BIOREACTORS CONTENTS 1 Introduction 2 O 2 uptake

BIOREACTORS

CONTENTS 1. Introduction 2. O 2 uptake and Stoichiometry 3. Surface aeration 4. Methods of aeration 5. Mechanically stirred bioreactors 6. Bubble driven bioreactors 7. Airlift bioreactors 8. Packed bed and trickle flow bioreactors 9. Fluidized bioreactors

BIOREACTORS- INTRODUCTION u. Importance of considering process engineering factors when culturing cells. u. Biological factors include the characteristics of the cells, their maximum specific growth rate, yield coefficient, p. H range and temperature range. u. The productivity of a fermentation is determined by the mode of operation of the fermentation process; eg. the advantages of fed-batch and continuous fermentations over batch fermentations.

Bioreactors- Introduction u. The oxygen demand of an industrial process is generally satisfied by aeration and agitation u. Productivity is limited by oxygen availability and therefore it is important to the factors that affect a fermenters efficiency in supplying O 2 u. O 2 requirement, quantification of O 2 transfer and factors influencing the transfer of O 2 into solution

BIOREACTORS- INTRODUCTION u. Mass transfer, in particular, oxygen transfer are important factor which determined how a reactor must be designed and operated. u. Cost was also described as an important consideration. The larger the reactor or the faster the stirrer speed, the greater the costs involved.

MASS TRANSFER AND PHASES The rate of oxygen transfer = driving force / resistance. E. g resistance to mass transfer from medium to mo`s 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)

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

PHASES PRESENT IN BIOREACTION / BIOREACTOR 1 = reactant supply and utilisation 2 = product removal and formation

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 resistance’s must be overcome

STOICHIOMETRY OF RESPIRATION To consider the Stoichiometry of respiration the oxidation of glucose may be represented as; C 6 H 12 O 6 + 6 O 2 6 H 2 O + 6 CO 2 = Atomic weight of Carbon Hydrogen Oxygen 12 1 16 Molecular weight of glucose is 180 How many grams of oxygen are required to oxidise 180 g of glucose? Answer 192 g

SOLUBILITY OF OXYGEN u. Both components oxygen and glucose must be in solution before they become available to microorganisms u. Oxygen is 6000 times less soluble in water than glucose u. A saturated oxygen solution contains only 10 mg dm-3 of oxygen u. Impossible to add enough oxygen to a microbial culture to satisfy needs for complete respiration u. Oxygen must be added during growth at a sufficient rate to satisfy requirements

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 10, 000 ppm Critical conc. 100 ppm Broth O 2 sat @ 25 o. C approx. 7 ppm 0. 8 ppm (growth stops) Rate of demand 2. 8 mmoles/ g cells /h 7. 7 mmoles/ g cells /h

MASS TRANSFER AND RESPIRATION (a) Mass balance Stoichiometry of respiration e. g glucose; C 6 H 12 O 6 + 6 O 2 6 H 2 O + 6 CO 2 Oxidation of 180 gms Glucose requires 192 gms O 2 Compare with a hydrocarbon (i. e 6 CH 2)

THE OXYGEN REQUIREMENTS OF INDUSTRIAL FERMENTATIONS Oxygen demand dependant on conversion 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 100 g of dry yeast C 3. 92 H 6. 5 O 1. 94

O 2 REQUIREMENTS 6. 67 CH 2 O + 2. 1 O 2 = C 3. 92 H 6. 5 O 1. 94 + 2. 75 CO 2 + 3. 42 H 2 O 7. 14 CH 2 + 6. 135 O 2 = C 3. 92 H 6. 5 O 1. 94 + 3. 22 CO 2 + 3. 89 H 2 O where CH 2 = hydrocarbon CH 2 O = carbohydrate From the above equations to produce 100 g of yeast from hydrocarbon requires three times the amount of oxygen than from carbohydrate

Compare solubility of Oxygen vs Glucose ( e. g. oxygen = 9. 0 mg/l @ 20 o. C, 11. 3 mg/l @ 10 o. C) 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

