Influence and control of pH during fermentation process

pH effects in fermentation process

Near pH is a very important parameter for microbial growth and product synthesis, and a comprehensive index of metabolic activity for fermentation.

The process is of great importance. Different species of microorganisms have different pH requirements. The optimum pH is 6.5~7.5 for most bacteria, 4.0~5.8 for mold, 3.8~6.0 for yeast, and 6.5~8.0 for actinomycetes. The range of pH adaptation depends on the ecology of the microorganism. If the pH circumference is not suitable, the growth and product synthesis of the microorganism will be inhibited. Moreover, it is also an effective measure to prevent the pollution of miscellaneous bacteria to control one of the main conditions to ensure the constant growth of micro-matter. For example, Cerevisiae petroleum grows well in the pH range of 3.5~5.0 and is not easy to infect bacteria. When the pH is higher than 50, the yeast morphology becomes smaller, the fermentation broth becomes black and pollutes a large number of bacteria. When pH was lower than 30, maternal growth was inhibited, cells were very irregular, and autolysis occurred. Bacillus thuringiensis has high pH tolerance and can grow well in a wide pH range. There was no significant difference in the number of P bacteria in the range of 6.0~10.0. A large number of bacteria grow after sterilization, but it is not conducive to the formation of crystals in the later stage. If pH>7.5, the germination period of buds will be prolonged, so the pH after sterilization should be around 6.8.

For the same microorganism, different fermentation products may be formed due to the different pH of the growth environment. For example, Aspergillus Niger produces acid at pH 2-3 and oxalic acid at pH near neutral. Yeast produces alcohol at pH 4.5 to 5.0, but at pH8, the fermentation products include not only alcohol, but also acetic acid and glycerol. For example, the glucose dehydrogenase conjugated to pyrrolidoquinine quinone (PQQ) in aerobacteria is strongly influenced by the pH of the culture medium. In potassium restricted cultures, no gluconic acid is produced at pH 80, but the most gluconic acid and 2-ketogluconic acid are produced at pH 5.0 to 5.5. When the pH of recombinant human serum protein is below 50, the protease activity increases rapidly, which is very adverse to albumin production. When the pH is above 56, the protease activity is very low, which can avoid the loss of albumin.

The optimum pH for microbial growth and product synthesis may not always be the same (but they are consistent, for example, the optimum pH for hyaluronic acid-producing bacteria is both 7.0). For example, the optimal pH for growth of acetone butanol bacteria was 5.5~7.0, while the optimal pH for fermentation was 4.3~5.3. The optimum pH for penicillin-producing bacteria was 6.5~7.2, while the optimum pH for penicillin-producing bacteria was 6.2~6.8. This is not only related to the strain characteristics, but also related to the chemical properties of the products. The optimal pH for biosynthesis of various antibiotics was as follows: streptomycin and erythromycin were 6.8~7.3; The values of aureomycin and tetracycline were 5.9~6.3. Penicillin is 6.2 to 6.8. Therefore, depending on the characteristics of different microorganisms and products, it is very important to control the pH during fermentation.

The main reasons why the change of fermentation liquid pH affects the growth and reproduction of microorganisms and the formation of metabolites are as follows:

i. Change the charge of plasma membrane of microbial cells

Plasma membrane with colloid properties, in a certain pH value can be positive, and is negatively charged when another pH, change in charge at the same time, will cause the change of the plasma membrane for some ion permeability, which affect the absorption of nutrients in culture media and metabolites leak, so as to affect the metabolism.

ii. Directly affects the enzyme activity

Since the enzyme has its most suitable pH, the activity of some enzymes in microbial cells will be inhibited at the unsuitable P, and the growth, reproduction and metabolism of microorganisms will also be affected.

iii. It directly affects metabolic processes

Fermentation broth P important nutrients and intermediate metabolites dissociate, thereby affecting the microbial use of these substances. Most of the substances that constitute microorganisms dissociate in water while maintaining a certain balance. The dissociation of water is related to hydrogen ions, so the concentration of hydrogen ions has a great impact on the dissociation of these substances, thus affecting the nutrient absorption, enzyme activity, decomposition and anabolism of microorganisms. Therefore, the change of pH will lead to the change of the metabolic process of microorganisms, so that the mass and proportion of metabolites are changed. In aerogenic bacilli, glucose dehydrogenase in the culture medium pH, potassium nutrient medium, H gluconic acid H0~55 to produce glucoic acid and 2-keto glucoic acid. In addition, in sulfur or ammonia nutrient restricted medium, the bacteria produced glucotaric acid and 2-ketoglucotaric acid at H5 but not at pH68.

