Cell Single Type; Batch Reactor
Substrate Enzyme Substrate + Cells X : cell mass
Product Product + More Cells N : cell number
Substrate Limited Cell Growth
E. coli growth on glucose (glucose is limiting substrate) How would you model this growth kinetics?
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) Conc. in bulk broth Critical conc Rate of demand 10,000 ppm 100 ppm 2.8 mmoles/g cells/h Broth O2 sat @ 25oC approx. 7 ppm 0.7 ppm 7.7 mmoles/g cells/h
Need to constantly supply!
Different Measures of Oxygen
ppm (parts per million) mg/L .. Pretty much similar to ppm mmole dm-3 .. 1/32 of mg/L % (partial pressure) Need Henrys law .. 21% is air ~ 7 mg/L at room temperature
Critical dissolved oxygen levels for a range of microorganisms
Organism TemperatureCritical dissolved oC Oxygen concentration (mmoles dm -3) 30 37 30 0.018 0.008 0.004 0.022
Azotobacter sp. E. coli Saccharomyces sp.
Penicillium chrysogenum 24
Azotobacter vinelandii is a large, obligately aerobic soil bacterium which has one of the highest respiratory rates known among living organisms
Tissue Ambient air Artery Vein Bone marrow Brain Epidermis Kidney Liver Muscle Oviduct Spleen Testis
Oxygen Concentration Percentage (%) 21 13 3.2 5 3-4 3-5 1.7-2.1 4.34 2.7-4 5 3.1 1.7
Mean Oxygen Concentration in Various Tissues
Air 21% Vein 3% Muscle 2.7-4% Alveoli 14%
Important for regulation Of function (growth, differentiation, Testis 1.7% death, Oviduct 5% Secretion, etc)
Artery 13%
Brain 3-4% Bone Marrow 5%
RESPIRATION RATE (Demand)
Oxygen Uptake Rate (OUR) = qO2*X
qO2 = mmoles of oxygen consumed per gram of dry weight (or mg O2/gm DCW hour) specific rate of oxygen consumption X = cell concentration (gm DCW/L)
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) (mg O2/g DCW hour) or = max. C/ (Ks + C) where C = oxygen conc.
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
Oxygen Requirement is Cell Dependent
Page 293 table.
Oxygen transfer (Supply)
Oxygen Transfer Rate
kLa and CO* Oxygen transfer coefficient (kL)(cm/h) and interfacial area (a)(cm2/cm3)
Because it not possible to accurately measure the total interfacial area of the gas bubbles (a), kL and a are combined into single term, referred to kLa (h-1).
= kLa( * CL ) OTR C
The kLa represents the oxygen transfer rate per unit volume. Oxygen concentration at the gas-liquid interface(Co*). P. 171-173; 292-297
Batch Growth - Oxygen Limitations
Oxygen transfer rate (mg O2/liter hour) (OTR) = kLa(C* - CL) Oxygen uptake rate (OUR) = qO2X Under oxygen limitation, OTR = OUR (negligible maintenance requirement w.r.t. growth):
OUR = qO2 X =
g X
YX O2
= k a( C
L
*
- CL )
How crucial is aeration?
Bioreactors
Sub ml to greater than 100 m3. Few cents to a few million dollars. Standing cultures Shake flasks Stirred tank reactors Bubble column and airlift reactors
Membrane and other novel bioreactors Micro-Bioreactors
Standing Culture
No power for aeration Simple equipment OTR poor Fine for low OUR experiments on small scales (few mL) Large-scale also performed with standing culture where electricity poor
Standing cultures - Surface cultures
Test Tubes & Fernbach Flasks
Which has better OTR?
Erlenmeyer flask Fernbach flask
Bacteria Low volume bacteria-based vaccine production
T-Flask
Which has better OTR? Mainly for small scale animal cell culture
Shake flasks
Shake flasks are commonly used for small scale cell cultivation. Through continuous shaking of the culture fluid, higher oxygen transfer rates can be achieved as compared to standing cultures. Shaking continually breaks the liquid surface and thus provides a greater surface area for oxygen transfer. Increased rates of oxygen transfer are also achieved by entrainment of oxygen bubbles at the surface of the liquid. Although higher oxygen transfer rates can be achieved with shake flasks than with standing cultures, oxygen transfer limitations will still be unavoidable particularly when trying to achieve high cell densities.
