Industrial Biotechnology lesson 6

1. Industrial Biotechnologylesson 6 BIOREACTOR DESIGN

2. Introduction • The function of the fermenter or bioreactor is to provide a suitable environment in which an organism can efficiently produce a target product—the target product might be • · Cell biomass • · Metabolite • · Bioconversion Product • The sizes of the bioreactor can vary over several orders of magnitudes. • The microbial cell culture (few mm3), shake flask ( 100 -1000 ml), laboratory fermenter ( 1 – 50 L), pilot scale (0.3 – 10 m3) to plant scale ( 2 – 500 m3) are all examples of bioreactors.

3. Introduction • The performance of any fermenter depends on the following key factors: • · Agitation rate • · Oxygen transfer • · pH • · Temperature • · Foam production • The design and mode of operation of a fermenter mainly depends on the production organism, the optimal operating condition required for target product formation, product value and scale of production. • The design also takes into consideration the capital investment and running cost.

4. Introduction • · Large volume and low value products like alcoholic beverages need simple fermenters and do not need aseptic condition. • · High value and low volume products require more elaborate system of operation and aseptic condition. • The Designing of a Bioreactor also has to take into considerations the Unique Aspects of Biological Processes: • A) The concentrations of starting materials (substrates) and products in the reaction mixture are frequently low; both the substrates and the products may inhibit the process.

5. Introduction • Cell growth, the structure of intracellular enzymes, and product formation depend on the nutritional needs of the cell (salts, oxygen) and on the maintenance of optimum biological conditions (temperature, concentration of reactants, and pH) within narrow limits. • B) Certain substances, inhibitors, effectors, precursors, metabolic products influence the rate and the mechanism of the reactions and intracellular regulation. • C) Microorganisms can metabolize unconventional or even contaminated raw materials (cellulose, molasses, mineral oil, starch, ores, wastewater, exhaust air, biogenic waste), a process which is frequently carried out in highly viscous media.

6. Introduction • D) In contrast to isolated enzymes or chemical catalysts, mo’s adapt the structure and activity of their enzymes to the process conditions, whereby selectivity and productivity can change. • Mutations of the microorganisms can occur under sub optimal biological conditions. • E ) Microorganisms are frequently sensitive to strong shear stress and to thermal and chemical influences. • F) Reactions generally occur in gas-liquid -solid systems, the liquid phase usually being aqueous. • G) Continuous bioreactors often exhibit complicated dynamic behavior.

7. Introduction • H) The microbial mass can increase as biochemical conversion progresses. • Effects such as growth on the walls, flocculation, or autolysis of microorganisms can occur during the reaction.

8. Installation of a fermenter; S-steam; C-condensate; W-water; A-air. The steam line permits inplace sterilization of valves, pipes and seals.

9. Requirements of Bioreactors • There is no universal bioreactor. • The general requirements of the bioreactor are as follows: • A) The design and construction of bioreactors must keep sterility from the start point to end of the process. • B) Optimal mixing with low, uniform shear. • C) Adequate mass transfer, oxygen. • D) Clearly defined flow conditions. • E) Feeding substrate with prevention of under or overdosing. • F) Suspension of solids. • G) Gentle heat transfer. • H) Compliance with design requirements such as: ability to be sterilized; simple construction; simple measuring, control, regulating techniques; scale-up; flexibility; long term stability; compatibility with up- downstream processes; antifoaming measures.

10. Fermenter Design • The basic points of consideration while designing a fermentor: • · Productivity and yield • · Fermenter operability and reliability • · Product purification • · Water management • · Energy requirements • · Waste treatment • Other few significant things to be taken in account: • · Design in features so that process control will be possible over reasonable ranges of process variables. • · Operation should be reliable • · Operation should be contamination free

11. Fermenter design • To achieve these the fermenter should have: • · Heat and oxygen transfer configuration • · Sterilization procedures • · Foam control • · Fast and thorough cleaning system • · Proper monitoring and control system • Traditional design is open cylindrical or rectangular vessels made from wood or stone. • Most fermentations are now performed in close system to avoid contamination. • It should be constructed from non-toxic, corrosion-resistant materials. • Small fermentation vessels of a few liters capacity are constructed from glass and/or stainless steel.