Factors affecting pH change during fermentation

In the fermentation process, pH is often dynamic change. This change in pH depends on the species of microorganism and the composition of the basal medium and fermentation conditions. In the process of thallus metabolism, it has the power to create its optimal growth pH, but the pH will fluctuate continuously when the external conditions change greatly. On the one hand, microorganisms secrete organic acids such as lactic acid, acetic acid, citric acid, etc., or some alkaline substances through metabolic activities, thus leading to the pH change of fermentation environment. On the other hand, microorganisms cause the pH change of fermentation liquid by using acidic substances or alkaline substances in fermentation medium.

The nitrogen source of basal medium has a great influence on the pH of fermentation broth. If ammonia is used as nitrogen source, NH; It exists in the form of NH in solution, and after it is used as R-NH, H+ is generated in the medium, so that the pH decreases; If NO is used as a nitrogen source, H+ is consumed, NO is reduced to R-NH, and pH rises; If amino acids are used as nitrogen sources, H+ is produced after amino acids are utilized, and pH decreases. The pH in the culture medium sometimes fluctuates. For example, protein in the medium, other nitrogenous organic matter or urea in glutamic acid fermentation is hydrolyzed by urine enzymes to release ammonia, the pH can rise rapidly, when ammonia is used by the bacteria, the pH will fall again. In the process of fermentation, adding nitrogen source will also appear the situation of pH fluctuation, such as adding ammonia, the pH of fermentation liquid first rose rapidly, when ammonia began to be used, the pH gradually decreased. In addition, too much sugar or oil is added at a time in fermentation, and incomplete oxidation will cause the accumulation of organic acids, resulting in a drop in pH. In fact, the measured pH change in the fermentation broth is the result of a combination of various reactions.

Control of pH during fermentation

The pH of the fermentation fluid is constantly changing during fermentation because microorganisms are constantly absorbing and assimilating nutrients and excreting metabolites. This is not only related to the composition of the medium, but also related to the physiological characteristics of the microorganism. Each microorganism has its own optimum pH for growth and fermentation. For example, rifamycin production, because all carbon units in rifamycin B molecule are derived from glucose, the utilization of glucose in the growth phase has a certain impact on rifamycin B production. It is proved that its optimum pH is in the range of 7.0~7.5.

When the pH is 7.0, the average yield coefficient reaches the maximum value. Among the various parameters of rifamycin B fermentation, the average yield coefficient is the most important from the economic point of view. Therefore, pH70 is the best condition for rifamycin B production. Under these conditions, the consumption of glucose is mainly used for product synthesis, and the appropriate amount of bacteria can also be ensured. The results showed that the yield of rifamycin B was 14% higher than that of the whole fermentation process when the pH of growth stage and production stage were maintained at 65 and 7.0, respectively. In Acetobacter cellulose fermentation, the pH at the early stage of batch culture is 4.0, and the pH at the production stage is adjusted to 55, which can greatly increase the cellulose yield and shorten the fermentation time. Therefore, according to the characteristics of microorganisms, in order to make microorganisms propagate in the optimal way, it is necessary not only to control the appropriate P in the original medium, but also to check the change of H at any time during the whole hair process and to carry out corresponding regulation.

In actual production, if the control condition is original) and the buffer capacity is not strong enough, attention must be paid to the effect of sterilization on P). If the ratio of energy class and carbon source is balanced, it is not necessary to add buffer. Can also be used in the fermentation process to add weak acid and weak base adjustment by acid or just outside adjustment can improve fermentation condition, to the material is a better way, can adjust the pH of the culture medium, and can supplement nutrition, increase the concentration of the medium, decreases the work of repression, so as to further improve the yield of fermented product, amino acid fermentation, the original medium, Generally adjust pH70 or so. In the inclined plane culture, seed culture and fermentation of the long bacteria stage, because the product is very few, pH change is not very big, generally do not adjust in the stage, because the nitrogen source is consumed and the accumulation of amino acids, pH change is larger, it must be regulated and controlled. For example, in the process of glutamate fermentation, the utilization rate of different fine pairs of P in different periods is high, and the change of pH fluctuates greatly. If the pH is low in the early stage of fermentation, the thalli grow vigorously, consume nutrients quickly, transfer to normal metabolism, and grow thalli without producing glutamate. When the pH is high, the growth of thallus is unfavorable, the glucose metabolism is slow, and the fermentation time is prolonged. However, in the early stage of fermentation, if the pH is slightly higher (pH 7.5~80), it is beneficial to inhibit the growth of miscellaneous bacteria. Therefore, the pH should be controlled at about 7.5 in the early stage of fermentation and 7 in the middle and late stage of fermentation. The reason is that the optimum pH of glutamate dehydrogenase is 7.0~7.2, and that of amino acid transferase is 7.2~7.4. In the fermentation of transglutaminase, a higher pH(7.0) in the early stage can shorten the delay period of cell growth, which is beneficial to the synthesis of the enzyme. In the middle and late stage of fermentation, a lower pH can further promote the growth of cells and maintain a higher product synthesis rate.

