Agitation and aeration are important for fermentation processes. Agitation provides improved mixing, heat and mass transfer but can also cause cell damage. Aeration is critical for aerobic fermentations as it supplies oxygen needed for cell growth and product formation. However, oxygen has low solubility in water and must be continuously supplied. Factors like agitation rate, medium composition and rheology, and biomass concentration impact the oxygen transfer rate between the gas and liquid phases. Maintaining optimal agitation and aeration is important for efficient fermentation.
Agitation and aeration are important for fermentation processes. Agitation provides improved mixing, heat and mass transfer but can also cause cell damage. Aeration is critical for aerobic fermentations as it supplies oxygen needed for cell growth and product formation. However, oxygen has low solubility in water and must be continuously supplied. Factors like agitation rate, medium composition and rheology, and biomass concentration impact the oxygen transfer rate between the gas and liquid phases. Maintaining optimal agitation and aeration is important for efficient fermentation.
Agitation and aeration are important for fermentation processes. Agitation provides improved mixing, heat and mass transfer but can also cause cell damage. Aeration is critical for aerobic fermentations as it supplies oxygen needed for cell growth and product formation. However, oxygen has low solubility in water and must be continuously supplied. Factors like agitation rate, medium composition and rheology, and biomass concentration impact the oxygen transfer rate between the gas and liquid phases. Maintaining optimal agitation and aeration is important for efficient fermentation.
Agitation and aeration are important for fermentation processes. Agitation provides improved mixing, heat and mass transfer but can also cause cell damage. Aeration is critical for aerobic fermentations as it supplies oxygen needed for cell growth and product formation. However, oxygen has low solubility in water and must be continuously supplied. Factors like agitation rate, medium composition and rheology, and biomass concentration impact the oxygen transfer rate between the gas and liquid phases. Maintaining optimal agitation and aeration is important for efficient fermentation.
in Submerged Culture Agitation in Fermentation Agitation provides Improved mixing Heat transfer Mass transfer Adverse effects of agitation Rupture of cell walls Change of morphology of filamentous MOs Variation in the efficient of growth & rate of growth Variation in the rate of product formation Power consumption Power consumption in agitation is a function of physical properties, operating condition and vessel and impeller geometry The power consumption P ( in mechanical agitation) can be calculated by P = 2 r N F Power number (N P ) is the ration of drag force on the impeller to inertial force is given by dimensional analysis: N P = N - agitation speed r - distance from the axis to the point of the force measurement Shear Sensitive Mixing Mechanically agitated fermenter is effective in mixing of fermenter contents suspension of cells breakup of air bubbles for enhanced oxygenation prevention of forming large cell aggregates Shear generated by the agitator can disrupt the cell membrane and eventually kill some microorganisms, animal and plant cells Shear is responsible for the deactivation of enzymes For optimum operation of an agitated fermentation system, understanding of hydrodynamics involved in shear sensitive mixing is important Aeration Supply of molecular O 2 to the surface of each microbial cell is of primary importance in aerobic fermentation MOs utilize O 2 for growth product formation oxidation of substances provision of energy through respiration Oxygen is poorly soluble in water The solubility of oxygen at 4 o C in pure water is only 8 mg l -1 Glucose is 6000 times more soluble in water than O 2 The solubility of oxygen decreases with increasing temperature and increasing concentration of solutes in the solution O 2 requirement of cells (depends on type of MO) is on the order of 1 g/L h. Dissolved Oxygen (DO) in a fully saturated fermentation medium is sufficient only for a less than a minute Hence continuous supply of O 2 is required O 2 supply to culture is generally by sterile air (20.8%O 2 ) In certain cases, O 2 , is mixed with air to increase the solubility Need appropriately designed gas mixing device Mixing is required to disperse air bubbles and to enhance O 2 transfer in the fermentation broth 2 Oxygen Demand Depend on, Type of culture Age of culture eg. Log phase cultures demand more