The impact of metabolic byproduct acetic acid on Escherichia coli fermentation and its solutions

Summarize the effects of metabolic byproduct acetic acid on Escherichia coli fermentation and propose corresponding solutions.

1、 Metabolic byproduct - acetic acid

Acetic acid is a metabolic byproduct during the fermentation process of Escherichia coli. There are different opinions on the concentration at which it produces inhibitory effects. It is generally believed that under aerobic conditions, a concentration of 5-10 g/L of acetic acid can have observable inhibitory effects on lag period, maximum specific growth rate, bacterial concentration, and final protein yield. When the concentration of acetic acid is greater than 10 or 20g/L, cells will stop growing, and when the concentration of acetic acid in the culture medium is greater than 12g/L, the expression of exogenous proteins is completely inhibited.

Measures to prevent the production of acetic acid:

1. Reduce the production of acetic acid by controlling the specific growth rate:

The higher the specific growth rate, the more acetic acid is produced. When the specific growth rate exceeds a certain value, acetic acid begins to be produced. The specific growth rate can be reduced by lowering the temperature, adjusting the pH, controlling the feeding, and other methods.

2. Dialysis cultivation:

During the cultivation process of Escherichia coli, dialysis technology can be used to remove harmful substances from the fermentation broth, reduce the acetic acid content, and achieve high-density fermentation and product expression of recombinant bacteria.

3. Control the concentration of glucose:

Glucose is one of the important carbon sources in the fermentation process of Escherichia coli, and its use as a carbon source should be controlled at a lower level to reduce the production of acetic acid.

The commonly used control methods include:

Constant pH method: Escherichia coli metabolizes grapes and other substances to produce acetic acid, causing a decrease in pH value. Therefore, the pH value can be used as an indicator to control glucose. The disadvantage of this method is that the change in pH is not entirely the result of glucose metabolism, which can easily cause errors in the feeding system.

Constant dissolved oxygen method: During bacterial metabolism, oxygen is consumed, causing a decrease in dissolved oxygen. When the glucose concentration drops to a certain level, bacterial metabolism decreases, oxygen consumption capacity decreases, and dissolved oxygen increases. Therefore, adding glucose based on the dissolved oxygen curve and maintaining a constant dissolved oxygen can control glucose at a certain level.

2、 Temperature

The optimal temperature for fermentation of Escherichia coli is 37 ° C. When the temperature is optimal for bacterial growth, the specific growth rate will increase. As the temperature increases, bacterial metabolism accelerates and the production of metabolic byproducts also increases. These by-products will have a certain inhibitory effect on the growth of bacterial cells. Rapid growth of bacterial cells can also affect the stability of plasmids. Lowering the cultivation temperature will decrease the uptake and growth rate of nutrients by the bacterial cells. At the same time, it also reduces the production of toxic metabolic byproducts and metabolic heat. Sometimes lowering the temperature is more beneficial for the correct folding and expression of the target protein. In the fermentation of recombinant Escherichia coli, the optimal temperature varies at different stages. In order to obtain a large amount of the target protein, the amount of bacterial cells must be ensured first. Therefore, in the early stage, the growth of bacterial cells can be prioritized, and in the induction stage, the expression of the target product should be prioritized.

3、 Cultivation method

The cultivation methods of microorganisms mainly include batch, continuous, and fed batch. The fermentation of Escherichia coli mostly adopts fed batch cultivation, which is an optimized method in modern fermentation technology and can effectively optimize the chemical environment during the microbial cultivation process. Ensure that microorganisms are in the optimal growth environment. On the one hand, this approach can avoid substrate inhibition caused by excessive initial concentrations of certain nutrients, and on the other hand, it can prevent restrictive nutrients from being depleted and affecting cell growth and product formation. Feeding batch cultivation has been widely used in the fermentation production of various primary and secondary biological products and proteins.

The two main goals pursued by biotechnology researchers are the development of new biological products and the search for more economical production methods for traditional or emerging biological products. In the past decade, the use of genetic engineering technology to produce some important biopharmaceuticals has been a rapidly developing direction in the field of biotechnology. In this research field, how to create more economical and effective methods to improve the economic efficiency of the production process and the market competitiveness of products has become a focal point of concern for scientists in the biotechnology field.

