Bioreactors For production of vaccines, proteins, organics acids, amino acids, antibiotics, enzymatic or microbial biotransformations, etc. Bioreactors (Fermenter) - Basic FunctionThe basic function of a fermenter is to provide a suitable environment in which an organism can efficiently produce a target product that may be - cell biomass, - a metabolite, - or bioconversion product. Commercially important Fermentation Microbial cells or Biomass as the product: Eg. Bakers Yeast, Lactic acid bacillus, Bacillus sp. Microbial Enzymes: Catalase, Amylase, Protease, Pectinase, Glucose isomerase, Cellulase, Hemicellulase, Lipase, Lactase, Streptokinase etc. Microbial metabolites : Primary metabolites Ethanol, Citric acid, Glutamic acid, Lysine, Vitamins, Polysaccharides etc. Secondary metabolites: All antibiotic fermentation Recombinant products : Insulin, HBV, Interferon, GCSF, Streptokinase Biotransformations: Eg. Phenyl acetyl carbinol,Steroid Biotransformation The stages of fermentation process Stage I : Upstream processing ( preparation of liquid medium, , sterilization, air purification etc.) Stage II: Fermentation (substrates desired product with the help of biological agents such as microorganisms Stage III: Downstream processing ( separation of cells from the fermentation broth, purification and concentration of desired product etc. ) Oxygen transfer is considered to be a more important problem than the supply of other nutrients ( why?) Oxygen has a much lower solubility in water than other nutrients (8 mg.l-1) Many cells are very sensitive to dissolved oxygen concentrations. Unlike sugars and proteins, cells cannot store oxygen. Fresh oxygen must be available for the cells at all times during the fermentation. OTR ( oxygen transfer rate ) critical oxygen demand (rate at which it is utilized by the microorganisms ). For oxygen to be transferred from an air bubble to an individual microbe, several independent partial resistances must be overcome Oxygen transfer from gas bubble to cell1. Transfer from the interior of the bubble to the gas-liquid interface2. Movement across the gas film at the gas-liquid interface3. Diffusion through the relatively stagnant liquid film surrounding the bubble4. Transport through the bulk liquid5. Diffusion through the relatively stagnant liquid film surrounding the cells6. Movement across the liquid-cell interface7. If the cells are in floc, clump or solid particle, diffusion through the solid of the individual cell8. Transport through the cytoplasm to the site of reaction. What are the major rate limiting steps in the transfer of oxygen from bubbles to cells? The movement of oxygen from through the boundary layer around a bubble The movement of oxygen through the bulk liquid when mixing is poor or when the medium is viscous The movement of oxygen through microbial slimes (when immobilized cultures are being used) The movement of oxygen through the boundary layer around a bubble is the slowest step rate limiting step It depends on molecular diffusion of O2 which is slow process We can apply Fick' s first law of diffusion. Oxygen Transfer Rate 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.KLa = the volumetric mass transfer coefficient (per hour). Must not allow oxygen levels to fall below CcritComputer simulation of response for dynamic measurement of Kl a. Easy Case for Measuring Kla When there are few microorganisms or when there is little nutrient, not much oxygen is needed. It may be possible to maintain a relatively high percentage of the saturation concentration. There will be big excursions when the air valve is opened and closed, and it is easy to draw a tangent to the rising curve. More Difficult Case Much more typical for industrial fermentations is low oxygen concentration because there are many organisms and high substrate concentration. This is the time when aeration is critical and when a good measure of Kla is needed. The D.O. may already be only slightly above its critical concentration, and excursions caused by turning off the air valve will be small. This sketch is not nearly bad enough; sometimes the rising curve is very poorly defined. Optimization Of Oxygen Transfer Rate Bioreactor configurations Stirred Tank Reactors(STR) Baffles off-set position Aspect Ratio height-to-diameter H/D Impellers Impellers choice often depends on viscosity of the liquid sensitivity of the system to mechanical shear. Examples of shear sensitive organisms as molds filamentous fungi plant cells. Animal cell cultures Addition of surfactants such as Pluronic F68 to the culture medium may prevent the attachment of cell to rising bubbles, reducing their exposure to shear stress. Speed of impeller Higher stirring rates can be used for microbial cultures. While less stirring rates are used for mycelial fungi and animal cell cultures Areation Speed of impeller Slow impeller speed when aeration rate is too high Flooded impeller poor oxygen transfer rates & Poor mixing Advantages of STR Disadvantages Power requirement: critical in large-scale bioreactor. The agitator assembly, including the seal, is often a potential route of contamination. No equalize shear forces throughout the reactor. STR with immobilized cells is not favored generally due to attrition problems. Bubble columns Classified as pneumatic reactors The height-to-diameter ratio is 4-6. Gas is