Industrial Microbiology

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Industrial Microbiology INDM 4005 Lecture
11 25/02/04
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5. REACTOR DESIGN AND PHYSIOLOGY

• TRANSPORT / Mass transfer, aeration and
  agitation
• OVERVIEW
• 1. Concepts of mass transfer through different
  phases using oxygen as an example.
• 2. Oxygen demand and respiration
• 3. Factors influencing mass transfer through
  gas\liquid interfaces
• 4. Kla - measurement, factors influencing.
• 5. Agitation, mixing patterns
• 6. Impeller design, fluid dynamics
• 7. Relationship of viscosity and agitation
• 8. Power input
• 9. Scale-up

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Lecture Overview
5. Reactor design and physiology 5.1. Mass
transfer and phases 5.1.1 different phases
present -introduction 5.1.2. Mass transfer and
respiration 5.1.3. Factors affecting oxygen
demand 5.1.4. Factors influencing oxygen
supply 5.1.4. (a) process factors 5.1.4. (b)
transfer through an interface (kla) 5.1.4. (c)
determination of kla 5.1.4. (d) factors
affecting bubble size 5.1.4. (e) gas hold-up
5.1.4. (f) economics of oxygen transfer
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Introduction

• The oxygen demand of an industrial process is
  generally satisfied by aeration and agitation
• Productivity is limited by oxygen availability
  and therefore it is important to the factors that
  affect a fermenters efficiency in supplying O2
• This lecture considers the O2 requirement,
  quantification of O2 transfer and factors
  influencing the transfer of O2 into solution

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5.1. MASS TRANSFER and PHASES

• 5.1.1 Different phases present -Introduction
• Fundamental concept in fermentation technology is
  transfer of materials (e.g nutrients, products,
  gases etc.) through different phases (e.g gas
  into a liquid).
• Major problem associated with provision of oxygen
  to the cell - is a rate limiting step and thus
  serves as a model system to understand mass
  transfer.
• The rate of oxygen transfer driving force /
  resistance. E.g resistance to mass transfer from
  medium to mos are complex and may arise from
• ? Diffusion from bulk gas to gas/liquid interface
• ? Solution of gas in liquid interface
• ? Diffusion of dissolved gas to bulk of liquid
• ? Transport of dissolved gas to regions of cell
• ? Diffusion through stagnant region of liquid
  surrounding the cell
• ? Diffusion into cell
• ? Consumption by organism (depends on
  growth/respiration kinetics)

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• The following diagram serves to illustrate the
  different phases and material that are relevant
  in general transport processes associated with
  fermentation technology

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Phases present in bioreaction/bioreactor
1 reactant supply and utilisation 2 product
removal and formation
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Mass Transfer

• One of the most critical factors in the
  operation of a fermenter is the provision of
  adequate gas exchange.
• The majority of fermentation processes are
  aerobic
• Oxygen is the most important gaseous substrate
  for microbial metabolism, and carbon dioxide is
  the most important gaseous metabolic product.
• For oxygen to be transferred from a air bubble
  to an individual microbe, several independent
  partial resistances must be overcome

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1) The bulk gas phase in the bubble 2) The
gas-liquid interphase 3) The liquid film around
the bubble 4) The bulk liquid culture medium 5)
The liquid film around the microbial cells 6)
The cell-liquid interphase 7) The intracellular
oxygen transfer resistance
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Stoichiometry of respiration
To consider the stoichiometry of respiration the
oxidation of glucose may be represented as
6H2O 6CO2
C6H12O6 6O2
Atomic weight of Carbon Hydrogen Oxygen
12 1 16
Molecular weight of glucose is 180
How many grams of oxygen are required to oxidise
180g of glucose?
Answer 192g
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Solubility of Oxygen

• Both components oxygen and glucose must be in
  solution before they become available to
  microorganisms
• Oxygen is 6000 times less soluble in water than
  glucose
• A saturated oxygen solution contains only10mg
  dm-3 of oxygen
• Impossible to add enough oxygen to a microbial
  culture to satisfy needs for complete respiration
• Oxygen must be added during growth at a
  sufficient rate to satisfy requirements

