Fermentation for Amino Acids

The market demand for amino acids has been increasing over the last 100 years. As bulk biochemicals, amino acids are used in the production of a wide range of products such flavor enhancers in nutritious foods. Amino acids are involved in regulating key metabolic pathways and processes, such as regulating protein metabolism and controlling the growth and immunity of organisms, which are essential for their growth and maintenance. Clinical studies have shown that amino acid deficiencies can lead to a series of serious diseases. As a result, the interest in producing amino acids in a more cost-effective and sustainable manner has increased significantly. Amino acids can be produced by different processes, such as extraction from protein hydrolysates, chemical synthesis or fermentation routes with the application of microorganisms. Thanks to the rapid development of genetic engineering technologies, fermentation production is becoming one of the most promising approach for the commercial production of amino acids. Under aerobic or anaerobic conditions, several microorganisms are used to convert sugars in a substrate into a broad range of amino acids. Amino acids are most commonly produced industrially by Corynebacterium glutamicum and Escherichia coli.

Fig 1. Amino acids production focusing on fermentation technologies. (D'Este, M.; et al. 2018)

What is the amino acid fermentation?

The fermentation process is the foundation of the majority of the commercial techniques used today to produce amino acids. Numerous microorganisms are employed in both aerobic and anaerobic environments to transform the sugars found in a substrate into a wide range of amino acids. When compared to other approaches (such extraction from protein-hydrolysates and chemical synthesis), this procedure has a number of benefits. It firstly just generates the l-form amino acids, skipping any additional purifying processes. The fact that it may be used in moderate environments to avoid product deterioration is another crucial feature. In addition, maintenance expenses are a great deal less than those associated with extraction procedures. On the other hand, fermentation impacts capital and operating expenses since it needs sterility, significant energy consumption for mixing and oxygen transfer (for aerobic fermentations), as well as water addition. Furthermore, compared to alternative techniques of producing amino acids, the demand for larger reactors results in a higher capital expenditure.

Comparison among different amino acid production methods. (D'Este M., et al., 2018)

Microorganisms are used for amino acid fermentation

Both C. glutamicum and E. coli are the most commonly used bacteria for fermentation-based amino acid production. They are able to generate a wide range of amino acids, and multiple metabolic engineering modifications have been made to enhance their effectiveness as amino acid generating organisms. C. glutamicum that has been genetically modified is employed to generate lysine or glutamic acid with high yields (up to 50% w w−1), while E. coli has been modified to allow for the production of aromatic amino acids like L-tryptophan, L-phenylalanine, and L-tyrosine.

(1) Corynebacterium glutamicum: As the primary bacterium responsible for manufacturing amino acids, C. glutamicum is an aerobic, non-pathogenic Gram positive soil bacterium that is extensively employed in the amino acid production sector. L-glutamate, L-lysine, L-phenylalanine, L-threonine, L-tryptophan, L-serine, L-proline, L-glutamine, L-arginine, and L-isoleucine are among the amino acids that are produced by C. glutamicum. It uses glucose as its preferred carbon source, but it can additionally utilize other sugars such maltose, sucrose, fructose, ribose, and mannose. 30 °C and a pH of seven are the ideal growing conditions for it. To find out if growth inhibition by substrate and product happens, inhibition experiments have been conducted. It has been shown that growth declines at concentrations of l-glutamic acid of 12 g/L and glucose more than 50 g/L.

(2) Escherichia coli: Aerobic Gram-negative bacteria, E. coli is frequently found in plants and is a typical component of the gut flora in animals. This well-known microbe is used to make a variety of amino acids, including the aromatic amino acids l-phenylalanine, l-tyrosine, and l-tryptophan, as well as the amino acids l-methionine, l-lysine, and l-threonine. Furthermore, site-specific mutagenesis, transcriptional attenuation regulations, and pathway modification through gene depletion allow metabolic engineering techniques to create a mutant strain of Escherichia coli that produces the branched chain amino acids l-valine, l-leucine, and l-isoleucine. These amino acids are highly intriguing due to their potential applications in feed additives, cosmetics, and pharmaceuticals. The primary substrates that E. Coli can metabolize are galactose, fructose, xylose, sucrose, mannose, and glucose. At 37 °C and a pH of 7, the ideal growing conditions are reached.

