The concentration of dissolved oxygen (DO) in fermentation liquid has important influence on the growth of microorganisms and the formation of generated products. During fermentation process, it is necessary to supply the appropriate amount of sterile air, so that strains can reproduce and accumulate the required metabolites. Generally speaking, oxygen is not easily dissolved. Under the atmospheric pressure of 25℃, the concentration of oxygen dissolved in water is only about 0.25mmol/L. The fermentation liquid in the fermentation process and the metabolites of microorganisms can significantly reduce the concentration of oxygen.
The demanded oxygen amount of different strains and different fermentation stages is different, and the DO value of fermentation liquid directly affects the activity of microbial enzyme, metabolic pathway and yield of generated product. During fermentation process, the mass transfer rate of oxygen is mainly affected by the concentration of dissolved oxygen in the fermentation liquid and the transmission resistance. It is of great significance to study the influence and control of dissolved oxygen on fermentation to improve production efficiency and quality of product.
Microorganisms can be divided into obligate aerobic microorganisms, facultative aerobic microorganisms, and obligate anaerobic microorganisms according to their oxygen requirements.
(1) Obligate aerobic microorganisms use oxygen as the final electron acceptor to obtain energy through aerobic respiration, such as mold; When such microbial fermentation is conducted, dissolved oxygen (DO) should generally be increased as much as possible to promote microbial growth and increase the bacterial volume.
(2) The growth of facultative aerobic microorganisms does not necessarily require oxygen, but if oxygen is supplied during culture process, the growth of bacteria is better, such as saccharomycetes; Typical fermentation,such as ethanol fermentation, the control of dissolved oxygen DO is divided into two stages.DO value is increased for cell expansion culture during initial stage, and DO value is strictly controlled for anaerobic fermentation during late stage.
(3) (3) Anaerobic and micro-aerobic microorganisms can tolerate oxygen in the environment, but their growth do not require oxygen, and these microorganisms are rarely used in fermentation production. However, for obligate anaerobic microorganisms, oxygen can be toxic to them, such as methanogens, and whether DO can be limited to a low value often becomes the key to the success of fermentation.
Under normal circumstances, the physical parameters that have a greater impact on the concentration of dissolved oxygen in the fermentation liquid include ventilation volume of the fermentation tank, stirring speed, temperature & pressure of the fermentation tank, etc.
2.1. Ventilation volume of fermentation tank
The influence on ventilation volume of fermentation tank has the most immediate effect. The most important factor that affects the concentration of dissolved oxygen is the amount of sterile air (or pure oxygen)which entering the fermentation tank. Because it enters into the logarithmic growth period along with the rapid growth of microorganisms during the fermentation process, the oxygen consumption volume of strains increased significantly, then if the intake of air supply is terminated, the oxygen in the fermentation liquid will be quickly exhausted by microorganisms. In industrial production, valves are usually installed on the intake pipe to adjust the ventilation volume.
2.2. Stirring speed
For the dissolved oxygen electrode itself, it will consume a lot of oxygen in the work. The signal of the dissolved oxygen electrode is proportional to the rate at which oxygen permeates the membrane to the surface of the electrode, and the transmission speed rate of oxygen is controlled by the transmembrane diffusion rate of oxygen. The transmembrane diffusion rate of oxygen is proportional to the concentration of oxygen dissolved in the fermentation liquid, and the ratio depends on the total mass transfer process. The transmembrane diffusion process of oxygen is the rate-limiting process of the whole mass transfer process. In the fermentation operation, the appropriate stirring operation can obtain a better transmembrane diffusion rate, so that the electrode response signal is better and the accurate reading is obtained. In addition, stirring prevents bubbles from gathering,which make the liquid form turbulence and increase the gas-liquid contact time; The air is broken into small bubbles by the high-speed shear force at the end of the stirring paddle, so as to significantly increase the effective gas-liquid transfer area to increase the concentration of dissolved oxygen in the fermentation liquid. It is important to emphasize that the fermentation tank must be stirred before the initial slope (100% dissolved oxygen) calibration of the dissolved oxygen electrode is performed.
2.3. Influence of temperature
The signal of dissolved oxygen electrode is significantly enhanced with the increase of temperature, which is mainly due to the influence of temperature on the rate of oxygen diffusion across the membrane. It is necessary to control the temperature of the fermentation tank during the fermentation process, because even a temperature change of about 0.5℃ will cause significant changes in the electrode signal (more than 1%). It has been reported that when the fermentation temperature is around 30℃, the deviation of the dissolved oxygen electrode measurement value caused by the temperature change is about 3%/℃. The periodic change of the dissolved oxygen indicator (observed every few minutes) reflects the influence of temperature change, and larger temperature fluctuation can cause larger drift during calibration. Therefore, extreme care should be taken when changing the temperature during fermentation process. The dissolved oxygen electrode needs to be calibrated before setting the operating temperature of the fermentation tank. Based on the existence of the above factors, some dissolved oxygen electrodes are originally equipped with instruments such as temperature sensors,etc to achieve automatic compensation of temperature. In addition, for fermentation tank with computer monitoring, independent temperature sensor signals can be used to achieve temperature compensation by relevant software.