The use of recombinant DNA technology to produce important biopharmaceuticals has epoch-making significance in the history of human civilization. Due to the direct impact of production costs and productivity on the survival of companies, optimizing the production process of recombinant biopharmaceuticals has become an important issue. It includes the following six aspects: (1) selection of suitable hosts; (2) Determination of recombinant protein accumulation sites (such as soluble intracellular accumulation, intracellular aggregation accumulation, periplasmic accumulation, or extracellular accumulation); (3) Molecular strategy for maximizing the expression of recombinant genes; (4) Optimization of cell growth and production environment; (5) Optimization of fermentation conditions; Optimization of post-processing procedures. Only when all six aspects are aimed at achieving high productivity can the optimization of the entire production process be achieved.

（1） Optimization strategies for cell growth environment

To improve cell density and productivity, it is first necessary to optimize the physical and chemical environment for microbial growth, including the composition of the growth medium, physical parameters (pH, temperature, and stirring), and product induction conditions. The purpose of optimizing these parameters is to ensure that cell growth is under optimal environmental conditions, avoiding excessive or insufficient nutrients, preventing product degradation, and reducing the formation of toxic products.

1. Optimization of the composition of the culture medium

The culture medium usually contains carbon (energy) sources, nitrogen sources, as well as micronutrients such as vitamins and trace elements. The concentration and proportion of these nutrients are important for achieving high-density fermentation of recombinant microorganisms. For example, excessive Fe2+and CaCO3 combined with relatively low concentrations of phosphate can promote the production of L-malic acid by Aspergillus flavus; Streptomyces can increase its serine protease production capacity by up to 10 times in the presence of 60-80 mmol/L CO32-; After achieving high cell density in recombinant microorganisms, limiting phosphate concentration can significantly increase the yield of antibiotics and heterologous interleukin b. In addition, it was found that although limiting the concentration of arginine can inhibit cell growth, the production of recombinant alpha amylase can be doubled compared to the excellent cell growth when arginine is sufficient.

The type of compound nitrogen source in the culture medium is also crucial for high-density fermentation of recombinant Escherichia coli. Generally, when yeast extract is present in the culture medium, the recombinant protein is unstable; When the feed medium contains peptone, Escherichia coli cannot reuse the acetic acid it produces. Adding yeast extract and peptone to the culture medium not only produces highly stable recombinant proteins, but also allows cells to reuse the synthesized acetic acid, which is a very interesting metabolic mechanism.

Henghua technology can be used to optimize the growth medium of arginine deficient Escherichia coli X90. The strain was grown at a specific growth rate of 0.4 h-1 on a basic culture medium containing arginine. After reaching a stable state, amino acids, vitamins, and trace elements were added to a homogenizer to investigate their effects on bacterial growth and arginine synthesis. The results showed that due to the inhibitory effect of end products in the amino acid biosynthesis pathway, the addition of certain amino acids actually inhibited cell growth. After adding NH4Cl, there was a dramatic increase in cell count. Adding vitamins has almost no effect on the growth of bacterial cells. By calculating the yield of biomass on each substrate, the composition of high-density fermentation medium can be determined. On this optimized medium, the cell density of Escherichia coli X90 can reach 92 g/L, while forming 56 mg/L of extracellular recombinant protease.

2. Addition of special nutrients

In some cases, adding some nutrients to the culture medium can improve productivity. The function of these nutrients may be as precursors to the product, or they may prevent the degradation of the product. For example, when cultivating recombinant E. coli to produce chloramphenicol acetyltransferase (a protein composed of many aromatic amino acids), adding phenylalanine can increase the enzyme's specific activity by about 2 times; Adding 60 g/L glucose and 100 mmol/L potassium phosphate to the culture medium for producing b-lactase from recombinant Bacillus subtilis can significantly improve the stability of the recombinant protein. The reason may be due to the inhibition of the activity of various extracellular proteases produced by host cells, thereby preventing the degradation of recombinant proteins.

