Fed-Batch Fermentation: Techniques, Applications, and Future Directions

What is fed-batch fermentation?

Industrial biotechnology makes extensive use of fed-batch fermentation, a controlled method of microbial fermentation, to optimize the production of an extensive variety of biochemicals, medications, and enzymes. This methodology entails a gradual augmentation of the bioreactor's culture capacity through the intermittent introduction of fresh substrate during the course of fermentation. In contrast to conventional batch fermentation, which may result in detrimental contaminant accumulation and nutrient depletion, fed-batch fermentation endeavors to maximize product yield while prolonging the microorganisms' exponential growth phase (see images below).

Advantages of fed-batch fermentation

By keeping the ideal circumstances for a longer period of time, it is able to greatly enhance the output of the target product.

Less Stress and Toxicity: Incorporating nutrients slowly avoids the accumulation of harmful byproducts that might stunt microbial development.

Efficiently utilizing nutrients helps to cut down on waste and can also bring down production costs.

Control feed rate of media containing substrate and nutrients and regulate the concentration of key substances to control product formation rate.

Achieve high cell density and increase production of metabolites not related to growth.

Extend the production duration of cell culture and operate with different feeding strategies to achieve maximum productivity.

Disadvantages of fed-batch fermentation

Relatively complex operation: It requires precise control of the timing, rate, and volume of feed additions, as well as other fermentation parameters such as temperature, pH, and aeration. This places high demands on equipment and control systems.

Increased risk of contamination: Due to the need for multiple feed additions, there are more opportunities for contact with the external environment, thereby increasing the risk of contamination by unwanted microorganisms.

Extended fermentation cycle: Compared to traditional batch fermentation, fed-batch fermentation generally has a longer cycle, which may lead to reduced production efficiency and increased production costs.

Applications of Fed-Batch Fermentation

Fed-batch fermentation has been widely used because its main advantages include controlling microbial growth rates, bioactive metabolites, and oxygen transfer limitation by feeding rate. Fed-batch fermentation can be used to produce the highest microbial biomass weight that forms bioactive metabolites. Various products produced by fed-batch techniques include proteins, amino acids, enzymes, antibiotics, vitamins, alkaloids, phenols, or other biochemical compounds extracted from bacterial, actinomycetes, fungal, and algal cells.

Pharmaceutical Field

Antibiotic Production: For example, in the production of penicillin, fed-batch fermentation allows for the continuous addition of essential nutrients, such as carbon sources and nitrogen sources, during the fermentation process. This enables the microorganism to grow and metabolize continuously, extending the synthesis time of penicillin and improving its yield. Additionally, the types and rates of feed can be adjusted according to the actual fermentation conditions to optimize fermentation parameters, ensuring the stability of penicillin quality.

Amino Acid Production: In the fermentation production of amino acids such as lysine and glutamic acid, fed-batch fermentation effectively addresses the issue of high nutrient consumption by microorganisms. For example, during glutamic acid fermentation, nutrients like sugar and nitrogen are consumed in large quantities as the microorganisms grow and metabolize. By timely feeding, a high metabolic rate can be maintained in the fermentation system, thus increasing the yield of glutamic acid.

Recombinant Protein Production: Pichia pastoris is an efficient cell factory. By using fed-batch fermentation technology, it can grow in a precisely controlled medium, significantly increasing cell density, thus obtaining high-yield, high-activity, and cost-effective recombinant proteins, such as vaccines, anticoagulants, and more.

Bacterial Ghost Vaccine Production: Bacterial ghost technology is a microbial expression system that generates empty shell cells from Gram-negative bacteria by integrating a lysis gene, which can serve as candidate vaccines. In fed-batch fermentation, optimizing the feed controller and specific substrate uptake rates during the lysis phase can significantly increase the yield of bacterial ghost vaccines. For example, the fermentation process of E. coli K-12/pHCl-InaN-GAPDH-ghost 27 SDM system is divided into four stages, achieving a maximum yield of 34.9 g dry cell weight (DCW)/L for bacterial ghost vaccines.

Virus-Like Particle (VLP) Vaccine Production: Virus-like particles (VLPs) are virus-like structures that do not contain genetic material but have excellent immunogenicity, making them suitable for vaccine production. In fed-batch fermentation, optimizing the host cell cultivation conditions and feeding strategy can improve the yield and quality of VLPs.

Bioactive Metabolite Production

Polyunsaturated Fatty Acid Production: For instance, Crypthecodinium cohnii is used for the fed-batch fermentation production of DHA (docosahexaenoic acid). By studying different carbon sources, such as glucose and acetate, and controlling the feeding rate of the medium, the overall volumetric productivity of DHA can be improved.

Other Bioactive Metabolite Production: Fed-batch fermentation can also be used to produce bioactive metabolites like enzymes, vitamins, alkaloids, phenolic compounds, and others. Optimizing fermentation conditions and feeding strategies can enhance the yield and quality of these metabolites.