2.4. Influence of pressure
In the process of microbial fermentation, the various biochemical reactions of microorganisms are generally carried out in the liquid phase, and only the oxygen dissolved in the fermentation liquid can be ingested by microorganisms. After sterile air enters into the fermentation tank, it appears as small bubbles which rise from the bottom to the upper side in the fermentation liquid. If the whole fermentation reaction system is regarded as an heterogeneous system, then there is a process of oxygen intake from the gaseous phase to the liquid phase, and then it is transferred to microorganisms from the bubble in the form of dissolved oxygen. For the dissolved oxygen electrode, under stable conditions, the output current (relative current) of the electrode is in direct proportion to the activity of oxygen in the liquid or the differential pressure of oxygen in the gas phase in equilibrium status. The electrode reading is usually expressed as the saturability (%) of air at atmospheric pressure, and 100% dissolved oxygen tension is equivalent to about 160mmHg (1mmHg~133Pa) of the oxygen partial pressure. If the total equilibrium gas pressure of the fermentation liquid changes, even if the gas composition does not change (because the partial pressure of oxygen will change proportionally), it will change the reading of the dissolved oxygen electrode.
Therefore, the change of pressure in bubbles in the fermentation liquid can affect the dissolved oxygen tension, which will further affect the electrode reading. In the fermentation tank, when the fermentation liquid is motionless ,the pressure of the fermentation liquid does not significantly affect the bubble pressure, but the change of the press head will lead to the significant effect. In general, the pressure drop when outside air enters into the air filter or pipeline can produce the pressure of about 7000 Pa, which is enough to increase the electrode signal by 7%. The changes of atmospheric pressure during the fermentation process can also cause changes in the reading, even in normal weather conditions, the reading can change as much as 5%.
3.1. Influence of dissolved oxygen on microbial growth
It is unfavourable for growth of microorganisms when concentration of dissolved oxygen is too high. Studies have shown that too high dissolved oxygen will not only produce too much power consumption, but also produce a large number of bubbles, while too high DO will also produce new oxygen, superoxide groups or hydroxyl radicals, which will destroy components of microbial cell. Some scholars have studied the relationship between the spore formation of Bacillus sphaerlcus and DO. The results show that within the certain DO range, the amount of spore formation in the fermentation liquid increases with the increase of DO, but the amount of spore formation in the fermentation liquid decreases when DO is too high. The possible reason is speculated that when Bacillus sphaerlcus is in the logarithmic growth stage and spore formation stage, sufficient ventilation volume uplifts the level of dissolved oxygen in the fermentation liquid, which is conducive to the formation of spores. However, when Bacillus sphaerlcus is in stable stage and declining stage, excessive ventilation volume will lead to excessive dissolved oxygen and autolysis of strains will occur, which will reduce the amount of formed spores.
DO in fermentation liquid will directly affect the activity of microbial enzymes and metabolic pathways, and then affect the growth of microorganisms and the accumulation of metabolites, and it ultimately determines the level of metabolite production yield. Some scholars have studied the influences of different dissolved oxygen on two key enzymes in glutamic acid fermentation (glutamate dehydrogenase GDH and lactic dehydrogenase LDH) and metabolic flow direction. Studies have shown that under the condition of very low DO, the cycle pathway of tricarboxylic acid is weakened, which is not enough to balance the rate of glucose glycolysis pathway, thus promotes the activity of LDH and leads the metabolism to the generation of lactic acid, which results in accumulation of lactic acid; However, under the condition of high DO, GDH activity declines significantly, cycle pathway of tricarboxylic acid is enhanced, and a large amount of CO2 is generated , which results in loss of carbon source , both above conditions are not conducive to generation of glutamic acid.
In the beer industry, the DO requirements are different in each fermentation stage of beer. For example, in the early stage, enough oxygen is needed to promote the growth and reproduction of yeast, and oxygen intake should be strictly controlled at each stage thereafter. Because the presence of oxygen will promote the metabolic pathway of aerobic respiration of yeast, thus its anaerobic metabolic process to ethanol fermentation will be inhibited. However, studies have shown that the ethanol produced under anaerobic condition is lower than the ethanol produced under the condition of 1-4% dissolved oxygen. This is mainly due to the fact that the amount of yeast under anaerobic condition is much lower than the amount of yeast under aerobic condition, and the production of ethanol has a great relationship with the amount of yeast.
3.2. Influence of dissolved oxygen on metabolites
Different microorganisms have different requirements for demanded amount of oxygen during different fermentation stages. In the study of the influence of dissolved oxygen on the bioconversion of tylosin into its acyl compound by streptomyces thermotolerans A80, scholars found that the biotransformation fermentation process is different from the fermentation stage of general antibiotics, and there are two peak stages of oxygen intake.