Adding special substances to growth media can sometimes improve productivity through an unknown mechanism. For example, adding sodium iodide during shake flask cultivation of Micromonospora cbersina can increase the yield of dynemicin A by 35 times, but this result cannot be replicated in small reactors.

3. Limit the accumulation of metabolic byproducts

The control of cultivation conditions has a significant impact on the formation of metabolic byproducts. In batch or fed batch cultures, excessive concentrations of certain nutrients can lead to the Crabtree effect. Under this effect, brewing yeast produces ethanol, while Escherichia coli produces excess acetic acid. Once acetic acid is produced, cell growth and the production of recombinant proteins are inhibited. The rate at which Escherichia coli forms acetic acid depends on the growth rate of the cells and the composition of the culture medium. It has been confirmed that adding complex nutrients (such as soybean hydrolysate) to the culture medium will increase the accumulation of acetic acid. Many researchers have conducted extensive work on how to alleviate the negative effects caused by the accumulation of acetic acid, such as using circular fermentation technology to limit the accumulation of acetic acid in high-density cultivation of recombinant Escherichia coli. Recent studies have also shown that adding certain amino acids can alleviate the inhibitory effect of acetic acid. Adding 10 mg/L glycine to the culture medium can significantly promote the synthesis of recombinant alpha amylase and beta lactase in Escherichia coli, and stimulate the release of enzymes from the periplasm into the culture medium, but acetic acid is still generated at this time.

（2） Training mode

Due to the inhibitory effect of many nutrients on cells at high concentrations, and the need to supply a large amount of nutrients to achieve high cell density, concentrated nutrients must be added to the reactor at a rate proportional to their consumption rate. Various forms of feeding strategies have emerged for this, ranging from simple linear feeding to complex strategies calculated using mathematical models to control feeding rates. Specifically, the selection of cultivation mode mainly depends on the following three factors: (1) the specific metabolic behavior of the cultured cells; (2) The potential of utilizing inhibitory substrates to synthesize the target product; (3) The ability to induce conditions and measure various parameters of cell culture.

1. Escherichia coli flow fermentation strategy

Escherichia coli is the strain with the clearest genetic background to date and is widely used in genetic engineering research. The most critical issue in high-density cultivation of Escherichia coli is how to minimize the production of acetic acid, as high concentrations of glucose or high growth rates can severely inhibit cell growth and the production of recombinant proteins. Research has found that even at glucose concentrations of only 0.25-0.5 g/L, Escherichia coli still produces acetic acid. Therefore, the flow addition strategy used in high-density fermentation must be formulated according to a certain algorithm to maintain the substrate concentration in the reactor at a low level. Nutrients are best added to the reactor at their consumption rate, which not only prevents substrate accumulation to toxic levels, but also prevents cells from being in a state of starvation.

In recent years, various methods have been reported to control the flow acceleration rate in Escherichia coli culture, most of which indirectly couple the flow acceleration rate with a physical parameter such as dissolved oxygen, pH, or CO2 release rate. Scholars have controlled dissolved oxygen at a predetermined value to ensure a lower growth rate, resulting in minimal production of acetic acid. The final cell dry weight reached 110 g/L, and it was found that a lower specific growth rate is beneficial for the high expression of recombinant proteins. In another high cell density culture that controls low growth rate, researchers used an exponential flow addition of glucose, ammonium salts, and inorganic salts, followed by a generalized linear flow addition culture strategy, effectively preventing the accumulation of acetic acid. The cell density of recombinant E. coli reached 66 g/L, and 19.2 g/L of active recombinant protein could be formed inside the cell through temperature induction.

If the glucose concentration is controlled at a sufficiently low level that is not toxic, it can also enable cells to rapidly grow to high cell density in the absence of restrictive matrix. This control strategy has high requirements for the instrument. Kleman et al. used an online glucose analyzer to determine the flow rate of glucose and other nutrients based on the microbial demand for glucose. This algorithm can automatically adjust the flow rate during the product induction stage according to changes in cell growth. Cultivate E. coli MV1190 carrying a plasmid containing a gene encoding 1,5-diphosphate ribulose carboxylase. The final cell dry weight reached 39 g/L, producing 1.7 g/L soluble active protein.

