Oxygen is normally supplied to microbial cultures in the form of air, i.e cheapest source of oxygen oxygen transfer is complex and it involves a phase change from its gaseous phase to the liquid phase. Oxygen should be maintained at optimal concentration for maximal yeild. Anaerobic conditions develop when the rate of oxygen utilization is > than OTR ( this limit growth and production)
Oxygen is normally supplied to microbial cultures in the form of air, i.e cheapest source of oxygen oxygen transfer is complex and it involves a phase change from its gaseous phase to the liquid phase. Oxygen should be maintained at optimal concentration for maximal yeild. Anaerobic conditions develop when the rate of oxygen utilization is > than OTR ( this limit growth and production)
Oxygen is normally supplied to microbial cultures in the form of air, i.e cheapest source of oxygen oxygen transfer is complex and it involves a phase change from its gaseous phase to the liquid phase. Oxygen should be maintained at optimal concentration for maximal yeild. Anaerobic conditions develop when the rate of oxygen utilization is > than OTR ( this limit growth and production)
Occurs in many processes such as evaporation, adsorption, drying, precipitation, filtration and distillation.
In bioprocess Concentration of compounds are not uniform
Mass transfer principle is made use of to achieve this
E.g., Oxygen transfer Solvent extraction
Aeration Oxygen is normally supplied to microbial cultures in the form of air, i.e cheapest source
Sterile air / oxygen which must be dispersed throughout the fermenter
Air introduced into the fermentor is filter sterilized and introduced via sparger which is located below the agitator
Sparger structure affects oxygen transfer in the medium as it influences the size of the gas bubble produced
Smaller the bubble, larger the surface area to volume ratio which provides greater oxygen transfer.
Spargers with small pore size are effective in producing small bubbles but are prone to clogging and require high energy
Oxygen transfer Oxygen transfer is complex and it involves a phase change from its gaseous phase to the liquid phase which is influenced by following factors
1. Temperature, pressure and surface area of oxygen bubbles 2. Chemical composition of the medium 3. Volume of gas introduced per unit reactor volume per unit time 4. Type of sparger system used to introduce air into the fermenter 5. Speed of agitation
In aerobic fermentation- oxygen should be maintained at optimal concentration for maximal yeild
Oxygen mass balance
-The rate at which O 2 can be delivered to the biological sysytem (OTR-Oxygen transfer rate) and the rate at which it is utilised by microorganism (COD- Critical oxygen demand) Anaerobic conditions develop when the rate of Oxygen utilization is > than OTR ( This limit growth and production)
OTR can be increased by elevating the pressure, enriching the inlet air with O 2 and increasing agitation and aeration Solubility of oxygen depends on temperature and pressure
Temperature in C
p(O2)=100 kPa Solubility in mg/l
p(O2)=20.9 kPa Solubility in mg/l
Determination of OTR OTR = Oxygen gradient
Resistance to oxygen transfer
= Oxygen gradient ( C*-C L ) eqn 1 K L a C* =is the saturated dissolved oxygen conc.(mmol/dm 3 ) C L =is the concentration of dissolved O 2 at time (t) (mmol/dm 3 ) K L =is the mass transfer coefficient at the gas to liquid phase (phase boundary)(cm/h) a =
is the gas/liquid interface area per liquid volume (cm 2 cm -3 ) Stages of resistance of oxygen transfer from gaseous phase to an individual cell 1. Resistance within the gas film to the phase boundary
2. Penetration of the phase boundary between gas bubble and liquid
3. Transfer from the phase boundary to the liquid
4. Movement within the nutrient solution
5. Transfer to the surface of the cell
6. Entry into the cell
7. Transport to the site of reaction within the cell Gas bubble Gas film Fluid film Fluid Fluid film Rate of O 2 transfer from air bubble to the liquid phase is described as dC L = k L a(C * - C L ) eqn. 2 dt Integration of eqn 1 gives C*-C L = e -KLat eqn 3
Interms of natural log, for k L a determination
ln (C * - C L ) = -K L a (t) eqn 4 K L a for the specific conditions is determined by plotting a semilog graph of ln(C * - C L ) against time where slope is mass transfer coefficient (K L a)
Therefore K L a is a measure of the aeration capacity of a fermentor and must be maintained above a minimum critical level to satisfy oxygen requirements
K L a K L = Is the mass transfer coefficient a = Is the gas/liquid interface area per liquid volume (cm 2 cm -3 )
These are difficult to measure individually and are generally linked to give as
K L a = volumetric mass transfer coefficient per hr
How to improve the oxygen transport? Increase of the O 2 -solubility Pressure increase from 100 to 200 kPa Increase the O 2 -content in the air enrichment of the aeration with O 2
Use of pure O 2
Change in the phase boundary (gas/liquid) size and distribution of the gas bubbles contact time between the gaseous phase and the liquid phase Viscosity of the nutrient solution viscosity reduction increases the relative velocity of the gas bubbles thinner liquid film higher k L a-value
Scale-up What is Scale-Up? Increasing the scale of a fermentation. (i.e. From lab scale to pilot scale and from pilot scale to industrial scale 3 Stages Bench Scale ( 2 20 L) Pilot Scale (100 500 L) Plant Scale (500 20,000 L)
What is a scale-up problem? A scale-up problem is something that we do not see in the small-scale experiment(lab scale) and are surprised and disappointed to find in the large scale process..
