Chapter 4: bioreactor considerations for suspension, immobilized, animal and plant cell cultures

Choosing the Cultivation Method Batch or Continuous culture? Do I need to modify the batch and continuous reactors? Chemostat with recycle Multistage chemostat systems Fed-batch operation Immobilization Solid-state Fermentation

What are the advantages and disadvantages of batch culture?

Advantages of continuous culture Growth rate can be controlled and maintained. Effect of changes in physical or chemical parameters can be examined. Biomass concentration can be maintained by varying the dilution rate. Find out more on the advantages.

Dilution Rate, D D = F/V, the number of culture volumes passing through the reactor per hour. dX/dt=-DX + X = X(-D) During steady state, dX/dt = 0 consequently  = D So, by varying the medium supply, growth rate can be varied. This holds until D  m Under these condition, the nutrient is no longer limiting Therefore, the expression (-D) becomes -ve

Critical Dilution Rate The lowest dilution rate at which wash out occurs. Dc is approximately equal to m. At dilution rates approaching Dc, the chemostat becomes less stable since slight fluctuations in the flow rate. A major drawback of chemostat is that they work best at lower dilution rates where the changes in X and S are small.

Modifications for batch and continuous bioreactor Choosing the cultivation method can affect: Product concentration and purity Degree of substrate conversion Yields of cells and products Capital cost in a process Modifications that can be done: Chemostat with recycle Multistage chemostat systems Fed-batch operation

Chemostat with recycle To keep the cell concentration higher than the normal steady-state level. Cells in the effluent can be recycled back to the reactor. Advantage of cell recycle: Increase productivity for biomass production A chemostat can be operated at dilution rates higher than the specific growth rate when cell recycle is used.

Multistage chemostat system Applicable to fermentations where the growth and product formation need to be separated into stages.

Fed-batch Operation Useful in antibiotic fermentation. Reactor is fed continuously (or intermittentlly), reactor is emptied periodically. Purpose is to maintain low substrate concentration, S Useful in overcoming substrate inhibition or catabolic repression, so that product formation increases.

Considerations for Microbial cell Bioreactor Adequate mixing is essential to ensure adequate supply of nutrients to the cells and removing any toxic materials from their vicinity. Mixing also affects the supply of oxygen by breaking large bubbles into smaller ones and dispersing them in the liquid so that their residence time in the reactor is increased. Accurate temperature control and efficient heat transfer also require good mixing. Mixing is also important to ensure quick dispersion of any added solutions such as acids, base, nutrients, so that there is no local high concentration buildup.

Considerations for Microbial cell Bioreactor At high growth rates, there is a need for sophisticated pH control and the addition of medium components, especially glucose when the fermentation is in fed-batch mode. High growth rates also result in a high output of metabolic energy, which requires high energy input for cooling in large fermenters. It is therefore necessary to evaluate the maximal specific heat production (cal/min-DCW) during a fermentation process so that the cooling requirements may be precisely determined. Cultures containing cells that grow relatively slow are much more susceptible to microbial contamination.

Considerations for Microbial cell Bioreactor Most microorganism grow in the pH 5.5-8.8 range, with fungi optimum at pH 5-7 and yeast pH 4-5. pH 4-5 has been used for yeast fermentation to facilitate growth and prevent contamination from other microorganisms. Production of foam is very common in microbial fermentations. It arises from the flow of air through the liquid medium and the subsequent formation of small bubbles as a consequence of mixing. If the bubble film is not strong enough, the bubble is easily destroyed and no foam is formed. This strength is dependent mainly on the surface tension of the liquid. Compounds that contribute to lowering the surface tension include proteins and protein hydrolysates, as well as oils and fats. In large scale industrial fermentation, using yeast with high concentration of corn steep liquor and molasses, the foaming is problematic.

Considerations for Microbial cell Bioreactor Even with less complex media, foam can form, usually in the last stage of fermentation involving high air-flow, high-speed mixing, and cell lysis. Except for beer production, foam is usually considered to be negative phenomenon because it can clog filters, which can cause dangerous increase in pressure. Wetting of the filters also stops them from functioning properly and the fermentor is more susceptible to contamination. The stable foam can also cause entrapment of oxygen so that the dissolved oxygen level increase substantially. However, the increased dissolved oxygen level is immediately decreased when the foam is broken. Such large and sudden changes in measured dissolved oxygen can play havoc if the fermentation is automatically controlled depending on the dissolved oxygen concentration.