O 2 than lag or stationary phase cultures Nature of end product eg. High O 2 level is required for gluconic acid and citric acid fermentation Step 1 - Diffusion through the bubble to the gas-liquid interface Step 2 - Diffusion across the gas-liquid interface Step 3 - Diffusion through the bubble boundary layer Step 4 - Movement through the bulk liquid by forced convection and diffusion Step 5-9: Movement through the floc Step 2 - Diffusion across the gas-liquid interface Movement of oxygen from the gas phase to the liquid phase Magnitudes of individual resistances depend on bubble and liquid phase hydrodynamics, composition and rheological properties, density and activity cells and gas liquid interfacial phenomena Major goals in aerobic fermenter design is to reduce O 2 transfer resistances Sum of all resistances (gas film, liquid film and interfacial resistances) = overall resistance to transfer of O 2 into the cell Resistances to oxygen transfer Oxygen Transfer Rate Assuming O 2 transfer from gas to liquid phase is controlled by the liquid film resistance around air bubbles oxygen transfer rate (OTR) is expressed as dC L /dt = OTR = k L a ( C * - C L ) k L = oxygen transfer coefficient a = gas - liquid interfacial area per unit liquid volume C * & C L = saturation and actual DO conc. in the liquid OTR depends on Oxygen transfer coefficient (k L ) Interfacial area (a) Concentration driving force (C * -C L ) Oxygen Transfer Coefficient - k L The mass transfer coefficient (k L ) represents the rate at which oxygen molecules move through the boundary layer to the bulk liquid k L increases with increasing turbulence k L can be increased by Reducing stagnant film thickness (reduce the thickness of the boundary layer) increasing the rate at which molecules travel through the boundary layer increase rate of surface renewal Mass transfer coefficient is also affected by interfacial phenomena k L is a function of physical properties and the vessel geometry Hydro-dynamics in multiphase mixing (fermentation broth) is complex Hence difficult/impossible to derive a theoretical correlation for k L So empirical correlations are obtained by fitting experiment data These correlations are expressed by dimensionless groups 3 Interfacial area - a The interfacial area is determined by the aeration rate bubble diameter The smaller the bubble size the larger the interfacial area Small bubbles rise slowly through the liquid and so spend longer time in contact with the liquid - enhances mass transfer However slow rise bubbles create less turbulence which in turn reduce k L and mass transfer Variables that influence interfacial area are sparger design, fermenter volume, airflow rate and bubble size Concentration driving force ( C* - C L ) C* is the concentration of oxygen in gas liquid interface which is equal to the saturation concentration of oxygen in the reactor The value of C* is determined by the partial pressure of oxygen in the gas phase (Po 2 ), on Henry's Law and on the medium's Henry's constant for oxygen (Ho) Relationship between gas partial pressure (P O2 ) and liquid-phase oxygen conc. (C * ) obeys Henry's law, P O2 = Ho C * H - Henry's law constant specific to a given gas component and liquid phase and temperature C * - is a function P O2 which also depends on the fraction of O 2 in the gas phase and its total pressure Therefore, OTR increases with pressure The value of a medium's Henry's constant for oxygen is dependent on the concentration of salts and sugars and on temperature Eg: Henry's constant for water at 25 o C and 35 o C is: Henry's constant increases with temperature and thus the solubility of oxygen decreases with temperature Temperature o C Henry's constant (atm.mg -1 .l) 25 0.0258 35 0.0299 Why k L a It is difficult to measure both k L and a in a fermentation The two terms are combined in the term k L a and termed volumetric transfer coefficient (VTC) Higher the value of k L a higher the aeration rate k L a = f ( , D, , etc.) Factors affecting k L a in fermentations Air flow rate Degree of agitation Medium Composition Effect of biomass Presence of anti-foam agents Rheological properties of the broth Air flow rate Air flow rate : 0.5 1.5 vvm If high air flow rates are used OTR falls rapidly due to impellers becoming flooded 4 Degree of agitation Agitation assists oxygen transfer in different ways Disperse air bubbles/break in to small bubbles Delays the escape of air bubbles from the liquid Prevents coalescence of air bubbles Decreases the size of the boundary layer Degree of agitation may be measured by the amount of power consumption k L a and Power Consumption k L a Gassed power consumption for unit volume is