sparged under high pressure near the base through perforated pipes, plates or glass or metal porous spargers. The disadvantages1. Small bubble coalescence into larger ones as they rise through the column uneven gas distriution (kla)2. use of high pressure air excessive foam formation Airlift Bioreactors (ALB) External-loop Airlift Bioreactor Airlift bioreactors For optimal mass transfer, the riser to downcomer cross-sectional area ratio should be between 1.8 and 4.3. Riser>> downcomer A gas-liquid separator in the head-zone (disengagement zone ). Airlift bioreactors have higher productivities than stirred tank reactors ( for shear sensitive cultures). Performance of airlift bioreactors depends on the gas injection rate and the resulting rate of liquid circulation The rate of liquid circulation increases with the square root of the height of the airlift device. Consequently, the reactors are designed with high aspect The tall design leads tov high gas hold-ups (air volume retained in the liquid ) v long bubble residence times va region of high hydrostatic pressure near the sparger at the base of the fermenter kLa and Co* enhanced oxygen transfer rates. GAS HOLD-UP Represents air volume retained in the liquidVh = V - V0 Where Vh = hold-up volume, V = vol. of gassed liquid, V0 = vol of ungassed liquid. Advantages of air-lift reactors with external risers as compared to those with internal risers? v the heating/cooling jacket is located on the walls of the airlift reactor greater turbulence near the jacket better heat transfer efficiency. The advantages of Airlift reactors Elimination of attrition effects generally encountered in mechanical agitated reactors. It is ideally suited for aerobic cultures since oxygen mass transfer coefficient are quite high in comparison to stirred tank reactors. Fluidized bed reactors (FBB) Packed bed bioreactor The biocatalyst (or cell) is immobilized on the non-moving solid surfaces (large particles) which may be rigid or compressible porous or non porous particles Environment of a packed bed is non-homogeneous but concentration variations along the depth can be decreased by increasing the flow rate. pH control by addition of acid and alkali is nearly impossible. Cake Compressibility Cake Compressibility Industrial applications of fixed bed reactors disadvantages of packed beds Clogging High liquid pressure drop due to bed compaction Channeling Comparing fluidised bed and fixed bed reactors Fluidized bed reactors are more efficient than fixed bed reactors . why?1) A high concentration of cells can be immobilized due to the larger surface area for cell immobilization 2) Mass transfer rates are higher due to the larger surface area and the higher levels of mixing in the reactor. 3) Fluidised bed reactors do not clog as easily as fixed bed reactors. Optimization of Oxygen Transfer Rate ChemostatDevice for maintaining a bacterial population in the exponential growth phase by controlling nutrient input and cell removal. Perfusion cells are retained within the bioreactor to achieve the highest level of product expression possible. Bioreactor design features The vessel must be strong Made of material that don't corrupt the fermentation product. Should be free of stagnant areas where pockets of solids or liquids may accumulate Provide good temperature Provide good pH control system Permit sterilization-in-place (SIP) Permit Clean-in-place (CIP) Headspace volume Typically, the working volume will be 70-80% of the total fermenter volume. high foam formation 50% headspace volume Foam Formation and the Requirement for Antifoam Addition Foam control Excessive foam formation can lead to blocked air exit filters and to pressure build up in the reactor. Addition of antifoam e.g. Vegetable oil , silicon oilAlso excessive antifoam addition can however result in poor oxygen transfer rates. (why?) The use of mechanical foam breakers High shear agitators these devices generate sit above the liquid and generate high shear forces which break the bubbles in the foam Ultrasonic foam breakers for small scale reactor The head space volume The larger headspace volume, then the greater the tendency for the foam to collapse under its own weight. -- 50% headspace volume Condenser The density of the foam increases when it moves from the warm headspace volume to the cold condenser region. This causes the foam to collapse Temperature control system 1. temperature probes 2. heat transfer system (jacket & internal coils)The jacket will typically be "dimpled" to encourageturbulence in the jacket and thus increase the heat transfer efficiency. pH control system The neutralizing agents used to control pH should be non-corrosive & non-toxic to cells KOH is preferred to NaOH -- Sodium carbonate is also commonly used in small scale bioreactor systems Hcl acid should never be used as it is corrosive to most stainless steel. Material of construction for bioreactors Bioreactor usually made of stainless steels Type 316L The less expensive stainless steels Type 304 (or 304L) is used for the jacket, the insulation shroud and other surfaces not coming into direct contact with the fermentation broth. The oxygen delivery system 1. Compressor 2. Inlet &Exit air sterilization system3. Air sparger Sterilisation of the air large bioreactors. (Heat sterilization) Steam can be used to sterilize the air. Air