Considerations for Microbial cell Bioreactor In large bioreactors, it may be advantageous to install mechanical foam breaker. In smaller bioreactors, chemical antifoam agents is generally used.

Considerations for Animal culture bioreactor Products of animal cell culture: enzymes, hormones, vaccines, monoclonal antibodies. Animal cells do not have cell walls, but are surrounded by a thin and fragile plasma membrane. This structure results in significant shear sensitivity. Mammalian cells grow at 37°C and pH 7.3. Usually 5% CO2-enriched air is used to buffer the medium pH. The culture medium needs to be gently aerated and agitated. Some mammalian cells are anchorage dependent and must grow on surfaces of glass or other support material. Some are not anchorage dependent and can grow in suspension culture.

Considerations for Animal culture bioreactor The reactor should be gently aerated and agitated. Some mechanically agitated reactors operating over 20 rpm and bubble column/airlift bioreactors operating at high aeration rates may cause shear damage to cells. Shear sensitivity is strain dependent. Well-controlled homogeneous environmental conditions (T, pH, DO) and supply of CO2-enriched air must be provided. A large support material surface-volume ratio needs to be provided for anchorage-dependent cells. The removal of toxic products of metabolism, such as lactic acid and ammonium, and high-value products such as Mab, vaccines should be accomplished during cell cultivation.

Considerations for Animal culture bioreactor Lab-scale cultivation: T-flasks (25-100 ml) – for anchorage dependent cell lines and shallow suspension culture. Spinner flask (100 ml – 1L) – with paddle type magnetic stirrers. Roller bottles (50 ml – 5L) rotating at about 1-5 rpm Trays containing shallow liquid suspension culture. These reactors are placed in a CO2 incubator at 37°C.

Considerations for Animal culture bioreactor Bioreactors for anchorage dependent cells: Hollow fiber reactors Ceramic matrix systems Weighted porous beads (immobilization) Bioreactors for suspension culture: Modified stirred bioreactors Airlift bioreactors Bubble column reactors

Considerations for Plant cell Bioreactor In the Western world, over 25% of pharmaceuticals are derived from extraction from whole plant. Examples of plant products: Pharmaceuticals: taxol, morphine, codeine Food colors: anthocyanins, saffron, shikonin Flavors: vanilla, strawberry, garlic Fragrances: jasmine, lemon, mint, rose Sweeteners: Miraculine, stevioside, thaumatin Whether the product is a chemical or a new plant, the biochemical engineer must become familiar with some basic characteristics of plants and their implications for bioreactor design.

Considerations for Plant cell Bioreactor Plant cells are large, and when they are exposed to turbulent shear fields where the eddy size approaches the cell size, the cells can be exposed to a twisting motion that can damage them. Lower level of shear appear to affect cell surface receptors and nutrient transport. Reactors of high shear must be avoided. Plant cells can withstand far more shear than animal cells. Stirred tanks designed for bacterial culture are not good choices, but modified stirred tanks can be suitable, eg change impellers.

Considerations for Plant cell Bioreactor Plant cell cultures can achieve high cell densities and viscosities. Their reduced respiration rate compensates in part of the need for vigorous agitation. Airlift reactors are better for low or moderate cell densities. Reactor with paddle type or helical ribbon impellers for high cell densities. Mixing depends on combination of sparging and mechanical agitation. Oversparging can be a problem for plant cells, because plants make at least one volatile hormone, ethylene, and its rapid removal can affect productivity.

Challenges in plant cell fermentation Low growth rates of plant cells presents problems for large-scale system: Maintenance of aseptic conditions for the 2-4 weeks of fermentation is difficult. Genetic instability of many cell lines.

Considerations for Solid State Fermentation (SSF) Fermentation of solid substrates at low moisture levels or water activities. Most SSF are mold fermentation producing extracellular enzymes on moist agricultural substrates. Low contamination risk due to low moisture level. Easy product separation. Poor mixing characteristics Difficult to control pH, DO, temperature within the fermentation mash. Usually a rotating-drug fermenter is used.

Considerations for Solid State Fermentation (SSF) Many SSF are shear sensitive due to mycelia disruption at high rotation speeds. At low agitation rate, oxygen transfer and CO2 evolution rates become limiting.