Fed-batch Culture- Definition, Principle, Process, Types, Applications, Limitations

Fed-batch culture is a semi-continuous bioprocess that involves the periodic or continuous addition of nutrients to a bioreactor, while the products are harvested only at the end of the process. It is a modification of batch culture, where all the substrates are added at the beginning of the fermentation and no nutrients are added during the process. Fed-batch culture is widely used in industrial applications because it offers several advantages over batch and continuous cultures, such as:

• It extends the productive duration of the culture by providing fresh nutrients and avoiding substrate depletion or inhibition.
• It can be used to switch genes on or off by changing the substrate concentration or composition, which can affect the expression of desired products.
• It can be manipulated for maximum productivity using different feeding strategies, such as discontinuous or continuous feeding, fixed or variable volume, and feedback control.
• It can achieve high cell densities and high product yields by limiting the growth rate and avoiding catabolite repression or Crabtree effect.
• It can reduce the broth viscosity and water loss by evaporation by adding concentrated or gaseous substrates.
• It can reduce the equipment footprint and operational costs by using smaller bioreactors and less medium compared to batch or fed-batch processes.

Fed-batch culture is suitable for bioprocesses that aim for high biomass density or high product yield, especially when the desired product is positively correlated with microbial growth. It is also useful for bioprocesses that involve substrate inhibition, metabolic regulation, or gene expression control. Some examples of products produced by fed-batch culture are antibiotics, enzymes, amino acids, organic acids, vaccines, and recombinant proteins.

The principle of fed-batch culture is to control the growth rate and metabolism of the microorganisms by adjusting the availability of the limiting substrate. The limiting substrate is the one that is consumed first by the microorganisms and determines the maximum growth rate. By adding the limiting substrate in small amounts or at a low rate, the concentration of the substrate in the culture broth can be maintained at a low level, which prevents the occurrence of catabolite repression or Crabtree effect. Catabolite repression is a phenomenon where the presence of a high concentration of a preferred carbon source (such as glucose) inhibits the utilization of other carbon sources by the microorganisms. Crabtree effect is a phenomenon where the excess glucose leads to the production of ethanol or other organic acids by some microorganisms, even under aerobic conditions. Both catabolite repression and Crabtree effect can reduce the yield and productivity of the desired product.

By using fed-batch culture, the microorganisms can be grown in a balanced growth state, where all the essential nutrients are available in sufficient amounts and no toxic metabolites are accumulated. This allows the microorganisms to express their full genetic potential and produce high amounts of biomass or product. The product can be either growth-associated or non-growth-associated. Growth-associated products are those that are synthesized in proportion to the biomass formation, such as enzymes or proteins. Non-growth-associated products are those that are synthesized independently of the biomass formation, such as antibiotics or secondary metabolites. Fed-batch culture can enhance the production of both types of products by optimizing the growth rate and substrate concentration.

The principle of fed-batch culture can be illustrated by using Monod`s equation, which describes the relationship between specific growth rate (µ), substrate concentration (S), maximum specific growth rate (µmax), and half-saturation constant (Ks). The equation is:

µ = (µmaxS)/(Ks + S)

According to this equation, when S is much higher than Ks, µ approaches µmax and the microorganisms grow at their maximum rate. However, this may also result in catabolite repression or Crabtree effect. When S is much lower than Ks, µ approaches zero and the microorganisms stop growing due to substrate limitation. However, this may also result in nutrient depletion or product inhibition. Therefore, the optimal condition for fed-batch culture is to maintain S at a level close to Ks, where µ is half of µmax and the microorganisms grow at a moderate rate with high efficiency and productivity. This can be achieved by using different feeding strategies based on online or offline measurements of various parameters related to cell growth and metabolism.