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 p. H, temp etc. ) Surface area/ volume ratio large vs small cells (bacteria v mammalian cells) hyphae, clumps, flocks, pellets etc. Nature of surface area (type of capsule etc)

METHODS OF AERATION u. A bioreactor is a reactor system used for the culture of microorganisms. They vary in size and complexity from a 10 ml volume in a test tube to computer controlled fermenters with liquid volumes greater than 100 m 3. They similarly vary in cost from dollars to a few million dollars. u. In the following sections we will compare the following reactors Standing cultures Shake flasks Stirred tank reactors Bubble column and airlift reactors Fluidized bed reactors

STANDING CULTURES u. In standing cultures, little or no power is used for aeration. Aeration is dependent on the transfer of oxygen through the still surface of the culture.

STANDING CULTURES u. The rate of oxygen transfer will be poor due to the small surface area for transfer. Standing cultures are commonly used in small scale laboratory systems in which oxygen supply is not critical. For example, biochemical tests used for the identification of bacteria are often performed in test-tubes containing between 5 -10 ml of media. u. T-flasks used in the small scale culture of animal cells are another example of a standing culture. T-flasks are normally incubated horizontally to increase the surface area for oxygen transfer.

u. The surface aeration rate in standing cultures can be increased by using large volume flasks. u. The following photograph shows a 250 ml Erlenmeyer flask containing 100 ml of medium and a 3 litre "Fernback" flask containing 1 litre of medium. Note how the latter has a large surface area.

STANDING CULTURES • Large Pyrex flasks are used for the small scale production of fermented products. • Standing culture aeration is not restricted to the laboratory. • In some countries, where the availability of electricity is unreliable, citric acid is produced using surface culture techniques. • In these cultures, the Aspergillus niger mycelia are grown on the surface of liquid media in large shallow trays. • The medium is neither gassed nor agitated.

ASPERGILLUS NIGER MYCELIA

STANDING CULTURES u. Aerobic solid substrate fermentations are another example of standing cultures. In these fermentations, the biomass is grown on solid biodegradable substrates. u. The solids may be continuously or periodically turned over to improve aeration and to regulate the culture temperature. One example of a commercial scale, solid substrate fermentation is the production of koji by Aspergillus oryzae on soya beans which is part of the soya sauce process. u. Another is mushroom cultivation. Considerable research is currently being invested into the feasibility of producing biochemicals by solid substrate fermentations.

SHAKE FLASKS

SHAKE FLASKS u. Shake flasks are commonly used for small scale cell cultivation. u. Through continuous shaking of the culture fluid, higher oxygen transfer rates can be achieved as compared to standing cultures. u. Shaking continually breaks the liquid surface and thus provides a greater surface area for oxygen transfer. u. Increased rates of oxygen transfer are also achieved by entrainment of oxygen bubbles at the surface of the liquid.

SHAKE FLASKS u. 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. u. The rate of oxygen transfer in shake flasks is dependent on the shaking speed the liquid volume shake flask design

SHAKE FLASKS O 2 TRANSFER k. La decreases with liquid volume k. La is higher when baffles are present k. L a k. La increases with liquid surface area

SHAKE FLASKS O 2 TRANSFER u. The k. La will increase with the shaking speed. u. At high shaking speeds, bubbles become entrained into the medium to further increases the oxygen transfer rate. u. The presence of baffles in the flasks will further increase the oxygen transfer efficiency, particularly for orbital shakers. u. The following photographs show baffles increase the level of gas entrainment in a shake flask being shaken in an orbital shaker at 150 rpm

Unbaffled flask Baffled flask

SHAKE FLASKS O 2 TRANSFER u. Note the high level of foam formation in the baffled flask due to the higher level of gas entrainment. u. The same improvement in oxygen transfer is not as evident with horizontal reciprocating shakers. u. The appropriate liquid volume is determined by the flask volume. For example, for a standard 250 ml flask, the liquid volume should not exceed 70 ml while for a 1 litre flask, the liquid volume should be less than 200 ml. u. Larger liquid volumes can be used with wide based flasks