12. Fermenter design • Pilot scale and many production vessels are normally made of stainless steel with polished internal surfaces • Very large fermenters are often constructed from mild steel lined with glass or plastic, in order to reduce the cost. • If aseptic operation is required, all associated pipelines transporting air, inoculum and nutrients for the fermentation need to be sterilizable, usually by steam. • Most vessel cleaning operations are now automated using spray jets, and called cleaning in place CIP. And located within the vessel. • Associated pipe work must also be designed to reduce the risk of microbial contamination. There should be no horizontal pipes or unnecessary joints and dead stagnant spaces where material can accumulate; otherwise this may lead to ineffective sterilization.

13. Fermenter design • Normally, fermenters up to 1000 L capacity have an external jacket, and larger vessels have internal coils. • Pressure gauges and safety pressure valves must be incorporated, (required during sterilization and operation). • For transfer of media pumps are used. Centrifugal pumps (generate high shear forces and path for easy contaminations), magnetically coupled, jet and peristaltic pumps. • Alternate methods of liquid transfer are gravity feeding or vessel pressurization. • In fermentations operating at high temperatures or containing volatile compounds, a sterilizable condenser may be required to prevent evaporation loss. • Fermenters are often operated under positive pressure to prevent entry of contaminants.

14. Control of Physicochemical Parameters • A) Agitation: • Agitation of suspended cell fermentations is performed in order to mix the three phases within a fermenter • liquid phase contains dissolved nutrients and metabolites • gaseous phase is predominantly oxygen and carbon dioxide • solid phase is made up of the cells and any solid substrates that may be present. • Mixing should produce homogeneous conditions and promote • a) Nutrient transfer • b) Gas transfer • c) Heat transfer • Heat transfer is necessary during both sterilization and for temperature maintenance during operation.

15. Control of Physicochemical Parameters • Transfer into liquid from the gaseous phase is enhanced by agitation: It prolongs retention of air bubbles in suspension, reduces bubble size to increase the surface area for oxygen transfer, prevents bubble coalescence and decreases the film thickness at the gas-liquid interface. • Maintenance of suitable shear conditions during the fermentation is very important: • Certain agitation systems develop high shear that may damage shear-sensitive cells. • Low shear systems can lead to cell flocculation or unwanted growth on surfaces, such as on the vessel walls, stirrer and electrodes. • The mixing of nutrients and gaseous exchange within any fermenter is influenced by:

16. Control of Physicochemical Parameters • a. medium density and rheology, • b. size and geometry of the vessel • c. the amount of power used in system. • CSTRs have agitators with multiple impellers to give a well mixed homogeneous environment. • Nevertheless, in reality, non-uniform conditions normally prevail in vessel greater than 500 liters capacity. • No direct contact exists between the cooling! Heating system and the fermentation medium. • The heat is conducted through the vessel wall, coils and baffles. • These systems are also used to sterilize the vessel and contents before inoculation, by the injection of pressurized steam contents before inoculation.

17. Control of Physicochemical Parameters • Automatic temperature control during the fermentation is accomplished by injecting either cold or hot water into the outer jacket and/or internal coils. • In some circumstances alternative cooling media may be used, e.g. glycol. • Mass transfer • Transfer of nutrients from the aqueous phase into the microbial cells during fermentation is relatively straightforward as the nutrients are normally provided in excess. • A. Transport of Nutrients • The performance of the reactor is affected if the rate of the transport of the limiting nutrients is slower than the rate of utilization by the cells. • Efficiency of the bioprocess could be increased by increasing the rate of transport of a limiting nutrient.