The process of fed-batch culture can be divided into four main steps:

1. Batch phase: The process will be initiated with batch fermentation and consume substrate, nutrients, and/or inducers. The microorganisms are inoculated and grown using a batch process for a set period, usually until the substrate is exhausted or the growth rate declines. This phase allows the cells to adapt to the culture conditions and reach a high cell density.
2. Feed phase: Fresh medium will be added through a variety of feed streams. By manipulating the feed rates during the run, carbon, nitrogen, phosphates, nutrients, precursors, or inducers are added intermittently or continuously to the culture. The common feeding strategies are:
   • Discontinuous feeding, achieved by regular or irregular pulses of substrates. This strategy is simple and easy to implement, but it may cause fluctuations in the substrate concentration and pH of the culture.
   • Continuous feeding, achieved by adding nutrients at a constant or variable rate. This strategy can maintain a steady-state condition and avoid substrate inhibition or catabolite repression. However, it requires accurate control and monitoring of the feed rate and culture parameters.
3. Production phase: As a result of the addition of fresh nutrients, extensive biomass accumulation normally occurs during exponential growth. Fed-batch fermentation is therefore very useful for bioprocesses that aim for high biomass density or high product yield when the desired product is positively correlated with microbial growth. The growth rate can be controlled by the substrate concentration to avoid catabolite repression. The products are harvested only at the end of the run or when the product concentration reaches a desired level.
4. Termination phase: The culture volume increases during the course of operation until the volume is full or the product quality deteriorates. Thereafter, a batch mode of operation is used to attain the final results. Alternatively, a certain amount of spent broth from the fermenter can be removed, followed by the addition of fresh nutrient medium. This is called repeated or cyclic fed-batch culture and it can prolong the production phase and increase the overall productivity.

The process of fed-batch culture can be illustrated by the following diagram:

Fed-batch culture can be classified into different types based on the feeding strategy, the volume change, and the number of cycles. Some of the common types of fed-batch culture are:

• Constantly-fed-batch culture: This is the simplest type of fed-batch culture, where the feed rate of a growth-limiting substrate is constant throughout the culture. This type of fed-batch culture can achieve high cell density and avoid substrate inhibition by maintaining a low substrate concentration in the bioreactor. However, it may not be optimal for some processes that require dynamic control of the feed rate based on the metabolic state of the cells or the product formation.
• Exponential-fed-batch culture: This is a type of fed-batch culture where the feed rate of a growth-limiting substrate is increased exponentially with time. This type of fed-batch culture can achieve higher cell density and productivity than constantly-fed-batch culture by matching the feed rate with the specific growth rate of the cells. However, it may require more sophisticated control systems and online measurements to adjust the feed rate accurately.
• Fixed-volume fed-batch culture: This is a type of fed-batch culture where the volume of the bioreactor remains constant during the culture. This can be achieved by feeding the growth-limiting substrate in a highly concentrated form or in a purified form by dialysis or radiation sterilization. This type of fed-batch culture can avoid dilution effects and maintain high cell density and product concentration in the bioreactor. However, it may require more complex feeding systems and higher costs for substrate preparation.
• Variable-volume fed-batch culture: This is a type of fed-batch culture where the volume of the bioreactor changes with time due to the addition of substrate feed. The volume change can be influenced by process requirements, time availability, and the objective of the fermentation process. The feeding strategy can vary depending on the composition and concentration of the substrate feed, as well as the initial concentration of other medium components. This type of fed-batch culture can offer more flexibility and versatility for different processes and products.
• Repeated or cyclic fed-batch culture: This is a type of fed-batch culture where a certain amount of spent broth is removed from the bioreactor and replaced with fresh medium at regular intervals. This can increase the substrate concentration and specific growth rate in the bioreactor, as well as reduce the accumulation of inhibitory products or toxins. However, it may also cause loss of biomass or product, as well as nutrient imbalance or depletion if not designed carefully.
• Single fed-batch culture: This is a type of fed-batch culture where only one feeding solution is added during the fermentation process, and no spent broth is removed. This can simplify the operation and reduce contamination risks, but it may also limit the duration and productivity of the fermentation process due to reactor volume constraints.

