Agitation

pH

TemperatureMedium Composition

In Situ Measurement of KLa QO2 and


54/96



(Contd)The Bioreactor Vessel is Equipped with:

The D.O. Probe, Connected to a D.O. Analyzer.

Chart Recorder:

To Measure Signal from D.O. Probe and

Measure On-line the D.O. Concentration in the

liquid phase of the Bioreactor.

In Situ Measurement of KLa QO2 and


55/96



(Contd) The D.O. Probe Measures the

PyO2 Partial Pressure (PyO2) of

dissolved O2 in the liquidphase, which means that itmeasures HO2CL.

Where:

HO2= Henrys Constant for O2 inWater

In Situ Measurement of KLa, QO2,


56/96


and CL* During Cell Growth in a

Bioreactor (Contd)

Fig. 2.10 Set up of a Stirred tank Bioreactor with Dissolved Oxygen Probe, pH probe andaccessories.

Acid

DO2

1

4 9

pH

7 8

1211

2

10

6

14

rpm

Alkali 13

15

15

16

5

3

1. Feed

2. Flow meter

3. Ring sparger4. Impeller

5. Motor

6. Shaft

7. pH probe

8. D.O. probe

9. Baffle

10. To Condenser

11. D.O. meter

12. pH meter

13. Speed controller

14. Water Jacket

15. Thermometer

16. Chart recorder

Water out

30 deg.

water in



57/96

L , QO2,

CL* During Cell Growth in a

Bioreactor (Contd)Turning air ON and OFF while Maintaining the

same R.P.M. we can:

Record the D.O. Probe Output in the ChartRecorder.

From these Data, we can get

KLa,QO2,

CL*

at given in-situ Bioreactor Conditions.



58/96



(Contd)

The ON-OFF Operation takes 5 min, during which time:

Cell Concentration X (g /L) Constant.We make sure that the D.O. Concentration CL

never falls below the critical oxygen concentration

CCRT,which means that the respiration rate

coefficient QO2 = QO2Max = Constant.

Using the D.O. probe output and a recorder we

measure directly the D.O. concentration as a

function of time t.



59/96

L , QO2,CL

* During Cell Growth in a Bioreactor

(Contd)

While we maintain the same R.P.M. of the bioreactor impeller, we

turn the AIR-OFF. During the AIR-OFF period the following

conditions apply:

Rate of Supply of O2 = 0 No Air Present in the Bioreactor

KLa = 0 because a = 0, no air bubbles present

Using Eq. 2.2 for O2 Mass Balance, we have:

We know cell concentration X by measuring it.

Therefore, we calculate QO2 because we also measure

the slope QO2X.

dCLdt

= 0 - QO2X



60/96

L , QO2,


(Contd)Fig. 2.11 Shows D.O. concentration CL inside thebioreactor = f(t) when Air is turned Off and On, alwayskeeping the R.P.M. of the impeller the same to providegood mixing of the liquid phase.

After a period of about 5 min, a liquid sample is takenfrom the bioreactor to measure the cell concentration X(g dry wt./L).

The KLa, QO2, and CL*values correspond to that

specific fermentation time and given cell growthconditions.

We can do many AIR-OFF and AIR-ONmeasurements to get all three parameters KLa, QO2,

and CL*

as a function of total batch fermentation time.



61/96

L , QO2,


(Contd)

TIME (MIN)

DO2

CONC.CL(mM

O2/L)

AIR-OFF

AIR-ONCL,CRIT

3 - 5

CL STEADY-STATE

FIG. 2.11. Transient Air-Off, Air-On Experiment in a Bioreactor System



62/96

L , QO2,


Bioreactor (Contd) During the AIR-OFF period the D.O. concentration CL is plotted

as a function of time t from which we get the slope = - QO2X, asshown in Fig. 2.12.

Time, t (min)

CL(mMO2/L)

0

1

2

3

4

0 1 2 3 4 5 6 7 8 9 10

SLOPE = - QO2X

FIG. 2.12. D.O. concentration CL as function of time during AIR-OFF period.



