Liquid State Fermentation: Definition, Process and Application

What is liquid fermentation?

Liquid fermentation (also known as submerged fermentation, SmF), a commonly used procedure in industrial biotechnology, is a fundamental method for growing microorganisms in a liquid nutrient media under controlled circumstances. This technique is essential for the large-scale manufacture of a wide range of biochemical products, including enzymes, antibiotics, biofuels, and organic acids, among others. Liquid fermentation provides microorganisms with a homogenous environment in which nutrients, oxygen, and other critical components are evenly distributed, allowing for maximum growth and efficient generation of the required metabolites.

Within this procedure, the liquid medium, abundant in dissolved nutrients including sugars, amino acids, vitamins, and minerals, facilitates the unrestricted mobility of microorganisms, thereby enabling uniform absorption of nutrients and metabolic activity. Liquid fermentation utilizes regulated aeration and agitation to maintain adequate oxygen levels for aerobic processes, minimize cell settling, and foster uniform dispersion of microbes in the medium. In order to maximize the manufacturing output and quality of the desired product, these parameters are carefully controlled.

Solid-state fermentation and submerged fermentation. (Nemes S A., et al., 2022)

What are the factors affecting liquid fermentation?

(1) Environmental temperature is a critical determinant since it directly impacts the metabolic processes of microorganisms. Optimal temperature ranges for growth and product generation are specific to each microorganism. Excessive departures from this ideal range might result in decreased growth rates or denaturation of crucial enzymes, therefore diminishing production or even stopping the fermentation.

(2) The pH of the fermentation medium has a major influence on microbial activity and enzyme stability. Most microorganisms require a certain pH range, and maintaining this range is critical for proper enzyme activity and microbial development. pH variations can limit microbial metabolism, resulting in lower yields or the formation of undesirable byproducts.

(3) Dissolved oxygen concentration is key in aerobic fermentation procedures. In order for aerobic microbes to respire and produce energy, there must be sufficient oxygen levels. Limitations in growth and productivity can occur in anaerobic conditions when there is insufficient oxygen availability. This might lead to the creation of various metabolites, which are not always desirable.

(4) Nutrients, oxygen, and microbes are distributed uniformly throughout the liquid media when the medium is agitated properly. Additionally, it aids in avoiding cell settling, which is a known contributor to uneven fermentation and localized nutrient depletion. But be careful, because too much movement might kill delicate microbes and shear cells.

Diagram of submerged fermentation process of ergothioneine. (Tang B., et al., 2022)

Fermentation technology at BOC Sciences

• Aerobic Fermentation
• Anaerobic Fermentation
• Continuous Fermentation & Fed-Batch Fermentation
• Solid State Fermentation (SSF)
• Submerged Fermentation (SmF)
• Fermentation CDMO

Liquid fermentation process

Medium preparation: The fermentation media is created by combining water with substrates that facilitate the proliferation of microorganisms. The usual components of this mixture include a carbon source (such as glucose or molasses), nitrogen sources (such as ammonium salts or urea), vitamins, and minerals. In order to avoid contamination, the medium is subjected to sterilization, often by autoclaving at elevated temperatures and pressures. This stage selectively eradicates undesired microbes that may disrupt the fermentation process.

Inoculum preparation: To acquire a robust and functional inoculum, the microorganism (bacteria, yeast, or fungus) is cultivated in a small-scale setting, such as a shaking flask or a laboratory fermenter. This process entails cultivating the microbe in a medium abundant in nutrients until it attains the intended maximum cell density.

Fermentation process: Large containers designed to cultivate microorganisms in a controlled environment are known as bioreactors or fermenters. The stirred-tank, airlift, and bubble column reactors are only a few of the many varieties of bioreactors. Phosphorus, temperature, dissolved oxygen, and substrate concentration are among the factors that undergo constant monitoring and modification. As a result, this ensures that the perfect conditions for fermentation are maintained all the way through.