MECHANICALLY STIRRED BIOREACTORS

MECHANICALLY STIRRED BIOREACTORS u. For aeration of liquid volumes greater than 200 ml, various options are available. u. Non-sparged mechanically agitated bioreactors can supply sufficient aeration for microbial fermentations with liquid volumes up to 3 litres. u. However, stirring speeds of up to 600 rpm may be required before the culture is not oxygen limited. u. In non-sparged reactors, oxygen is transferred from the headspace above the fermenter liquid. Agitation continually breaks the liquid surface and increases the surface area for oxygen transfer.

MECHANICALLY STIRRED REACTORS - SPARGED STIRRED TANK BIOREACTORS u. For liquid volumes greater than 3 litres, air sparging is required for effective oxygen transfer. u. The introduction of bubbles into the culture fluid by sparging, leads to a dramatic increase in the oxygen transfer area. u. Agitation is used to break up bubbles and thus further increase k La. u. Sparged fermenters required significantly lower agitation speeds for aeration efficiencies comparable to those achieved in nonsparged fermenters. u. Air-sparged fermenters can have liquid volumes greater than 500, 000 litres.

BUBBLE DRIVEN BIOREACTORS u. Sparging without mechanical agitation can also be used for aeration and agitation. Two classes of bubble driven bioreactors are bubble column fermenters and airlift fermenters. u. Bubble driven bioreactors are commonly used in the culture of shear sensitive organisms such as moulds and plant cells. An airlift fermenter differs from bubble column bioreactors by the presence of a draft tube which provides better mass and heat transfer efficiencies. u. Airlift fermenters are however considerably more expensive to construct than bubble column reactors. There are several designs for air-lift fermenters although the most commonly used design is one with a central draft tube.

BUBBLE DRIVEN BIOREACTORS

BUBBLE DRIVEN BIOREACTORS u. An airlift fermenter differs from bubble column bioreactors by the presence of a draft tube which provides better mass and heat transfer efficiencies more uniform shear conditions. u. Bubble driven fermenters are generally tall with liquid height to base ratios of between 8: 1 and 20: 1. u. The tall design of these fermenters leads to high gas hold-ups, long bubble residence times and a region of high hydrostatic pressure near the sparger at the base of the fermenter. u. These factors lead to high values of k. La and Co* thus enhanced oxygen transfer rates

AIRLIFT BIOREACTORS u. An airlift fermenter differs from bubble column bioreactors by the presence of a draft tube. u. The main functions of the draft tube are to: Increase mixing through the reactor The presence of the draft tube enhances axial mixing throughout the whole reactor Reduce bubble coalescence. This presumably occurs due to circulatory effect that the draft tube induces in the reactor. The circulation occurs in one direction and hence the bubbles also travel in one direction.

AIRLIFT BIOREACTORS Small bubbles lead to an increased surface area for oxygen transfer.

AIRLIFT BIOREACTORS u. Equalize shear forces throughout the reactor. Major reason why the productivity of cells grown in airlift bioreactors have higher productivities than those grown in stirred tank reactors.

AIRLIFT BIOREACTORS u. The major disadvantages of air-lift fermenters are u high energy requirements u excessive foaming u cell damage due to bubble bursting; particularly with animal cell culture

AIRLIFT BIOREACTOR AIR-RISER AND DOWN-COMER u. An air-lift reactor is divided into three regions: - the air-riser - down-comer - disengagement zone.

AIRLIFT BIOREACTOR

AIRLIFT BIOREACTOR u. The region into which bubbles are sparged is called the air-riser. The air -riser may be on the inside or the outside of the draft-tube. The latter design is preferred for large scale fermenters as it provides better heat transfer efficiencies. u. The rising bubbles in the air-riser cause the liquid to flow in a vertical direction. To counteract these upward forces, liquid will flow in a downward direction in the down-comer. This leads to liquid circulation and thus improved mixing efficiencies as compared to bubble columns. u. The enhanced liquid circulation also causes bubbles to move in a uniform direction at a relatively uniform velocity. This bubble flow pattern reduces bubble coalescence and thus results in higher k La values as compared to bubble column reactors.