2. Flow addition fermentation of recombinant yeast

The yeast strain widely used in genetic engineering research is brewing yeast. However, using brewing yeast as a recombinant host also has the following drawbacks: (1) the level of recombinant protein production is relatively low; (2) Plasmid instability; (3) Generate ethanol. Among them, the generation of ethanol is the least desirable for researchers as it inhibits the formation of recombinant proteins. Recent studies have shown that other yeasts, such as Pichia pastoris, also have the potential to serve as recombinant hosts. Clare et al. compared the ability of recombinant Pasteur Pichia pastoris and brewing yeast to express and secrete mouse epidermal growth factor at high cell densities. Cultivate Pasteur Pichia pastoris with 19 copy numbers per genome, and ultimately obtain 447 mg/L of intracellular recombinant protein; The highest level obtained from cultivating brewing yeast is only 6-7 mg/L.

By first using exponential flow addition and then adopting a linear flow addition control method based on CO2 release and RQ value, the dry weight of recombinant Pichia pastoris cells can reach 80-90 g/L and secrete high levels of recombinant human serum protein. However, when cultivating brewing yeast, the dry weight of cells and the yield of recombinant protein were only 25 g/L and 20 mg/L, respectively. Even if the growth rate of brewing yeast is maintained at 0.12-0.18 h-1, it will still form 10-13 g/L ethanol, resulting in a decrease in yield. But the production of ethanol by brewing yeast is not uncontrollable. Shimizu et al. used a complex flow addition system to control the growth rate of yeast at 0.3 h-1, which maximizes the production of glutathione (GSH) and minimizes the generation of ethanol.

3. Control of flow cultivation

A good flow control system must avoid two tendencies: one is excessive flow control, where feed components accumulate in the reactor and inhibit cell growth and product formation; The second is insufficient flow, which may lead to a lack of essential nutrients for cells. The rapid development of computer technology has provided more effective means for the control of flow training. In recent years, there have been numerous reports on the application of computer technology to monitor and control fermentation processes. With the help of modern computer technology, people are able to use various growth parameters and mathematical models to control the addition of nutrients in flow addition culture, thus enabling complex control systems to be implemented. Among various artificial intelligence technologies, fuzzy reasoning is the most widely used. Fuzzy logic control partially relies on mathematical growth models and also uses a "linguistically defined rules system" to help the system respond to the nonlinear and dynamic behavior of the fermentation process. Alfafara et al. used a fuzzy logic control system to control the acceleration of glucose flow in their study on the production of glutathione by flow addition cultivation of brewing yeast. After optimizing the system, the specific rate of glutathione production reached 6.2 h-1. At present, the biggest problem in applying fuzzy logic control technology in flow addition culture is how to reduce the number of adjustments required for substrate and product concentration oscillations. The development of adaptive fuzzy logic control algorithms is expected to be helpful in this regard.

（3） Induction strategy

For many recombinant microorganisms with inducible promoters, maximum productivity can only be achieved by separating the growth phase and product formation phase. In flow addition culture, the separation of these two periods can be achieved by delaying induction until cell growth has reached high density. In addition, if the plasmid is stable and the product is non-toxic to the culture, a repeated feeding batch culture system can be used to improve productivity. Scholars have used the technique of repeated feeding and batch cultivation to cultivate brewing yeast. By changing 50% of the culture medium every 24 hours for 30 days, the yield of hirudin can be increased by three times compared to continuous cultivation systems.