63/96



(Contd)AIR-ON PeriodDuring this period the following oxygen mass balance

equation applies:

From the CL vs. time (t) data we can get

dCLdt = KLa (CL* - CL) - QO2X

dCL

dt~

t

CL



64/96

L , QO2,


(Contd) Re-arranging Eq. 2.2 and solving for CL we get Eq. 2.9

By plotting CL vs. at a givenfermentation time, t,

wecan get the slope which is equal to

dCL

dt

+ CL*.....(2.9)CL =

KLa

1- QO2X +

dCLdt

+QO2X

KL

a1

-



65/96

L , QO2,


(Contd)and therefore, the value of KLa is found, and theintercept also gives the value of

During the Air-On Period:

CL* = ConstantQO2 = Constant

KLa = Constant

CL, dCL/dt vary with time t

PyO2HCL* =



66/96

L , QO2,


Bioreactor (Contd)

[dCL/dt+QO2X]

CL(mgO2/L

)

0.8

1.4

2.0

2.6

3.2

3.8

4.4

0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8

SLOPE = -1/kLa

Intercept = CL*

FIG. 2.13.D.O. concentration CL as function of [dCL/dt + QO2X] during AIR-ON period.



67/96

, Q ,CL

* During Cell Growth in a Bioreactor

(Contd) Figures 2.8 and 2.9 show batch aerobic fermentation results in a

stirred tank bioreactor system for the production of thebiopolymer poly(glutamic acid) produced by Bacil lus subtil isobtained by A. Richard and A. Margaritis.

Reference: A. Richard and A. Margaritis (2003), Rheology,Oxygen Transfer, and Molecular Weight Characteristics ofPoly(glutamic acid) Fermentation by Bacil lus subtil is,Biotechnology and Bioengineering, Vol. 82, No. 3, p. 299-305 .

Please read chapter 8, Bioproducts and Economics pp. 609-685,

in Book Biochemical Engineering by H.W. Blanch and D.S.Clark, Marcel Dekker, Inc., New York (1996). This material isuseful for the Plant Design Course, CBE 497 (4th year).



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, Q ,and CL

* During Cell Growth in a

Bioreactor (Contd)

FIG. 2.14. Batch fermentation kinetics ofBacil lus subtili sIFO 3335 during polyglutamic acid production. Biomass, X (); dissolvedoxygen concentration, CL (); Polyglutamic acid (PGA) concentration, P ().

Taken from A. Richard and A. Margaritis, Rheology, Oxygen Transfer, and Molecular Weight Characteristics of Poly(glutamic ac id)

Fermentation by Bacil lus subtili s, Biotechnology and Bioengineering, Vol. 82, No. 3, p. 299-305 (2003).



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and CL* During Cell Growth in a

Bioreactor (Contd)

FIG. 2.15. Dynamic air-on/air-off data during Poly(glutamic acid (PGA) production by Bacil lus subtili sIFO 3335

(fermentation time = 26 h). Dissolved oxygen concentration CL () as a function of time.Taken from A. Richard and A. Margaritis, Rheology, Oxygen Transfer, and Molecular Weight Characteristics of

Poly(glutamic acid) Fermentation by Bacil lus subtili s, Biotechnology and Bioengineering, Vol. 82, No. 3, p. 299-305(2003).


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2.3. EMPIRICAL CORRELATIONS

OF KLa

A large number of Empirical


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Correlations Exist for KL and KLa forAgitated and Aerated Bioreactor

Vessels. General Background Reading:

Textbook by H.W. Blanch and D.S.

Clark Biochemical Engineering,Chapter 5. Transport Processes,

pp. 343-415. Publisher: Marcel Dekker,

Inc., New York, 1996.

Consider a Stirred Tank BioreactorVessel at a given:

Pg


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g

VL

DT

LH

AIR, Q

Q = Vol. air flow rate

@S.T.P.

DT = Tank diameterHL = Liquid height (un-

gassed)

VL = Working Liquid

volume (un-gassed)Pg = Gassed power

P = Un-gassed power

Impeller Speed R.P.M.

Aeration Rate QWorking Liquid Volume V

L

of the Vessel

FIG. 2.16. Typical stirred tank bioreactor vessel

Most Empirical Correlations for KLa have the


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Most Empirical Correlations for KLa have the

following form

Where:

KLa = Vol. mass transfer coefficient Pg = Gassed power supplied by

mechanical impeller for mixing of

bioreactor vessel. VL = Liquid working volume ofbioreactor vessel

KLa = C PgVL

m Ugk................(2.10

EMPIRICAL CORRELATIONS


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OF KLa

Ug = Superficial air velocity

m, k = Exponents, constants

The values for C, m, and k depend greatly on the ionic strength of theaqueous phase in the bioreactor.

Ionic strength, I, of the solution in the bioreactor is defined by Equation 2.11.