sterilisation system - Positive pressure During sterilization the concept of "maintaining positive pressure" will often be used. It is very important that positive pressure is maintained when the bioreactor is cooled following sterilization. Without air being continuously pumped into the reactor, a vacuum will form and contaminants will tend to be drawn into the reactor. Air sterilisation system - Positive pressure Sparger The air sparger is used to break the incoming air into small bubbles. Various designs : porous materials made of glass or metal- the most common type of filter used in modern bioreactors is the sparger ring. Oxygen delivery system - Sparger A sparge ring consists of a hollow tube in which small holes have been drilled. A sparge ring is easier to clean than porous materials and is less likely to block during a fermentation. The sparge ring must located below the agitator and will have approximately the same diameter as the impeller. Thus, the bubbles rise directly into the impeller blades, facilitating bubble break up. Oxygen delivery system - Sparger Downstream processing The isolation and purification of biotechnological product to a form suitable for its intended use. it is preferred to use filtration as a clarification step for small scales (less than 2,000 L of culture harvest), whereas centrifugation might be the choice for larger scales of operation. Why cells need to bedisrupted ? to get the desired product inside the cells available for further processing if the product is secreted disruption step isnot needed Methods for Disruption of Microbial Cells1) Chemical Disruption of Microbial Cells by Alkali Organic solvents Chelating agents-Detergents2) Biological Disruption of Microbial Cells by enzymes ( specific for the major cell wall constituent of the organism).3) Physical Disruption of Microbial Cells the usual choice for large volume disruption physiological state of cell affects the strength animal cells lack cell wall easy to disrupt fungal mycelium is mechanically weak easy to disrupt using mechanical methods yeast cell wall is thick cells are resistant to disruption methods bacterial cells are difficult to disrupt resistance to disruption:G-negative cells < G-positive rods < G-positive cocci Physical Disruption of Microbial Cells Sonication High frequency sound waves Cavitations/ shear forces Commonly used on a small scale for cell disintegration Impractical for large batches when a liquid is subjected to ultrasonic vibrations regions of compression and rarefaction are produced in the liquid. Cavities are formed in these regions of rarefaction, which subsequently collapses in the regions of compression. This results in the generation of great shear forces . Wet milling Glass bead milling+ high disruption efficiency+ no negative impacts on further processing+ continous operation possible- energy requirements and capital investmentare high- denaturates enzymes (at low rate, coolingneeds) CONTINUOUS DYNO-MILL DISRUPTION Dyno Laboratory Mill KDL High-pressure homogenization High pressure homogenization Disruption by passing cell suspension under high (55-120 Mpa) pressure through an adjustable restricted orifice discharge valve Cell disrupted mainly by Impingement of a high velocity jet of suspended cells Cavitation Sudden pressure drop upon discharge Liquid shear in the orifice used to break microorganisms like Aspergillus niger, Escherichia coli, and Bacillus megatherium. Impingement French press Clarification of Disrupted Cellular Materials Concentration Concentrate the clarified protein solution quickly while keeping it in a stable state by using ammonium sulfate (salting out). Affinity Chromatography Affinity Chromatography Affinity Chromatography The ligand must be readily (and cheaply) available Ligand must be attachable (covalently) to the matrix (typically sepharose) such that it still retains affinity for protein Binding must not be too strong or weak Ideal KD should be between 10-4 & 10-8 M Elution involves passage of high salt or low pH buffer after binding Size Exclusion Chrom. Molecules are separated according to differences in their size as they pass through a hydrophilic polymer Polymer beads composed of cross-linked dextran (dextrose) which is highly porous (like Swiss cheese) Large proteins come out first (cant fit in pores), small proteins come out last (get stuck in the pores) Size Exclusion Chromatography (SEC) Electrophoresis is a technique used to separate and sometimes purify macromolecules - especially proteins and nucleic acids - that differ in size, charge or conformation ion-exchange, affinity-, immunoaffinity, size exclusion (gel filtration), reversed-phase, and hydrophobic interaction chromatography can result in a selective fractionation of a crude mixture Protein Isolation & Purification After cells (or media) are harvested proteins may be purified/isolated Intracellular (inside cell) proteins are harder to purify Require cell disruption, separation, removal of cell debris, DNA, RNA, lipid Extracellular (outside cell) proteins are easier to purify No cell disruption needed, just isolate Protein Isolation Methods Differential salt precipitation Differential solvent precipitation Differential temperature precipitation Differential pH precipitation Two-phase solvent extraction (PEG) Preparative electrophoresis Column chromatography