If both inducers and products are toxic to cells, the induction and growth phases should be artificially separated. For this situation, two-stage continuous cultivation is the most suitable cultivation method. Control the conditions of the first tank to ensure optimal cell growth, while induction and product formation occur in the second tank. For example, cultivating a recombinant Escherichia coli strain capable of producing beta lactase in a homogenizer, and introducing the fermentation broth from the first tank into the second tank, forms a two-stage cultivation system. Add nutrients and IPTG as inducers to the second tank. As a result, 300 mg of active beta lactase (equivalent to 25% of total protein) was obtained, of which 90% was secreted extracellular. This system can run stably for at least 50 days. Another similar system was used to cultivate E. coli for producing recombinant protein A-EcoRI protein fusion. The cultivation was carried out in a constant turbidity apparatus, and the second tank was thermally induced, resulting in a specific productivity six times higher than that of batch fermentation. Researchers also attempted to combine a two-stage continuous culture system for producing recombinant proteins with affinity chromatography columns, in an attempt to achieve continuous production and purification of recombinant proteins. However, due to some technical reasons, this combination has not yet been successful.

The growth rate plays an important role in both cell growth and product formation. A common situation is that the optimal growth rate for cell growth is not suitable for the formation of products or the realization of other characteristics. We found that when cultivating bread yeast, the cell yield was highest at a specific growth rate of 0.2 h-1, while the yeast fermentation activity was optimal at a specific growth rate of 0.178 h-1. We proposed a two-stage flow addition culture strategy to control the specific growth rate in response to this phenomenon, and achieved a unity of high fermentation activity and high cell yield in one reactor. （4） Cell cycle fermentation is considered from the perspective of reactors to achieve high cell density, and cell cycle bioreactors are commonly used. This reactor uses a tangential flow or hollow fiber filter to separate cells from the mash, and the cells return to the container. The cell-free mash is continuously transferred at a given rate, while fresh culture medium is used at the same time. By utilizing cell cycle technology, cells can be retained in the reactor and achieve high cell density, while toxic waste products and extracellular products are continuously transferred, which can delay or prevent feedback inhibition caused by cell growth or product formation. Cell cycle bioreactors can be applied to various organisms and production systems, but their application also has many limitations, mainly including: (1) excessive shear stress acting on cells entering the filtration unit; (2) There are many practical difficulties in amplifying the system.

When operating a cell cycle bioreactor, two factors must be considered: dilution rate (flow rate/volume); The second is the circulation rate (referring to the rate of medium passing through the filtration system). The size of dilution rate affects the growth rate of cells, and different experimental purposes have different requirements for dilution rate; A high circulation rate can ensure uniform mixing of components, especially suitable for situations where cells are prone to aggregation or clumping. However, a high circulation rate can result in excessive shear forces acting on the cells and rapid damage to the filtration unit membrane. Therefore, it is difficult to simultaneously determine the appropriate dilution rate and circulation rate, which is also an important factor limiting the application of cell cycle technology.

Cell cycle technology is expected to achieve high volumetric productivity, which is very advantageous for product extraction. In recent years, circular fermentation technology has been widely used to produce cellular metabolites such as fuel alcohol, organic acids (such as butyric acid), and 2,3-butanediol. Lee and Chang used cell cycle fermentation technology to achieve a dry weight of 145 g/L for recombinant Escherichia coli cells, and their recombinant penicillin acylase productivity was nearly 10 times higher than batch culture. For the cultivation of live cells, which are the desired products, cell cycle fermentation can also play a role. In the food industry, different lactobacilli need to be cultured for the production of milk, cheese, and yogurt. Using a cell cycle bioreactor can easily increase the density of these organisms.

With the help of various control measures, people can now easily achieve cell densities exceeding 100 g/L. However, existing research results indicate that growth conditions corresponding to optimal biomass formation typically result in lower specific productivity. For example, using a cell cycle reactor to produce 2,3-butanediol increased biomass by approximately 6 times, but the volumetric productivity only increased by 2-3 times. Similarly, flow addition culture can achieve a cell dry weight of 43 g/L for Streptomyces, but with zero protease activity. However, when the cell dry weight is 18 g/L, the protease activity can reach as high as 3500 U/mL. We often encounter similar problems in our research. To solve this problem, on the one hand, it is necessary to study how to promote the efficient expression of recombinant proteins and improve the stability of recombinant strains, and on the other hand, to investigate the formation conditions of high-level products associated with high cell density.