I = (Zi2Ci)(2.11) Where:

I = Ionic strength of solution, (g ions/L)

Zi = Electric charge of ionic species i, present in the solutione.g.

SO4-2 = has Zi = -2

Na+ has Zi = +1

Ag+ has Zi = +1

Ci = Concentration of ionic species in the solution = (g-ions/L)

Cross-sectional area of

bioreactor vessel

Vol. air flow rate @ S.T.P.=



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OF KLa

Constants C, m, and k also depend on: Temperature, T

pH

Physical properties of the solution Presence of other nutrients

For Pure Water at pH = 7, T = 25 oC, the following

empirical correlation applies:

KLa = (0.026)PgVL

0.4

Ug

0.5....(2.12)



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OF KLa

Where:KLa = Vol. mass transfer coefficient (s

-1)

Pg = Gassed power (W)

Ug = Superficial air velocity (m s-1)

Note: The values of C = 0.026, exponents

0.4 and 0.5 in Eq. 2.12 can be usedonly with the units of KLa, Pg and

Ug specified above.

A log-log plot of experimental data according to Equation2.10 is shown in the following figure.


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Taking the log on both sides of Eq. 2.10, we get

log (KLa) = log (C) + k log (Ug) + m log (Pg/VL).

log (Pg/VL)

logKLa

SLOPE = m

Ug = CONSTANT

FIG. 2.17. A log-log plot of experimental data according to Equ. 2.10.

Definition of gas-holdup, Ho, in an agitated and

aerated essel


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aerated vessel

TV

AIR

LIQUID PHASE,

VL

AIR BUBBLES,

Vg (DISPERSEDPHASE)

Ho = gas hold-up =Volume occupied by gas phas

Total volume

(VT) Total volume = Liquid Volume (VL)+Gas volume (Vg)

Ho =Vg

Vg +VL

.........................(2.13

FIG. 2.18. Typical agitated and aerated stirred tank bioreactor vessel

Assuming a monodispersed size distribution of airbubbles each having the same diameter dB, then the


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g B,gas hold-up Ho is related to the interfacial specificgas-liquid area and dB according to Eq. 2.14.

Where:

Ho = dimensionless dB = bubble diameter, m

a = interfacial specific area, m2/m3 = m-1

Eq. 2.14 can be used as an approximation for arough estimate of specific interfacial area a (m2/m3of total volume)

.........................(2.14dB

6Hoa =


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3. AGITATION OF BIOREACTOR

SYSTEMS

Fig. 3.1 shows the dimensions of what is called astandard stirred tank bioreactor vessel with


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standard stirred tank bioreactor vessel with

Baffles.

FIG. 3.1. Standard Stirred Tank Bioreactor Geometry [Adopted from S. Aiba, A.E.

Humphrey and N.F. Millis. Bubble Aeration and M echanical Agitation. In Biochemical

Engineering, 2nd Ed., Academic Press, Inc., New York (1973) 174].

Geometric Ratios for a Standard BioreactorVessel


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VesselImpeller Di/Dt HL/Dt Li/Di Wi/Di Hb/Di Wb/Dt No. Baffles

Type

Flat-Blade 0.33 1.0 0.25 0.2 1.0 0.1 4Turbine

Paddle 0. 3 3 1.0 - 0.25 1.0 0.1 4

impeller

Marine 0.33 1.0 pitch = Di 1.0 0.1 4

Propeller

Where:

Dt = tank diameter,

HL = liquid heightDi = impeller diameter

Hb = impeller distance from bottom of vessel

Wb = baffle width

Li = impeller blade length

Wi = impeller blade height


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FIG. 3.2 A. Different Impeller Types. (a) Marine-type propellers; (b) Flat-blade

turbine, Wi = Di/5. Disk flat-blade turbine, Wi = Di/5, Di = 2Dt/3, Li = Di/4; (d)

Curved-blade turbine, Wi = Di/3; (e) Pitched-blade turbine, Wi = Di/8; and (f)

Shrouded turbine, Wi = Di/8.


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FIG. 3.2 B. Mixing Patterns for Flat-Blade Turbine Impeller. Effect of Baffles. Liquid

agitation in presence of a gas-liquid interface, with and without wail baffles: (a) Marine

impeller and (b) Disk flat-blade turbines; (c) in full vessels without a gas-liquid interface

(continuous flow) and without baffles.