Engineering strategies for enhancing L-valine production in model organisms. (Gao H., et al., 2021)

Advantages of Fermentation for Amino Acids Production

Due to economic and environmental advantages, fermentation is currently the most used amino acid production technology at industrial scale. The fermentation process has several advantages over other methods:

It can avoid further purification steps
It can be operated under mild conditions, preventing product degradation
Compared to the extraction processes, the maintenance costs are significantly lower
Fermentation technologies can maximize the yield, specificity and production of target amino acid compounds

Fig 2. Biosynthesis of glutamic acid from glucose. (Sanchez, S.; et al. 2017)

The Fermentation Process

The Selection of Amino Acid Producing Bacteria

The most commonly used bacteria for amino acid production by fermentation are C. glutamicum and E. coli. Both are capable of producing a wide range of amino acids and several metabolic engineering changes have been applied to improve their performance as amino acid producing organisms. Modified C. glutamicum has been used to produce lysine or glutamic acid with high yields, while E. coli has been modified to produce various aromatic amino acids such as L-tryptophan, L-phenylalanine and L-tyrosine.

Fermentation Process Design in Amino Acid Production

Process monitoring

During the fermentation, continuous monitoring of key parameters and process variables such as inoculum mass, pH, feed rate, aeration intensity, and process temperature is required. The inoculum preparation is a critical step in the biological process, as it significantly affects productivity and yield. Therefore, to ensure the optimal inoculation, the stability and yield of the inoculum should be thoroughly tested prior to transfer. To avoid contamination, sterility must be maintained throughout the process. Continuous sterilization systems have been integrated into the fermentor configuration to ensure asepsis during all the stages of the process.

Fed-batch production

In the fed-batch production process, cells and products are left in the reactor in order to obtain a higher yield or productivity. The nutrients required to carry out the fermentation, such as ammonium sulfate or pure ammonia, biotin, pure ammonia, and other vitamins, are provided at the beginning along with the inoculum.

Continuous production

Compared to fed-batch technology, this operating mode can yield productivity and process outputs that are 2.5 times greater. The primary disadvantages of this configuration procedure, however, are the potential strain instabilities brought on by the frequent changes in operating conditions and the elevated danger of contamination resulting from the continual flows into and out of the reactor. Previous research has shown the continuous processes' potential. Specifically, the continuous setup of Brevibacterium lactofermentum resulted in twice as much l-glutamic acid productivity as the batch procedure, reaching 8 g/L/h. Furthermore, a cascade bioprocess may be used to enhance the continuous configuration's performance. This technology allows for the growing phase of the microorganisms to be carried out in a different reactor from the manufacturing process itself, providing optimal conditions for both stages. In addition, faster growth rates provide shorter residence times, which lead to smaller bioreactors and increased productivities.

Downstream Separation and Purification

Effective downstream and purification processes are essential to reduce the costs related to the amino acid production. The separation of amino acids from fermentation broth is usually accomplished by centrifugation or filtration, followed by purification using chromatographic techniques of choice depending on product characteristics such as solubility, purity, etc.

Fermentation Process Modelling and Analysis Technology

Scale down method

Scale down devices are gaining increasing attention as a tool to mimic the conditions of large-scale bioreactors. In order to have an appropriate predictive model for the performance of large-scale reactors, the scaled down design has to be simulated the reaction conditions of industrial processes

Computational Fluid Dynamics (CFD)

Process modeling can support the design and the optimization of fermentation processes. CFD, considering the process operating parameters, process stoichiometry, and the environmental aspects, enables to estimate the process efficiency, the product titer, selectivity, and optimum yield.

Process intensification

One key approach for increasing the productivity of an amino acid fermentation process is process intensification (PI). PI is described as any technology advancement that results in a safer, cleaner, and more energy-efficient process. In this sector, two approaches can be taken: intensifying the equipment, such as new reactors, heat exchangers, or mass transfer units, or intensifying the process itself, utilizing novel separation tactics or procedures. For example, switching the reactor configuration (operating mode) to repeated batch or fed-batch reactors can result in double the lysine productivity of the batch. According to this setup, after the first batch or fed-batch operation, between 60% and 90% of the finished broth is sent to the downstream, while the rest remains in the vessel. The reactor is subsequently replenished with new medium to its initial working capacity, and the next fermentation cycle begins on schedule. By reducing the time required to introduce new inoculum into the system as well as the downtime required to prepare the sterile bioreactor, fermentation time may be reduced, productivity can be increased, and process economics can be significantly improved.

Our Services

BOC Sciences provides fermentation CDMO service for amino acids. We use the fermentation method to produce amino acids. We are able to provide large scale fermentation capacity in excess of 2,000,000 liters and offer our customers fast turnaround times and a seamless manufacturing process.

Workflow of Our Service

References

D'Este, M.; et al. Amino acids production focusing on fermentation technologies - A review. Biotechnology Advances.2018. 36(1): 14-25.
Sanchez, S.; et al. Our microbes not only produce antibiotics, they also overproduce amino acids. Journal of Antibiotics. 2017. 71: 26-36.