affected by large number of variables Impeller speed No of impellers Impeller size Culture rheology Superficial air velocity Gassed power consumption per unit volume k L a (P g /V) k L a and Power Consumption Gassed power consumption per unit volume k L a = k (P g /V) x V s y P g power absorption in an aerated system V liquid volume V s Superficial air velocity k, x, y empirical factors specific to the system under investigation. x depends on the size/scale of the fermentation (laboratory, pilot or production) eg: k L a = k (P g /V) 0.95 V s 0.67 cooper et. al. (1944) One impeller Sulphite oxidation technique Influence of medium composition Inorganic Electrolytes Inorganic salts as solutes increase the surface tension of pure water These solutes make the whole solution, interior and surface, very much more cohesive or strongly bound together Increase K L a Alcohol Addition alcohol to water reduces surface tension. Cause increase in surface area and a corresponding decrease in average bubble diameter. Thus, increase in K L a Antifoam Agent All antifoams are surfactants and they decrease the surface tension of pure liquid by adsorbing strongly at relatively low bulk concentration Antifoam agents which accelerate the thinning promote bubble coalescence Addition of the antifoam agent causes the bubble size to grow and the interfacial area is reduced Many antifoams which are necessary to control foaming in fermentation reduce OTR by as much as 50% Therefore antifoam additions must be kept to an absolute minimum Effect of the addition of antifoam on bubble size in a bubble column With no antifoam added With antifoam added 5 Rheological properties of the broth Fermentation broth consists of Liquid medium Microbial mass Product produced Air bubbles Rheology of the broth is affected by Composition of the medium Concentration of biomass Morphology of biomass Concentration of the product Rheology of the broth has a great influence on k L a and agitation Problems with O 2 transfer from gas to liquid Coalescence of air bubble Mycelia may form agglomerates Mixing problems Medium containing starch Viscosity increases Degrade starch Viscosity decreases Development of mycelia MO growth Depending on rheological properties of the broth agitation can increase or decrease viscosity and then k L a k L a 1/viscosity Buckland et. al. (1988) k L a decreases proportionately with the square root of broth viscosity Viscosity of the Newtonian broth does not vary with agitation but Non-Newtonian does Microbial biomass Biomass concentration and its morphology has a profound effect on fermentation Most bacterial and yeast fermentations give rise to non- viscous Newtonian broths turbulent flow can be achieved Fungal fermentations become highly viscous and non Newtonian broths Method to increase OTR in fermenter Increase total pressure Enrich air with pure O 2 Use pure O 2 Only low flow rates is required to meet demand Will not strip CO 2 and other toxic gasses Unable to maintain positive pressure Oxygen free radical formation- toxic to essential enzymes/MOs High Cost of production Important Excess aeration cause problems such as excessive foam and high N 2 N 2 has sweeping effect in moving CO 2 N content of air for aeration is enhanced removal of gases and inhibitory metabolic products, esp. CO 2 from culture Addition of antifoaming agents may be necessary 6 Oxygen as a growth limiting nutrient DO is an important substrate in aerobic fermentations and may be a limiting substrate Oxygen transfer problem arises at high cell densities - growth is limited by O 2 availability in liquid phase DOC can affect the specific growth rate Therefore, oxygen concentration should be maintained at a value above C crit Effect of Dissolved Oxygen (DO) on specific oxygen uptake rate (QO 2 ) follows Michaelis-Menten type relationship QO 2 increases with increasing DO with a first order relationship up to C crit No further increase in oxygen uptake rate occurs above C crit DO must be maintained > C crit in order to obtain maximum growth/biomass production Above this value is independent of DO Even temporary depletion of dissolved oxygen below C crit may cause irreversible cell damage O 2 only slightly soluble - needs to be supplied continuously to culture to ensure that oxygen absorption rate oxygen consumption rate In cultures with limiting substrate, Monod equation can be used to expressed the specific growth rate ( ) of microorganisms; = max (C S / K S + C S ) - specific growth rate K S - saturation constant C S - substrate concentration So for oxygen limitation = max C L /(K 0 +C L ) For bacteria, yeast and fungi, C crit ranges from 0.0003 to 0.05 mMol/L depending on the