3.1 Mixing and Power Requirements for


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Newtonian Fluids in a Stirred Tank

FIG. 3.3 NP vs. NRe; the power characteristics are shown by the power number, NP, and the

modified Reynolds number, NRe, of single impellers on a shaft. [Adopted from S. Aiba, A.E.

Humphrey and N.F. Millis. Bubble Aeration and M echanical Agitation. In Biochemical

Engineering, 2nd Ed., Academic Press, Inc., New York (1973) 174].

Fig. 3.3 shows relationship between NP and


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NRe at three different flow regimes:

Laminar Transient

Fully Turbulent

for three different impeller types:

Six-bladed flat blade turbine

Paddle impeller

Marine Propeller

The power number is given by Equ.3 1


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3.1

NP = Pgc/n3Di5(3.1)The impeller Reynolds number is given

by Equ. 3.2

NRe = nDi2/..................(3.2)

Where:NRe = dimensionless Reynolds number

NP

= dimensionless Power number

P = Un-gassed power for liquid (no air), W


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gc = 1, for SI units system

n = Impeller rotational speed, revolutions per

sec., (s-1)

Di = Impeller diameter, m = Density of liquid, kg/m3 = Viscosity of liquid, (N.m)/(s)For six-bladed flat-blade turbine impeller (cf.

Fig. 3.3), the mixing becomes fully turbulent atan impeller Reynolds number NRe = 3,000.

Power number NP = 6 (constant) at NRe > 3,000

Different Types of impellers have


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different power characteristics Fig. 3.3.

For six-bladed flat turbine and forturbulent conditions:

NP

= 6 = Pgc/n3D

i

5or P = (6)(n3Di

5)/(gc)..(3.3)At NRe = 3,000 the corresponding

impeller speed is:

n = (3,000)()/(Di2)()(3.4)

Eq. 3.4 is an estimate of the minimum impeller

d f 6 fl bl d bi i ll f h


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speed, n, of a 6-flat blade turbine impeller for the

on-set of turbulent flow within the stirred tank

bioreactor vessel.

Eq. 3.3 shows that for a fluid of a given density,

:P n3Di5

This is an important consideration for bioreactorvessel scale-up.

Eq. 3.1 is used to find the un-gassed power, P, at

i


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a given:

impeller diameter, Di and

impeller speed, n.

For aerobic fermentation (aerated) bioreactors:

Pg (gassed) < P (un-gassed) power

since eff(effective density) < Pg/P < 1

The aeration number, Na, is defined by Equ. 3.5 and is

d t tif th ti P /P f ti f


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used to quantify the power ratio Pg/P as a function of

aeration rate Qg, as shown in Fig. 3.4.

For water:Na = Qg/nDi

3(3.5)

Where:

Na = aeration number (dimensionless)Qg = Volumetric flow rate of air (m

3 at STP/s)

n = impeller rotational speed, revolutions per

second (s-1).

Di = impeller diameter (m).


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FIG. 3.4 Power requirements for agitation in a gassed system. The ordinate and abscissa are

degree of power decrease, Pg/P, and the aeration number, Na. Parameters are the types of

impellers, whose representative geometrical ratios in agitated vessels are also shown in the

figure. [Adopted from S. Aiba, A.E. Humphrey and N.F. Millis. Bubble Aeration and

Mechanical Agitation. In Biochemical Engineering, 2nd Ed., Academic Press, Inc., New

York (1973) 176].

Fig. 3.4 shows the relationship between


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Pg/P ratio and Aeration Number, Na,

for three types of mechanical impellers:

Flat-blade turbine (A)

Vaned disk impeller withdifferent vanes (np = 4, 6, 8, 16)

curves, B, C, D, E

Paddle impeller

Calculation of the Required Volumetric


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Mass Transfer Coefficient, KLa, During

Fermentation, and Gassed Power, Pg.

At Steady-State Operation of an AerobicFermentation:

OTR = OURKLa[CL

* - CL] = QO2X.(3.6)

For a given QO2, X, and (CL* - CL), KLa canbe calculated using Eq. 3.6.


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be calculated using Eq. 3.6.

For a given VL and Ug, Pg can be calculatedusing the empirical correlation for KLa given

by Eq. 3.7.

KLa = C [Pg/VL]m [Ug]k3.7

Figs. 3.3 and 3.4 are used in combination to find the

correct rotational impeller speed, n, to deliver therequired Pg at a given Ug, for the required value of

KLa.

cbe320b slides(part 1)

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