Shake flasks- baffles
The presence of baffles in the flasks will further increase the oxygen transfer efficiency, particularly for orbital shakers. The following photographs show how baffles increase the level of gas in entrainmentment a shake flask being shaken in an orbital shaker at 150 rpm
Unbaffled flask
Baffled flask
Note the high level of foam formation in the baffled flask due to the higher level of gas entrainment. The same improvement in oxygen transfer is not as evident with horizontal reciprocating shakers.
Shake flasks- factors affecting kLa
shaking speed the liquid volume shake flask design
kLa decreases with liquid volume
kLa increases with liquid surface area
kLa is higher when baffles are present
- The kLa will increase with the shaking speed. - The appropriate liquid volume is determined by the flask volume. - Larger liquid volumes can be used with wide based flasks.
Mechanically Stirred Reactor
Agitation (used for large liquid volumes > 3L). Sparging dramatically increases oxygen transfer area Sparged reactors require significantly lower agitation speeds
PROS Nearly Perfect Mixing High OTR Page 286 CONS High Power Internal Moving Parts High Shear
Stirred tank reactor
Non-sparged
Sparged
Degree of agitation
forms small bubbles delaying escape of air bubbles from liquid prevents coalescence of air bubbles decreasing thickness of stagnant liquid film at the gas/liquid interface
Culture Rheology
Consumption/formation of polysaccharides Microbial biomass
Bubble Column & Airlift Fermenters
Airlift fermenters have a draft tube. Because of the draft tube (guides flow) Airlift fermenters have better mass and heat transfer efficiencies Airlift fermenters have more uniform shear conditions. Airlift fermenters require no internal moving parts Has good mixing Reduces bubble coalescence High viscosity can limit bulk circulation
Bubble Column & Airlift Fermenters
EFFECT OF DRAFT TUBE Fluid flows up in Air Riser; down in Down-Comer Air riser on outside gives better heat transfer Liquid movement better mixing and less bubble coalescence More bubbles higher kLa
EFFECT OF DISENGAGEMENT ZONE Widening slows liquid, disengages bubble from flow And reduces aerosol formation Stretches bubble Burst with flow Reduces Foaming
Which reactor gives higher productivity? Why?
Oxygen transfer
The mass transfer coefficient (kL) The value of kL can be increased by
size of boundary layer rate molecules travel through the boundary layer size of boundary layer determined by level of mixing.
The diffusivity of the molecule through the boundary layer is determined by
Medium viscosity Temperature Increasing temperature also reduces the medium viscosity but it will also decrease the solubility of oxygen.
Oxygen transfer
Factors affecting the interfacial area (a) The primary method of enhancing the value of kLa is by increasing the area of the gas-liquid interface (a). The interfacial area is determined by the aeration rate and the bubble diameter Airlift bioreactors are good Microbioreactors have an advantage
Determination of kLa
Determination of kLa in a bioreactor is important in to establish its aeration efficiency and quantify effects of operating variables on oxygen supply Used to compare bioreactors before scale up or scale down A number of different methods are available Without cells growing With cells growing
1. kLa Sulphite Oxidation
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
Cu++ or Co++
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
2. kLa Gassing Out
unsteady state method
ln(C*-CL) = -kLat + ln(C*-C0)
3. kLa Steady-State Direct Method
where:
PO = Partial pressure of oxygen R = gas constant (0.08206 dm3.atm.K-1.mol-1) Vl = medium volume (measured) Vg = gas volume (measured) T = temperature (Kelvin) (measured)
4. kLa Dynamic Method
A B Off then on C
qO2X
Utilises the growing culture to reduce O2 levels Slope of AB is a measure of the respiration rate
(OTR is zero, so dC/dt = - qO2X)
BC is difference between transfer of oxygen into solution and uptake by the culture (and from AB we know qO2X)
Practice
Example 10.1 page 297.
Use the data (dynamic method) from figure above to estimate kLa. The DCW is 2g/L, what is the respiration rate?
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