form of growth and on the extent of pellet or aggregate formation 0 2 4 6 8 10 12 14 0 2 4 6 8 10 12 CL Q Q max C crit Q max/2 The dissolved oxygen concentration in a reactor is thus determined by the balance between the oxygen transfer rate (OTR) and oxygen uptake rate (OUR) dC L /dt = OTR - OUR x 2 L * L L .C QO - ) C a(C k dt dC x L o L m x/o L * L L C . C K .C Y 1 - ) C a(C k dt dC 7 When oxygen transfer is the rate limiting step, assuming energy requirement for cell maintenance is negligible OUR = OTR QO 2 = K L a (C * - C L ) (1) Y x/O = X/O = (dCx/dt)/(dO 2 /dt) dO 2 /dt = (dCx/dt)/ Y x/O = . C x /Y x/O . C x /Y x/O = K L a (C * - C L ) dC x /dt = Y x/O . K L a (C * - C L ) Q max = maximum specific O 2 uptake rate (specific O 2 demand by microorganism) The value is constant for growth of MO in a well defined environment. Value of Q max for some microorganisms A. niger - 30 mMole/g cell/hr P. chrysogenum - 3.9 mMoles/g cells/hr E. coli - 10.8 mMoles/g cells/hr O 2 demand for hydrocarbon is 2.5 higher than carbohydrate Simple sugar easy to metabolize High O 2 demand Complex sugar difficult to metabolize low O 2 demand Q max for Penicillium spp. grown on different sugars; Glucose - 13.4 mMol/L.h Sucrose - 6.7 mMol/L.h Lactose - 4.9 mMol/L.h Aerobic Fermenter Design Aerobic fermenter must has adequate aeration and agitation to meet the metabolic requirements of mo. The mixing should not cause damage to MO and power consumption should be as low as possible Most aerobic fermenters employ spargers underneath an impeller and mechanical agitation to reduce air bubble size and to increase gas liquid contact area Non-mechanical agitated fermenter (e.g. bubble column and air lift fermenters) have advantage of lower equipment and power cost. May not be efficient in viscous fermentation broths such as fungal and actinomycete fermentations Mechanical agitation (STR) - enhances heat transfer, reduces inhomogenity within the liquid and aids suspension of solid particles In STR, large diameter and low speed impeller with a small number of blades is required for mixing while high speed and small diameter impeller with many lades is required to increase interfacial area Non-agitated fermentations are normally carried out in tall vessel where aeration is sufficient to produce high turbulence but greater energy input in the production of compressed air is required Another consideration in determining choice - many industries need to process different products in same plant - flexible systems that can be modified is favoured Measurement of k L a Sodium Sulfite Oxidation Method Dynamic Gassing-out methods Static Gassing-out method Dynamic Gassing-out method Oxygen balancing method 8 Fermenter scale up Scale up should be done with out a decrease in yield Model and prototype should be similar to each other Factors involved in scale up Inoculum development Sterilization Environmental conditions Nutrients pH Temperature DO Shear Foam Affected by aeration and agitation Conditions that should be satisfied 1. Geometric similarity of the physical boundaries: The model and the prototype must be the same shape All linear dimensions of the model must be related to the corresponding dimensions of the prototype by a constant scale factor Theoretically scale-up is done based on geometric and dynamic similarity of the two systems 2. Dynamic similarity of the flow fields: Ratio of flow velocities of corresponding fluid particles is the same in model and prototype Ratio of all forces acting on corresponding fluid particles is the same in model and prototype forces act on a fluid element in a fermenter during agitation are: viscosity force F V drag force on impeller F D gravity force F G When dynamic similarity of two flow fields with geometrically similar boundaries is achieved, the flow fields exhibit geometrically similar flow patterns According to Newtons equation of viscosity Which can be assumed to be Further and So, Dynamic similarity between model (m) and prototype (p) is achieved when or when Further Reynolds number and similarly Therefore dynamic similarity when the values of non- dimensional parameter are the same at geometrically similar locations However, it is difficult to achieve this similarity when more than one dimensionless groups are involved and hence scale up criteria should be applied 9 Scale-up Criteria Power consumption per unit volume P mo /v is employed as a criterion for scale-up Since liquid volume is proportional to D I 3 for geometrically similar systems, P mo /D I 3 - Power per unit volume For constant P mo /D I 3 Impeller tip speed Reynolds Number and Model (20L) Prototype (2500 L) P o /v 1 1 D I 1 5 N 1 0.34 N Re 1 8.5