Metabolic Engineered Compound Production

Polyhydroxybutyrate (PHB) Production: For example, fed-batch fermentation using Collotnocorus pp. strain MV10 for cyclic fed-batch fermentation has achieved high cell density growth and high polyhydroxybutyrate (PHB) yield.

Other Metabolic Engineered Compound Production: Fed-batch fermentation can be applied to produce other metabolic engineered compounds, such as biofuels and bioplastics. Optimizing feeding strategies can enhance the yield and quality of these compounds.

Food Industry

Lactic Acid Production: In lactic acid fermentation, fed-batch fermentation can control the substrate concentration and avoid the inhibitory effects caused by adding a large amount of substrate at once. For example, when fermenting with lactic acid bacteria to produce lactic acid, slow addition of carbon sources like lactose helps the bacteria to ferment continuously at an appropriate substrate concentration, increasing the yield and production efficiency of lactic acid while also helping to control the pH value during fermentation.

Alcoholic Beverage Production: Such as wine and beer brewing. In wine production, fed-batch fermentation can control yeast growth and the rate of alcohol production. During the early fermentation stage, appropriate amounts of sucrose and other nutrients are added to promote yeast growth. As fermentation progresses, the feeding rate can be adjusted based on the sugar consumption and alcohol concentration changes, extending the fermentation time, increasing alcohol yield, and enriching the flavor of the wine.

Energy Industry

Bioethanol Production: In bioethanol fermentation production, fed-batch fermentation can efficiently utilize large amounts of biomass feedstock. For example, in bioethanol production from cellulose-based biomass such as corn stover, nutrients like hydrolyzed cellulose sugar liquid can be fed in stages to increase the substrate concentration in the fermentation tank, extend the fermentation time, and boost ethanol yield.

Biological Hydrogen Production: Some microorganisms produce hydrogen during fermentation. Fed-batch fermentation can continuously supply nutrients such as organic acids and sugars to hydrogen-producing microorganisms, maintaining their high hydrogen production activity throughout the fermentation process. For example, in hydrogen production using hydrogen-producing clostridia, timely feeding can extend the growth phase and hydrogen production phase of the microorganisms, improving hydrogen yield.

Environmental Protection Field

Wastewater Treatment: When using microorganisms to treat high-concentration organic wastewater, fed-batch fermentation can control the microbial growth environment, improving wastewater treatment efficiency. For example, in treating food processing wastewater rich in sugars, proteins, and other organic substances, feeding wastewater or other nutrients in batches helps microorganisms grow and metabolize under suitable substrate concentrations and environmental conditions, breaking down the organic substances in the wastewater into harmless substances like carbon dioxide and water, thereby achieving wastewater purification.

Fed-batch fermentation process

1. Pre-Fermentation Preparation

Medium Preparation: Select and prepare an appropriate fermentation medium containing key nutrients such as carbon sources, nitrogen sources, inorganic salts, and growth factors.

Seed Culture: Perform seed cultivation and scale-up to obtain a sufficient quantity of microbial culture in a healthy growth state.

Fermentation Equipment Preparation: Clean, disinfect, and sterilize the fermenter to ensure cleanliness and integrity. Install and calibrate sensors and instruments such as thermometers, pH meters, and dissolved oxygen probes.

2. Fermentation Stage

Fermentation Initiation: Inoculate the prepared seed culture into the fermenter to start the fermentation process. The fermenter contains an initial volume of medium at this point.

Feeding Operations: As fermentation progresses, fresh medium or specific nutrients are added to the fermenter either intermittently or continuously.

Intermittent Feeding: Medium or nutrients are added at regular or irregular intervals during fermentation-for example, a specific amount of carbon or nitrogen source is added at designated time points.

Continuous Feeding: Nutrients are added continuously according to a preset program or adjusted in real time based on online monitoring data.

Fermentation Monitoring: Continuously monitor key parameters such as temperature, pH, dissolved oxygen concentration, substrate concentration, product concentration, and microbial growth. Adjust fermentation conditions—such as temperature, aeration rate, agitation speed, and feeding rate—based on these parameters to maintain optimal fermentation efficiency.

3. Fermentation Endpoint Determination and Product Harvesting

Endpoint Determination: Evaluate whether fermentation has concluded based on changes in process parameters, fermentation time, product concentration, and microbial growth status.

Product Harvesting: At the end of fermentation, separate and extract the target product from the fermentation broth or residue using methods such as centrifugation, filtration, extraction, or precipitation.

4. Post-Fermentation Processing

Equipment Cleaning and Maintenance: After product harvesting, promptly clean, disinfect, and maintain the fermenter and associated equipment to prepare for the next use.

Waste Management: Treat waste liquid, exhaust gas, and solid waste generated during fermentation to ensure compliance with environmental regulations.

Advantages of Fed-Batch Fermentation vs. Traditional Fermentation

Batch Fermentation vs. Fed-Batch Fermentation

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