After determining the appropriate pH for a certain fermentation process, various methods are used to control it. Firstly, it is necessary to consider and test the basic formula of fermentation medium to ensure that the pH change in the fermentation process is within the appropriate range. Because the medium contains substances metabolizing acid production (such as glucose, (NH)SO) and alkali production (such as urea, NaNO) as well as buffering agents (such as CaCO;) And other components in the fermentation process to affect the change of pH, especially CaCO, can react with keto acid, and play a buffer role. In batch fermentation, this method is commonly used to control the change of pH.

In the process of fermentation, if the above method can not reach the control target, the following method can be used to adjust the pH:

i. Adding calcium carbonate method

When physiological alkaline ammonium salt is used as a nitrogen source, the pH of the liquor will drop due to the remaining acid root after NH is used by the bacteria. Calcium carbonate can be added to the culture medium to adjust the pH. In the fermentation of lactic acid, calcium carbonate is often used to adjust the pH to prevent the decrease of lactic acid production caused by the decrease of pH.

ii. Ammonia water flow addition

In the process of fermentation, ammonia is added to adjust the pH according to the change of pH, and as a nitrogen source, to supply NH. Ammonia is cheap and easily sourced. However, the effect of ammonia water is fast, which has a great influence on the pH fluctuation of fermentation broth. A small amount of multiple flow addition should be used to avoid P inhibiting growth, and can also prevent the appearance of insufficient H. The specific flow adding method should be determined according to the characteristics of bacteria, bacteria, sugar consumption and other conditions, the general control pH 7.0~8.0 is good to use automatic system flow method. The use of ammonia in the process of fermentation to adjust P caution, the amount of ammonia will cause microbial poisoning, leading to a rapid decline in respiratory intensity. Therefore, in the fermentation process where ammonia gas is needed to adjust pH or replenish nitrogen sources, the bacteria can be prevented from ammonia overdose poisoning by monitoring the change of dissolved oxygen concentration. For example, streptomycin fermentation, using ammonia water flow addition control of pH, P suitable for the range of streptomycin synthesis, filled with the product synthesis required nitrogen source.

iii. Addition of urea flow

This method is widely used in our country at present. Using urea as nitrogen source to adjust pH, the pH change has a certain regularity, and easy to control. Firstly, due to ventilation, agitation and the action of urinary enzymes in the bacteria, the elemental fractions liberate ammonia, and the pH rises; Ammonia and medium components are used by the bacteria and form organic acids and other intermediate metabolites, which are reduced. At this time, urea needs to be added in time to adjust pH and supplement nitrogen sources. When urea is added to the flow, urea is decomposed by thallus urine enzyme, and ammonia is released. Ammonia is used to make the pH drop by combining metabolites, and repeated flow is carried out to maintain a certain pH. In addition to the change of pH, a small amount of multi-flow addition should be taken into account in the different stages of bacterial growth and sugar consumption fermentation to maintain a slightly lower pH, so as to facilitate the growth of microorganisms. When the growth of bacteria is accelerated, sugar consumption is accelerated, the amount of flow can be more appropriate, pH can be slightly higher, later fermentation is conducive to promote glutamate production, maintain pH72 or so is good. When the amount of residual sugar is very small, close to the pot, with no or less added for the best, so as not to cause waste.

iv. Control pH by feeding

The pH control is combined with metabolic regulation, and the pH is controlled by feeding control. For example, in the process of penicillin fermentation, according to the physiological and metabolic needs of producing bacteria, the pH is controlled by adjusting the sugar rate, and the penicillin yield is increased by more than 25% compared with the balanced rate of sugar and pH control.