18. Control of Physicochemical Parameters • B. Transport of Oxygen • Compressed air entering a fermenter is usually stripped of moisture and any oil vapors that may originate from the compressor. • To prevent the risk of contamination, gases introduced into the fermenter should be passed through a sterile filter. • A similar filter on the air exhaust system avoids environ-mental contamination. • Sterile filtered air or oxygen normally enters the fermenter through a sparger system, and airflow rates for large fermenters rarely exceed 0.5-1.0 volumes of air per volume of medium per minute (v/v/m). • To promote aeration in stirred tanks, the sparger is usually located directly below the agitator.

19. Control of Physicochemical Parameters • Sparger structures can affect the overall transfer of oxygen into the medium, as it “influences the size of the gas bubbles produced. • Small bubbles are desirable because the smaller the bubble, the larger the surface area to volume ratio, which provides greater oxygen transfer. • However, spargers with small pores that are effective in producing small air bubbles are more prone to blockage and require a higher energy input. • The availability of the oxygen depends on: • · Solubility • · Mass transfer rate of oxygen in the fermentation broth • · Rate of utilization of DO by microbial biomass.

20. Control of Physicochemical Parameters • To enhance the rate of bioconversions, sometimes the inoculum concentration is increased. • This is adversely affects the oxygen availability to the cells. • High density of cells causes rapid depletion of dissolved oxygen in the fermentation media, as there is a misbalance between the oxygen consumption rate and the rate of oxygen transfer. • In such cases the rate of oxygen transfer from the gas phase into the liquid media need to be enhanced to improve the rate of bioconversion.

21. Control of Physicochemical Parameters • The major resistance in oxygen transfer to cells are: • · Gas film resistance between the bulk gas and gas-liquid interface • · Interfacial resistance at the gas-liquid interface • · Liquid film resistance between the interface and bulk liquid phase • · Liquid phase resistance for the transfer of oxygen to the liquid film surrounding a microbial cell • · Liquid film resistance around cells • · Intracellular resistance

22. Control of Physicochemical Parameters • The total oxygen transfer resistance is the sum of the individual resistance. • The gas film resistance is almost negligible. • Liquid film around a single cell has negligible resistance to the diffusion of oxygen but when the cells are in pellets form, then the liquid film resistance around the cell is significant. • Intracellular oxygen transfer resistance is usually negligible compared to other factors. • If there is pellet formation, intrapellet resistance may be important since oxygen has to diffuse through the intercellular space to available cells. • Size of pellet is important to avoid formation of anaerobic regions. • The critical size of the clumps (pellets) depends upon:

23. Control of Physicochemical Parameters • a. The rate of consumption of oxygen, • b. Diffusivity of oxygen • c. The concentration of dissolved oxygen in the medium • The major resistance is due to the liquid film around the gas bubble. • In a well mixed fermenter, the concentration of dissolved oxygen in the bulk liquid phase is constant and the concentration gradient in the bulk liquid will thus be negligible. • When proper mixing in the fermentation media is difficult to achieve, there may be significant concentration gradient within the bulk liquid and hence the oxygen transfer resistance in the bulk liquid may not be negligible. • Bulk fluid mixing is thus taken into consideration in the design of aerobic fermenters to reduce the oxygen transfer resistance.

24. Oxygen mass transfer from an air bubble to a microbial cell

25. Physical Factors Affecting Oxygen Transfer • Temperature: Temperature affects the solubility and diffusivity of oxygen in the fermentation broth. • The solubility of oxygen decreases but diffusivity increase with the rise in temperature. • Pressure: The partial pressure of oxygen in the gas phase mainly affects the solubility of oxygen. In certain fermentation systems, increasing the total pressure of air supplied to the fermenter or else by operating the system under a constant high pressure head of air improve the rate of oxygen transfer. • In aerobic fermentors , oxygen is supplied to the fermentation medium by sparging air bubbles underneath the impeller of an agitated fermentor. • Oxygen from a rising air bubble is first dissolved in the fermentation medium and then taken up by the cells.