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Comparison of conc. driving forces and uptake
rates for glucose and oxygen by yeast

• Problems encountered in oxygen transport can be
  illustrated by comparing transport of glucose vs
  oxygen
• 1 Sugar (glucose) Broth O2 sat _at_ 25oC
• Conc. in bulk broth 10,000 ppm approx. 7 ppm
• Critical conc 100 ppm 0.8 ppm
• (growth stops)
• Rate of demand 2.8 mmoles/ g cells /h 7.7
  mmoles/ g cells /h

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5.1.2. MASS TRANSFER and RESPIRATION

• (a) Mass balance
• Stoichiometry of respiration e.g glucose
• C6H12O6 6O2 ? 6H2O 6 CO2
• Oxidation of 180 gms Glucose requires 192 gms O2
• Compare with a hydrocarbon (i.e 6 CH2)

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The Oxygen requirements of industrial
fermentations

• Oxygen demand dependant on convertion of Carbon
  (C) to biomass
• Stoichiometry of conversion of oxygen, carbon and
  nitrogen into biomass has been elucidated
• Use these relationships to predict the oxygen
  demand for a fermentation
• Darlington (1964) expressed composition of 100g
  of dry yeast C 3.92 H 6.5 O 1.94

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O2 Requirements

• 6.67CH2O 2.1O2 C 3.92 H 6.5 O 1.94 2.75CO2
  3.42H2O
• 7.14CH2 6.135O2 C 3.92 H 6.5 O 1.94 3.22CO2
  3.89H2O
• where CH2 hydrocarbon
• CH2O carbohydrate
• From the above equations to produce 100g of yeast
  from hydrocarbon requires three times the amount
  of oxygen than from carbohydrate

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• Compare solubility of Oxygen vs Glucose ( e.g.
  oxygen 9.0 mg/l _at_ 20oC, 11.3 mg/l _at_ 10oC)
• How would salt water influence oxygen conc. i.e.
  sea water?
• Thus must consider
• ?Requirement for oxygen important in
  biotechnological processes
• ?Quantification of oxygen transfer (to avoid rate
  limiting step) important
• ? Factors influencing rate of transfer (e.g.
  viscosity) important

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• Case Study
• Give the chemical properties of oxygen, why is it
  so important to life?
• From your notes on physiology give examples of
  biochemical pathways (of commercial significance)
  influenced by oxygen (i.e aerobic vs anaerobic).
• What type of bioreactor is used in the production
  of the products chosen?

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(b) OXYGEN CONC. vs RESPIRATION RATE (growth
rate)

• The effect of dissolved oxygen on the specific
  uptake rate (i.e respiration or growth) is
  described by
• Michaelis Menton or Monod type relationship
• Respiration rate (QO2) QO2 max . O2 conc / (
  Ks O2 conc)
• or
• ? ?max. C/ (Ks C) where C oxygen conc.
• QO2 mmoles of oxygen consumed per gram of dry
  weight

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Effect of dissolved O2 concentration on the QO2
of a microorganism
QO2
Ccritical
Dissolved Oxygen Concentration
Specific O2 uptake increases with increase in
dissolved O2 levels to a certain point Ccrit
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Critical dissolved oxygen levels for a range of
microorganisms
Organism Temperature Critical
dissolved oC Oxygen concentration (mmol
es dm -3)
Azotobacter sp. 30 0.018 E.
coli 37 0.008 Saccharomyces
sp. 30 0.004 Penicillium chrysogenum 24 0.02
2 Azotobacter vinelandii is a large, obligately
aerobic soil bacterium which has one of the
highest respiratory rates known among living
organisms
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Critical dissolved oxygen levels

• To maximise biomass production you must satisfy
  the organisms specific oxygen demand by
  maintaining the dissolved O2 levels above Ccrit
• Cells become metabolically disturbed if the level
  drops below Ccrit
• In some cases metabolic disturbance may be
  advantageous
• Or high dissolved O2 levels may promote product
  formation
• Amino acid biosynthesis by Brevibacterium flavum
• Cephalosporium synthesis by Cephalosporium sp.