Harvesting: The product is extracted from the fermentation broth once the fermentation process is finished. This might entail using centrifugation or filtration to separate the microbial biomass from the liquid, depending on the kind of product.

Complete overview of liquid state fermentation processes. (Yatmaz E., et al., 2019)

What are the functions of liquid fermentation?

Liquid fermentation, a kind of "submerged fermentation," is a bioprocess in which microorganisms are cultured in a nutrient-rich fluid media with an ideal concentration of oxygen. Bioconverting microorganisms engaged in fermentation into extra high-value molecules called secondary metabolites is the goal of the SmF bioprocess, which is similar to the SSF bioprocess. Bioactive chemicals attached to the bran matrix are released during this process. After 24 hours of rice bran SmF, lactic acid bacteria biotransformed the phenolic derivatives 4-ethylphenol, vanillin, vanillic acid, and vanillyl alcohol into these compounds. To make the bioprocess more efficient, enzymes called ferulic acid esterase and ferulic acid decarboxylase were used. The most important thing that came out of the study was that lactic acid bacteria could be able to make biovanillin on the spot using Smf from agro-industrial lignocellulosic waste, like cereal bran. In today's food, beverage, pharmaceutical, cosmetic, and tobacco industries, synthetic vanillin (4-hydroxy-3-methoxy benzaldehyde) is the flavoring of choice for vanilla. Thus, new pathways for vanilla synthesis via strain metabolism can be developed through SmF and SSF biotechnological processes that use inexpensive substrates, such as agro-industrial by-products. Compared to SmF, SSF has several benefits, the most important of which are a high yield, improved product stability, reduced protein degradation, and low contamination risk. The decreased production cost is an additional benefit. Economic analysis shows that obtaining a cellulase enzyme from SSF (15.67 USD/kg) is three times cheaper than getting the same enzyme from SmF (40.36 USD/kg). This is very beneficial because the enzyme is sold for 90 USD/kg. Conversely, SmF has several benefits that make it useful in a variety of contexts. For example, it allows for precise regulation of fermentation variables including temperature, humidity, and pH, as well as appropriate aeration as a result of continuous homogenization. Additionally, the risk of fungal hyphae drying up during mold fermentation can be reduced using the SmF bioprocess.

Application of liquid fermentation

A number of antibiotics, including tetracycline, streptomycin, and penicillin, rely on liquid fermentation processes. The procedure entails growing microbes like Streptomyces griseus or Penicillium chrysogenum in a nutrient-rich environment to generate these antimicrobial chemicals. They are subsequently collected and refined for use in combating bacterial diseases. Insulin, HGH, and monoclonal antibodies are only a few examples of the recombinant proteins and enzymes produced by liquid fermentation. To produce these proteins, which are essential for the treatment of a wide range of illnesses and disorders, scientists utilize genetically modified microbes like bacteria or yeast. Food processing enzymes such as amylases, proteases, and lactases are produced by liquid fermentation. Products' texture, taste, and longevity in the fridge are all improved by these enzymes. For instance, lactose-free dairy products are made possible by using lactase enzymes that are created by fermentation.

Integration of Solid State and Submerged Fermentations for the Valorization. (Martău G A., et al., 2021)

References

1. Nemes S A., et al., Integrated technology for cereal bran valorization: perspectives for a sustainable industrial approach, Antioxidants, 2022, 11(11): 2159.
2. Tang B., et al., Optimization of submerged fermentation conditions for biosynthesis of ergothioneine and enrichment of selenium from Pleurotus eryngii 528, Food Science and Technology, 2022, 42: e40022.
3. Yatmaz E., et al., Liquid State Bioreactor, Essentials in fermentation technology, 2019: 135-168.
4. Martău G A., et al., Integration of solid state and submerged fermentations for the valorization of organic municipal solid waste, Journal of Fungi, 2021, 7(9): 766.
5. Subramaniyam R, Vimala R. Solid state and submerged fermentation for the production of bioactive substances: a comparative study, Int J Sci Nat, 2012, 3(3): 480-486.