26. Physical Factors Affecting Oxygen Transfer • In CSTR, the rate of oxygen transfer varies with the power supplied for agitation of fermentation broth, hence estimation of the power requirement for effective agitation and oxygen transfer is essential for the design of aerobic bioreactors. • When high biomass concentrations are used to increase productivity it also creates an enormous demand for oxygen. • The operation of aerobic processes is generally more demanding, as it is difficult to prevent oxygen from becoming a rate-limiting factor. • Oxygen transfer is complex, as it involves a phase change from its gaseous phase to the liquid phase, and is influenced by the following factors: • 1. the prevailing physical conditions; temperature, pressure and surface area of air/oxygen bubbles;

27. Physical Factors Affecting Oxygen Transfer • 2. the chemical composition of the medium; • 3. the volume of gas introduced per unit reactor volume per unit time; • 4. the type of sparger system used to introduce air into the fermenter; • 5. the speed of agitation; or • 6. a combination of these factors. • During aerobic fermentations molecular oxygen must be maintained at optimal concentrations to ensure maximum productivity. • The two steps associated with an oxygen mass balance are the rate at which oxygen can be delivered to the biological system (oxygen transfer rate, OTR) and the rate at which it is utilized by the microorganisms (critical oxygen demand).

28. Physical Factors Affecting Oxygen Transfer • If the rate of oxygen utilization is greater than die OTR, anaerobic conditions will develop, which may limit growth and productivity. • OTR may be raised by elevating the pressure, enriching the inlet air oxygen, and increasing both agitation and airflow rates. • In order for oxygen to transfer from the gaseous phase to an individual cell or site of reaction, it must pass through several points of resistance. • 1. resistance within the gas film to the phase boundary. • 2. penetration of the phase boundary between the gas bubble and bulk liquid; • 3. transfer from the phase boundary to the bulk liquid; • 4. movements within the liquid; • 5. transfer to the surface of the cell;

29. Physical Factors Affecting Oxygen Transfer • 6. entries into cell; and • 7. transport to the site of reaction within the cell. • The rate-limiting step (controlling factor) in oxygen transfer is the movement of oxygen from the gaseous phase to the gas-liquid boundary layer, particularly for viscous media, • Gaseous oxygen molecules move rapidly, due to their kinetic energy. • However, to enter the liquid they have to cross this boundary layer at the surface of the bubble. • This is composed of a thin layer of oxygen molecules that line the inside of the bubble and a thicker layer of water molecules coating the bubble surface. • Diffusion across this boundary is particularly influenced by temperature, solutes and surfactants.

30. Physical Factors Affecting Oxygen Transfer • Once in the liquid, the rate of oxygen acquisition by cells depends on the oxygen gradient between the oxygen in the bulk liquid and at the site of utilization. • Movement in the bulk liquid is aided by good mixing. • The rate of use by the biological system will be determined by the affinity and saturation characteristics of the terminal oxidase. • As microorganisms exhibit different oxygen requirements, the level of aeration necessary will vary from fermentation to fermentation.

31. Transfer of Heat in Bioreactors • Microbial growth is usually accompanied by the release of metabolic heat into the fermentation medium. • Metabolic activities can generate as much as 100-200 BTU gal-1h-1 of thermal energy, while mechanical energy inputs of 0.5 and 2.5 HP per 100 gal can generate an additional 10-60 BTU gal-1h-1. • To maintain a constant temperature in the fermenter, heat is either supplied or removed from the fermentation broth during the course of fermentation. • Heat transfer takes place in well stirred fermenters by forced convection. • In fixed bed microbial reactors heat transfer takes place by natural convection or phase change (evaporation-condensation).

32. Heat Transfer Configurations: • The primary heat transfer configurations in fermentation vessels are: • i. External jackets • ii. Internal coils • iii. External surface heat exchanger • The internal coils though provide better heat transfer capabilities, but they cause problems of microbial film growth on coil surfaces, alteration of mixing patterns and fluid velocities. • The external surface heat exchangers, the media is pumped through an external heat exchanger where the heat transfer takes place through the surface of exchanger tubes.