Problems associated with scale up are due to the different ways in which process parameters are affected by increase in size of the unit 1) Inoculum development when a scale is increased -extra stages have to be incorporated in the inoculum development programme
2)Sterilization It is a scale dependent factor When there is increase in scale of a fermentation process, the sterilization regime should be adjusted according to the scale This may result in change in quality of the medium after sterilization
Major factors involved in Scale-Up 3. Environmental parameters Increase in scale may results in a change in the environment for the microorganism. Environmental parameters may changed due to increase in scale: i) Nutrient availability ii) pH iii) Temperature iv) Shear conditions v) Dissolved oxygen concentration vi) Dissolved CO2 concentration vii) Foam production
Agitation Aeration agi tati on aerati on shear cost foam mi xi ng oxygen CO 2 Scale up window based on AGITATION / AERATION This illustrates the "scale-up" window defining the operating boundaries for aeration and agitation in the scale- up of a typical fermentation.
Agitation and aeration rate must fall between a minimum and maximum value Problems that may arise, when these values are not falling within the limits.
ACTION RESULT Minimise aeration below the limit Decrease in CO2 and O2 levels Maximise aeration above the limit Increased Foam formation takes place Minimise agitation below the limit Bulk mixing poor
Maximise agitation above the limit Shear and cost increased Solution for scale up problem Identify the environmental parameter affected by aeration and agittaion. eg: Oxygen concentration, Shear, bulk mixing
Identify the process variable or variables which affects the identified environmental parameter
Calculate the value of the process variable to be used on large scale which results in the same environmental conditions on both scales Process variables which affect the mixing and mass transfer
Process Variable Mass transfer or Mixing property affected Power consumption per Unit volume Oxygen transfer rate Volumetric air flow rate Oxygen transfer rate Impeller tip speed Shear rate Pumping rate Mixing time Reynolds number Heat transfer Scale-Up Parameters First scale-up criterion is the preservation of Geometrical similarity when building a new bioreactor vessel, the geometry is usually scaled linearly. height to diameter ratio (H/D) or aspect ratio is kept constant to ensure the tanks will operate similarly.
Vessel 1 Vessel 2 The aspect ratio of the vessels is 1.5. The height and diameter between the two vessels is scaled linearly.
By multiplying the dimensions of Vessel 1 by 6.4, the dimensions of Vessel 2 are determined.
Note: the volume of the vessels does not scale linearly (volume of vessel 1 is 147 ft 3 and vessel 2 is 38,603 ft 3 , 262 times larger.) To scale-up a manufacturing process from one bioreactor to another, the process parameters are scaled based on the following :
NOTE: Cannot keep all parameters constant during scale up because they scale by different values
Scale-Up Parameters Common Scale-Up Parameters Agitation-based scaling parameters: Mixing Time Power Input per Volume (P/V) Tip Speed Notes: These three parameters are all dependent on agitation rate, so all three cannot be held constant when scaling-up. For example: Keeping mixing time constant might cause a high P/V that the cells cannot handle. Scaling based on constant tip speed might cause a low agitation rate that will not deliver oxygen adequately. - Thus all three scaling parameters must be evaluated and the final scale-up agitation rate must produce acceptable values for all three parameters.
Gassing-based scaling parameters Vessel Volumes per Minute, VVM Superficial Gas Velocity, V s
Note: The two gas flow rate scaling parameters are both dependent on the dimensions of the vessel. Scaling based on one will greatly affect the other. Scale-Up Parameters Agitation Parameters Parameter Definition Scale-Up Factor Why is this Important?
Mixing Time
Amount of time it takes the bioreactor to create a homogeneous environment N 2 =N 1 (D 1 /D 2 ) 1/4 N 2 agitation speed in scale-up N 1 agitation speed in scale- down D 1 impeller diameter of scale - down D 2 impeller diameter of scale- up
Want to ensure that the materials are well-mixed in a timely manner
Power Input per Volume (P/V)
Amount of power transferred to a volume of cell culture through the agitator shaft and impellers P/V N 3 /D 2 P- power supplied V- Volume of Bioreactor N- Agitation Speed D- Impeller Diameter Mammalian cells cannot handle a lot of power introduced into the culture media as it can cause small eddies that will shear the fragile cell membranes
Tip Speed
Related to the shear rate produced from the impellers moving through the cell culture media N 2 =N 1 (D 1 /D 2 )
N 2 agitation speed in scale-up N 1 agitation speed in scale- down D 1 impeller diameter of scale - down D 2 impeller diameter of scale- up High shear rates can cause the cell membrane to tear and the cells to die. If scale-up based on constant tip speed is attempted, P/V and mixing time will decrease Gassing Parameters Parameter Definition Scale-Up Factor Why is this Important?
Vessel Volumes per Minute (VVM) means the volume of gas flow per bioreactor volume per minute.
Volume of Gas Flow/time necessary to ensure that enough oxygen will be supplied to the cells
Superficial Gas Velocity (V s )
volume of gas per cross-sectional area of the vessel. V s = Q gas /A v
V s - superficial gas velocity Q gas - gas volumetric flow rate A v - inside cross-sectional area of vessel increasing V s causes
An increase in foam generation A decrease in P/V An increase in oxygen transfer
Scale-Up Review Overall, scaling up process parameters is tricky
Each scale-up parameter is dependent on another. Scaling-up based on constant P/V will affect the mixing time and the tip speed in the bioreactor. As well, for gas flow rates, scaling-up on constant V s will affect the VVM.
Cannot keep all constant during scale-up
Not one scale-up process is correct
Technicians determine which parameter is critical to the process and try to find a happy medium between each of the remaining parameter