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5.1.3. FACTORS AFFECTING OXYGEN DEMAND

• ? Rate of cell respiration
• ? Type of respiration (aerobic vs anaerobic)
• ? Type of substrate (glucose vs methane)
• ? Type of environment (e.g pH, temp etc.)
• ? Surface area/ volume ratio
• large vs small cells (bacteria v mammalian
  cells)
• hyphae, clumps, flocs, pellets etc.
• ? Nature of surface area (type of capsule etc)

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O2
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5.1.4. FACTORS INFLUENCING OXYGEN SUPPLY
5.1.4 (a) Process factors
Gas composition, volume velocity
Foam/antifoam Temperature Type of
liquid Height/width ratio Hold up
Design of Impeller size, no. of blades rotational
speed
Baffles width, number
Size of sparger gas bubble
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5.1.4(b) Transfer through an interface (Kla)
Ci O2 conc at interface CL O2 conc in
liquid Pg Partial pressure of gas Pi
Partial pressure at interface
Bubble Gas
Liquid of Gas film
film Pg Pi (1/k2)
Ci (1/k4) (1/k1)
(1/k3) CL
Bulk Liquid
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Overall mass transfer is (Whitman theory)

• dC/dt kg (Pg - Pi) KL
  (Ci - CL)
• (Driving force) (Resistance)
• Note kg 1/k1 1/k2, KL 1/k3 1/k4
• Use conc rather than partial pressure (measure?)
• ? dC/dt KL (Csat - CL) ......assume that
  Csat substitutes for Ci (measure?)
• This is per unit interface!
• Overall then dC/dt KLa( Csat - CL)

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5.1.4. (c) Determination of KLa

• Determination of KLa in a fermenter is important
  in to establish its aeration efficiency and
  quantify effects of operating variables on oxygen
  supply
• Used to compare fermenters before scale up or
  scale down
• A number of different methods are available

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5.1.4.(c) Determination of KLa

• (1) The Sulphite oxidation technique
• Measures the rate of conversion of a 0.5m
  solution of sodium sulphite to sodium sulphate in
  the presence of a copper or cobalt catalyst
• Na2SO3 1/2 O2 Na2SO4
• Oxidation of sulphite is equivalent to the
  oxygen-transfer rate
• Disadvantages i) slow,
• ii) effected by surface active agents
• iii) Rheology of soln not like media

Cu or Co
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5.1.4.(c) Determination of KLa

• (2) Gassing out techniques
• Estimation of KLa by gassing out involves
  measuring the increase in dissolved O2 of a
  solution during aeration and agitation
• The OTR will decrease with the period of aeration
  as CL approaches CSAT due to resultant decrease
  in driving force (CSAT - CL)
• The OTR at any one time will be equal to the
  slope of the tangent to the curve of dissolved O2
  conc against time of aeration

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The increase in dissolved O2 conc of a soln over
a period of agitation
Y
Dissolved oxygen concentration
X
Time
The OTR at Time X is equal to the slope of the
tangent drawn at point Y
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• 5.1.4.(c) Determination of KLa
• (2) Gassing out techniques
• involve initially lowering the oxygen value to a
  low level
• (i) Static Method
• O2 concentration of the solution is lowered by
  gassing out with liquid N2
• The deoxygenated liquid is then aerated,
  agitated and increase in dissolved O2 is
  monitored with oxygen probe
• Rapid method 15 mins
• May utilise fermentation medium and dead cells
• Require membrane -type electrode which doesnt
  have response time required for true changes in
  oxygenation rate
• Main disadvantage on industrial scale are
  quantities of liquid N2 required and single point
  measurements not representative of the bulk
  liquid

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5.1.4.(c) Determination of KLa

• (ii) Dynamic Method
• Involves measuring oxygen levels in growing
  culture in the fementer
• Utilises the growing culture to reduce O2 levels
• Correction factors must be used
• Slope of AB is a measure of the respiration rate
• BC is observed increase in dissolved oxygen is
  the difference between transfer of oxygen into
  solution and uptake by the culture

