Continuous Fermentation & Fed-Batch Fermentation

Continuous fermentation & fed-batch fermentation both allow open systems and require the addition of fresh medium during the strain culture in advancing high product yield. As a leading CDMO, BOC Sciences has the ability to offer continuous fermentation and/or batch fermentation services and to optimize the processes.

Based on our expertise in microbial fermentation and extensive supporting facilities, BOC Sciences provides one-stop fermentation services, including strain development, fermentation process optimization, GARS service, and more. We are committed to providing our customers with customized services, high-quality products, and expert consultation on microbial fermentation projects.

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).

Fed-batch fermentation process. (Valdez-Castro L., et al., 2003)

The fed-batch fermentation process. (Wang X L., et al., 2020)

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.

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.

Batch fermentation process

An inoculum, a little volume of culture containing the microbes that will be utilized for fermentation, is prepared first. To make sure this culture is strong and can expand quickly in the fermenter, it is usually cultured in a controlled environment.

In order to ensure that the fermentation medium remains free of any germs that would be detrimental to the microbes that would be growing in it, it is first prepared and then sterilized.

The inoculum is created and then added to the fermenter's sterile media. An inoculum typically occupies 1% to 10% of the fermenter's total capacity.

The fermentation is permitted to continue under controlled circumstances after inoculation. For aerobic processes, it is crucial to monitor and manage important factors including temperature, pH, oxygen, and agitation in order to maximize microbial growth and product generation.

Measuring microbial growth, substrate consumption, product production, and pH variations are just a few of the many culture parameters tracked throughout fermentation. By keeping an eye on things, we can learn about the kinetics of fermentation and find out when it finishes.

Fermentation is considered complete when the substrate is drained or the target product concentration reaches its maximum, at which point the culture is harvested. Using centrifugation or filtering, the cells may be isolated from both the product and the medium.

Based on the product type, the following downstream processing processes are used to extract and purify the product: distillation, crystallization, or chromatography.

What is continuous fermentation?

The process stimulates microbial fermentation by continuously adding substrate (feed containing nutrients) to the bioreactor and then removing the used medium and product. This technique ensures a constant fermentation environment, as opposed to fed-batch or batch fermentation, which includes changing variables throughout the process. There are several benefits to using continuous fermentation for the manufacture of biochemicals, biofuels, and other goods that need efficient and consistent manufacturing (see image below).

Schematic diagram of continuous fermentation. (Guo Y., et al., 2022)

Advantages of continuous fermentation

• Continuous systems have the ability to produce better productivity in comparison to batch systems due to the fact that the microorganisms are consistently kept in their exponential development phase.
• Continuous systems can optimize the use of substrates and nutrients, resulting in less waste and the possibility for decreased production costs.
• Product quality consistency is achieved by maintaining continuous operating conditions, resulting in a more consistent outcome compared to batch fermentation.
• Scaling up a continuous process for industrial production is typically simpler since the process dynamics remain consistent regardless of scale.
• Avoided repetitive fermentation preparation, cleaning, and sterilization between batches in the discontinuous process; required no additional manpower, effort, and time.
• Extended the exponential microbial growth phase of the culture process compared to the batch process, thereby reducing processing time and ensuring a high production level of final products.
• Eliminated inhibition of substrates or by-products as final products are harvested continuously.

Continuous fermentation process

Continuous fermentation necessitates a specifically engineered bioreactor that is equipped with inlet and outlet streams to continuously introduce substrates and eliminate products and waste, respectively. To maintain sterility and promote the growth of certain bacteria, both the bioreactor and the input substrates undergo sterilization procedures.

A specialized group of microorganisms, which have adapted to the specific conditions required for fermentation, is put into the bioreactor that has been thoroughly sterilized. The culture is let to proliferate and establish a stable presence within the bioreactor, attaining an ideal level of biomass concentration required for uninterrupted functioning.

The temperature, pH, oxygen levels (for aerobic processes), and agitation, which are critical process factors, are constantly monitored and modified in order to maximize microbial activity and optimum product creation.

As the microorganisms break down the substrates, they constantly create the required product(s) through metabolism. The architecture of the bioreactor enables the uninterrupted extraction of the culture medium that contains the product. This process is carried out at a rate that maintains a balance between the addition of new substrate and the prevention of dilution or buildup of products and by-products.

The discharge from the bioreactor consists of the product along with microbial cells and other constituents. The desired product is isolated and purified using further processes, including centrifugation, filtering, and purification. During the whole process, the product's quality and the functioning of the bioreactor are consistently checked to guarantee constant product quality and optimal system efficiency.

Regular sampling and microbiological testing are essential. Regular cleaning and maintenance are necessary, however they are needed less often compared to batch operations. The frequency of these tasks depends on the system's design and operating lifespan.

Applications of continuous fermentation

Continuous fermentation systems offer important economic advantages with improved fermentation rates, especially when continuous fermentation is combined with cell immobilization techniques. The continuous fermentation process can utilize mixed cultures to afford chemicals, and this technology holds great promise for the efficient production of fermented beverages such as beer, wine, cider, and bioethanol. Continuous fermentation is also used to produce certain bioproducts, including polyhydroxyalkanoates (PHA), lactic acid (LA), butanol, citric acid, and 1, 3-propanediol.

Batch vs continuous fermentation

(a)During batch fermentation, no more nutrients are introduced and no final products or biomass are extracted until the fermentation is completed. The process of continuous fermentation involves a constant supply of nutrients and the continual production of products (see image below).

(b) Batch fermentation often necessitates less complex equipment and setup in comparison to continuous fermentation. Continuous fermentation necessitates sophisticated methods for monitoring and regulating process parameters to avert departures from the intended condition. (c) Batch fermentation is a frequently employed method for manufacturing items that are manufactured in several batches, such as medications. This approach allows for the customization of conditions to suit the individual requirements of each product. Additionally employed in less extensive projects when the attributes of simplicity and adaptability have greater significance. Continuous fermentation is most suitable for the efficient and reliable manufacture of large quantities of commodities such as biofuels and bulk chemicals, where maintaining a high volume and consistent output is of utmost importance.

Simplified scheme of (a) batch, (b) fed-batch, and (c) continuous fermentation. (Paulová L., et al., 2013)

What Can We Do?

Microbial strain culture can be manipulated in a variety of ways. One of the most effective ways is by varying the feeding method. BOC Sciences is able to select the most appropriate feeding strategy based on the nature of the target product or different production purposes. We have the ability to perform continuous fermentation or fed-batch fermentation and provide the most cost-effective customized fermentation service to our customers.

Reference

1. Valdez-Castro L., et al., Neural networks applied to the prediction of fed-batch fermentation kinetics of Bacillus thuringiensis, Bioprocess and Biosystems Engineering, 2003, 25: 229-233.
2. Wang X L., et al., Sequential fed-batch fermentation of 1, 3-propanediol from glycerol by Clostridium butyricum DL07, Applied Microbiology and Biotechnology, 2020, 104: 9179-9191.
3. Guo Y., et al., Continuous Fermentation by Lactobacillus bulgaricus T15 Cells Immobilized in Cross-Linked F127 Hydrogels to Produce ᴅ-Lactic Acid, Fermentation, 2022, 8(8): 360.
4. Paulová L., et al., Advanced fermentation processes, Engineering aspects of food biotechnology, 2013: 89-110.