During different fermentation stages of the same microorganism, the demanded amount of oxygen of microorganism is also different. In the process of producing hyaluronic acid (HA) by fermentation, the influence of DO on the molecular weight of HA is the result of the balance of two opposite effects. Firstly, the increase of dissolved oxygen is beneficial to the synthesis of high molecular weight HA. The reason that the appropriate increase of dissolved oxygen promotes the synthesis of high molecular weight HA is that HA is one component of the strains membrane, which can protect group A and Group C streptococcus from the invasion of external oxygen free radicals; The other reason is that too high DO can promote the production of oxygen free radicals and degrade HA macromolecules. 50% dissolved oxygen value is a critical point, if dissolved oxygen value is higher than this concentration, HA molecular weight will decline instead of rising.
DO is an important factor in microbial fermentation, and aerobic fermentation usually requires a large amount of air to meet the needs of microorganisms for dissolved oxygen. Studies have shown that DO has the greatest influence on the production of antibacterial substances by Bacillus subtilis B47, and it has a negative effect, that is, the more DO, the less antibacterial substances produced by the strains. DO not only affects the synthesis pathway of secondary metabolites, but also affects the synthesis speed of metabolism. In the process of amino acid fermentation, because most of the bacteria produced by amino acid are aerobic bacteria, that is, the bacteria must be supplied with appropriate amount of sterile air, then the strains will reproduce and accumulate the required metabolites. The researchers use L-threonine producing strains TRFC as the strains for batch fermentation to produce L-threonine. The influences of different DO on synthesis of L-threonine are studied by researchers. The results show that when DO is more, that is, oxygen supply is sufficient, the metabolism of strains is vigorous, which is conducive to the growth of the strains. In addition, oxaloacetic acid, as the precursor of L-threonine, is mainly produced by tricarboxylic acid cycle and phosphoenolpyruvate carboxylation reaction, which has high requirements for DO. Adequate oxygen supply can make the strains breathe enough, and it is also conducive to acid production and amino conversion.
The main purpose of adjusting & controlling dissolved oxygen concentration is to maintain the DO in the fermentation liquid within the certain limitation degree or range, to ensure the optimal growth and metabolic conditions of microorganisms, and to obtain as many metabolites with good quality as possible. Dissolved oxygen control mainly refers to DO measurement value, monitoring value of ventilation volume for fermentation tank, stirring speed of fermentation tank, pressure of fermentation tank, fermentation temperature and so on for comprehensive consideration. During the process of microbial fermentation , the relationship between DO and parameters of other fermentation processes is extremely complex, which is affected and restricted by a variety of physical, chemical and microbial factors in the bioreactor. The control of DO is mainly concentrated in the following three aspects:
4.1. Controlling amount of dissolved oxygen
Controlling amount of dissolved oxygen (C*-CL) is the driving force of oxygen dissolution, and the primary factor for controlling amount of dissolved oxygen is to control oxygen partial pressure (C*). High density culture often uses the way of passing pure oxygen to increase the oxygen partial pressure, while anaerobic fermentation uses various ways to control the oxygen partial pressure at the lower level. Such as beer fermentation, during wort oxygenation stage and yeast inoculation stage, the oxygen content is generally required to reach 8~10PPM; While during beer fermentation stage, the regular oxygen content in beer shall not exceed 2PPM.
4.2. Controlling oxygen transfer rate
The oxygen transfer rate mainly considers the influencing factors of KLa. In the certain sense, when the KLa is larger, the mass transfer performance of the aerobic bioreactor is better. The pathways of controlling KLa can be divided into three parts which are operating variables, physicochemical properties of reaction liquid and structure of reactor. Operating variables include temperature, pressure, ventilation volume and rotating speed (stirring power), etc. The physical and chemical properties of the fermentation liquid include the viscosity of the fermentation liquid, surface tension, oxygen solubility, the composition of the fermentation liquid, the flow state of the fermentation liquid, the type of fermentation,etc; The structure of reactor refers to the type of reactor, the proportion of the size of each part of the reactor, the form of the air distributor, etc. Of course, some factors are interrelated to each other.
4.3. Controlling the oxygen uptake rate of microorganisms
Factors related to the oxygen uptake rate of microorganisms include microbial species, the growth situation of microorganisms at the time of inoculation, the amount of inoculation, whether it is infected with miscellaneous bacteria, etc. For aerobic fermentation, when the amount of inoculation is more, the amount of oxygen consumption is more, and the oxygen uptake rate is higher, then the DO will decline faster. When DO fluctuates sharply during the fermentation process, the pH of the fermentation liquid also changes sharply, which indicates that the fermentation liquid is contaminated by other miscellaneous bacteria.
The oxygen content in fermentation liquid has an important influence on the growth of strains and the formation of products. The controlling of dissolved oxygen amount is mainly considered in three aspects, which include the dissolution of oxygen, the transmembrane transfer rate of oxygen and the oxygen uptake rate of microorganisms. The controlling of DO during fermentation process is a dynamic process, which should be based on the actual fermentation situation, and then use various adjustment means to make the DO in the fermentation liquid maintain at an appropriate level. In fermentation industry, it is gradually moving towards automatic and control integration mode from DO measurement to analysis and control. In short, the use of DO as the online control signal of supplementary medium will greatly improve the accuracy of fermentation control and automation performance.