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Dynamic gassing out for the determination of Kla
values. Aeration terminated at point A and
recommenced at point B
C
X
A
Slope AB gives RX (Respiration rate) Slope BC
gives dC/dt
Dissolved oxygen concentration
B
Time
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Dynamic Method

• Expressed as the equation
• dC/dt Kla (Csat - CL) - RX
• R respiration rate (mmoles of O2 g-1 biomass
  h-1),
• X concentration of biomass
• Turn off air supply, monitor dissolved O2
• dC/dt - RX ... thus the slope of the trace
  gives RX
• Resume aeration and monitor,
• Supply term can be calculated (from slope
  substitute calculated value of RX)

dC/dt KLa (Csat - CL) - RX (slope
BC) (solve) (Literature) (Observe) (slope AB)
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Dynamic Method

• Advantages
• Can determine KLa during an actual fermentation
• Rapid technique
• Can use a dissolved oxygen probe of the membrane
  type
• Limitations
• Limited range of dissolved oxygen levels can be
  studied
• Must not allow oxygen levels to fall below Ccrit
• Difficult to apply technique during a
  fermentation with a high oxygen demand
• Relies on measurements taken at one point

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• CASE STUDY
• Identify why the following factors influence KLa
• ? Rheological properties of broth (e.g viscosity)
• ? Air flow rate and volume
• ? Degree of agitation
• From a process point of view outline the
  relationship between KLa and
• ? Power consumption (cost)
• ? Operating variables (liquid density, impeller
  design and speed, aeration, and its importance

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5.1.4.(d) FACTORS AFFECTING BUBBLE SIZE

• (a) Influence of gas velocity on bubble
  formation

slow medium fast
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• b) Influence of liquid properties on bubbles

Liquid can change from A ? B when salts are
added. Implication for mass transfer in different
media. Will this property of liquids influence
Kla - why?
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5.1.4.(e) GAS HOLD-UP

• Represents air volume retained in the liquid
• Vh V - V0
• Where Vh hold-up volume, V vol. of gassed
  liquid, V0 vol of ungassed liquid.

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• Correlations exist that relate hold-up to power
  input , for example,
• (P/V)0.4 . Vb 0.5
• P/V power input per unit vol ungassed liquid,
  Vb linear velocity of air bubble (ascending
  velocity).
• Ascending velocity of bubble (Vb)
• Vb FHl/H0V
• Where H0 hold-up of bubble, F aeration rate,
  Hl liquid depth, V liquid volume.

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CASE STUDY Show an application of optimising
hold-up in a reactor i.e. through mixing or
length to width ratio (increasing path length)
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• How does height (h) of a reactor vary with radius
  (r) when volume (v) is kept constant?
• volume of a cylinder is v ? r 2 h
• Let us fix the volume as 1 then
• h 1/ ? r 2
• If r 1 then h 1/?
• r 2 then h 1/4?
• r 3 then h 1/9?
• Therefore as the radius increases the height (or
  path length) decreases as the square of the
  radius

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5.1.4.(f) ECONOMICS OF OXYGEN TRANSFER
Fermentation e.g Penicillin - ?high KLa Waste
treatment -? economy Kla . Csat maximum rate
at which oxygen can be dissolved Economy and
capacity related through power input per unit
volume (P/V) ECONOMY KLa. Csat / (P/V)
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CASE STUDY Compare a pumped air, sparged system
of aeration with a surface aerator (as used in
waste treatment i.e. What are the
advantages of each system
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The balance between OXYGEN DEMAND and
SUPPLY Must consider how processes may be
designed such that O2 uptake rate of the culture
does not exceed the oxygen transfer rate of the
fermentor. Uptake rate QO2.X QO2 O2 uptake
rate, X Biomass dC/dt KLa(Csat - CL )
supply rate Dissolved O2 conc. should not fall
below the critical dissolved O2 conc. (Ccrit) A
fermentation will have a max Kla dictated by
operating conditions thus it is the demand that
often has to be adjusted. Achieved by Control
of biomass conc. Control of specific O2 uptake
rate Combination of both