Fed-batch culture is a widely used technique in industrial biotechnology, as it offers several advantages over batch and continuous culture methods. Some of the applications of fed-batch culture are:

• Production of antibiotics: Fed-batch culture is used to produce antibiotics such as penicillin, cephalosporin, chlorotetracycline, griseofulvin, streptomycin and tetracycline. These antibiotics require controlled feeding of nutrients to avoid substrate inhibition, catabolite repression, product inhibition and oxygen limitation. By adding the limiting substrate in small amounts, fed-batch culture can optimize the growth and productivity of the antibiotic-producing microorganisms.
• Production of baker`s yeast: Fed-batch culture was used in the production of baker`s yeast as early as 1915. It was recognized that an excess of malt in the medium would lead to too high a growth rate resulting in an oxygen demand in excess of that which could be met by the equipment. By adding malt gradually, fed-batch culture can achieve high cell density and high yield of baker`s yeast.
• Production of enzymes: Fed-batch culture is used to produce various enzymes such as cellulases, pectinases, xylanases, beta-glucanase and glutamine synthetase. These enzymes are often produced by filamentous fungi or bacteria that have complex nutritional requirements and are sensitive to environmental conditions. By adjusting the feed rate and composition, fed-batch culture can enhance the enzyme synthesis and secretion.
• Production of recombinant proteins: Fed-batch culture is used to produce recombinant proteins such as insulin, human growth hormone, interferon and monoclonal antibodies. These proteins are usually expressed by genetically modified microorganisms or animal cells that need specific inducers or precursors to activate the gene expression. By controlling the feed of these inducers or precursors, fed-batch culture can regulate the expression level and quality of the recombinant proteins.

Fed-batch culture is a widely used technique for enhancing the productivity and yield of various bioprocesses. However, it also has some limitations that need to be considered and overcome. Some of the major limitations are:

• Complexity and cost: Fed-batch culture requires more sophisticated equipment, monitoring, and control systems than batch culture. It also involves higher operational and maintenance costs. The design and optimization of the feeding strategy can be challenging and time-consuming, as it depends on various factors such as the microorganism, the substrate, the product, the reactor, and the environmental conditions. Moreover, fed-batch culture may require additional steps for sterilization, aeration, agitation, and harvesting of the broth.
• Risk of contamination: Fed-batch culture involves the addition of fresh medium to the reactor during the fermentation process. This increases the risk of contamination by unwanted microorganisms or foreign substances. Therefore, strict aseptic conditions must be maintained throughout the process. The feeding system must be properly sealed and sterilized to prevent any leakage or entry of contaminants. The quality and purity of the feed medium must also be ensured to avoid any adverse effects on the culture.
• Accumulation of by-products: Fed-batch culture may result in the accumulation of by-products or metabolites that are not desired or useful for the process. These by-products may have negative impacts on the growth, metabolism, and productivity of the microorganisms. They may also interfere with the downstream processing and purification of the product. Therefore, fed-batch culture requires careful monitoring and control of the concentration and composition of the broth. Some by-products may be removed or diluted by partial harvesting or washing of the culture. Alternatively, some by-products may be utilized or converted by co-culturing with other microorganisms or by adding specific enzymes or chemicals to the reactor.
• Limitation of other nutrients: Fed-batch culture typically involves the addition of a single limiting substrate to control the growth rate and metabolism of the microorganisms. However, this may lead to the limitation or depletion of other essential nutrients such as nitrogen, phosphorus, vitamins, minerals, etc. These nutrients are required for maintaining the cell viability, activity, and integrity. Therefore, fed-batch culture must ensure that all the necessary nutrients are available in sufficient amounts for the optimal performance of the culture. This may require adjusting the composition and concentration of the feed medium or supplementing it with additional nutrients at appropriate intervals.

These are some of the main limitations of fed-batch culture that need to be addressed and overcome for achieving better results and efficiency in bioprocessing. Fed-batch culture is a powerful technique that can offer many advantages over batch culture, but it also requires more careful planning, execution, and optimization to ensure its success.

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