AIRLIFT BIOREACTORS DISENGAGEMENT ZONE

AIRLIFT BIOREACTORS DISENGAGEMENT ZONE u. The roles of the disengagement zone are to add volume to the reactor, reduce foaming and minimise recirculation of bubbles through the down comer.

AIRLIFT BIOREACTORS DISENGAGEMENT ZONE u. The sudden widening at the top of the reactor slows the bubble velocity and thus disengages the bubbles from the liquid flow. u. Carbon-dioxide rich bubbles are thus prevented from entering the downcomer. u. The reduced bubble velocity in the disengagement zone also leads to a reduction in the loss of medium due aerosol formation. u. The increase in area will also helps to stretch bubbles in foams, causing the bubbles to burst. The axial flow circulation caused by the draft tube also helps to reduce foaming

PACKED BED AND TRICKLE FLOW BIOREACTORS u. The topic of packed bioreactors was discussed in another lecture on immobilisation.

PACKED BIOREACTORS u. The rate of mass transfer between the cells and the medium depends on the flow rate and on the thickness of the biomass film on or near the surface of the solid particles. u. Packed bed reactors often suffer from problems caused by poor mass transfer rates and clogging. Despite this they are used commercially with enzymatically catalysts and with slowly or nongrowing cells. u. They are also used in the anaerobic treatment of high strength wastewaters (eg. food processing wastes). Large plastic blocks are used as solid supports for the cells. These blocks have a large surface area for cell immobilization and when packed in the reactor are difficult to clog.

TRICKLE FLOW BIOREACTORS u. Trickle bed reactors are a class of packed bed reactors in which the medium flows (or trickles) over the solid particles. In these reactors, the particles are not immersed in the liquid. The liquid medium trickles over the surface of the solids on which the cells are immobilized They are used widely in aerobic treatment of sewage.

TRICKLE FLOW BIOREACTORS u. Oxygen transfer is enhanced by ensuring that the cells are covered by only a very thin layer of liquid, thus reducing the distance over which the dissolved oxygen must diffuse to reach the cells.

TRICKLE FLOW BIOREACTORS u. Because stirring is not used, considerable capital costs are saved. u. However, oxygen transfer rates per unit volume are low compared with spared stirred tank systems. u. Trickle flow systems are used widely for the aerobic treatment of sewage. u. They are used to polish effluent from the activated sludge or anaerobic digestion process and for the nitrification of ammonia.

FLUIDIZED BED REACTORS

FLUIDIZED BED REACTORS u. Fluidised bioreactors are one method of maintaining high biomass concentrations and at the same time good mass transfer rates in continuous cultures. u. Fluidised bioreactors are an example of reactors in which mixing is assisted by the action of a pump. In a fluidised bed reactor, cells or enzymes are immobilised in and/or on the surface of light particles. u. A pump located at the base of the tank causes the immobilised catalysts to move with the fluid. The pump pushes the fluid and the particles in a vertical direction. The upward force of the pump is balanced by the downward movement of the particles due to gravity. This results in good circulation.

FLUIDISED BED REACTORS u. For aerobic microbial systems, sparging is used to improve oxygen transfer rates. u. A draft tube may be used to improve circulation and oxygen transfer. Both aerobic and anaerobic fluidised bioreactors have been developed for use in waste treatment. u. Fluidised beds can also be used with microcarrier beads used in attached animal cell culture. u. Fluidised-bed microcarrier cultures can be operated both in batch and continuous mode. In the former the fermentation fluid is recycled in a pump-around loop.

FLUIDIZED BED REACTORS

SUMMARY u. Methods of aeration in different bioreactors u. Aeration in standing cultures u. Oxygen transfer in shake flasks u. Advantages and applications of mechanically stirred bioreactors u. Bubble driven bioreactors u. Airlift bioreactors u. Packed bed and trickle flow bioreactors u. Fluidised bioreactors