The main factors affecting seed tank culture include: nutrition condition, culture condition, infection control, determination of expansion stage and determination of inoculation amount.
The acquisition of culture-related nutrients has a direct effect on the growth, reproduction, enzyme activity and yield of microorganisms. The medium can be divided into C/N based growth nutrition, inorganic salt osmotic pressure control, trace element maintenance composition, and pH enzyme activity regulation system. The requirement of medium is simple composition, abundant source, cheap price and convenient material.
Seed medium is the basic environment for seed growth and reproduction, which provides nutrients, energy sources and suitable physical and chemical conditions for microorganisms. First of all, adequate and balanced nutrients are the key to ensuring seed vitality and quantity. For example, carbon sources are the energy base of microbial growth, and nitrogen sources are important components of cellular material. If carbon or nitrogen sources are in short supply, seed growth will be inhibited and will not achieve the desired quantity and quality.
Inorganic salts in the medium, such as phosphorus, sulfur, magnesium, potassium, etc., play an indispensable role in maintaining the osmotic pressure balance of cells, enzyme activity, and cell structural integrity. Take phosphorus as an example, it involves in the synthesis of nucleic acids and phospholipids, if the lack of phosphorus element, the cell's genetic material replication and the formation of cell membranes will be hindered, thus affecting the growth and reproduction of seeds.
The purity and impurity content of medium raw materials will affect the growth of microorganisms. If the raw material contains toxic substances or inhibiting factors, it may have a toxic effect on the growth of the seed, and even lead to the death of the seed.
It is of great significance for age of seed requirement to be transplanted during logarithmic growth period.
Because the young seeds has not fully grown and matured, various enzyme systems and metabolic mechanisms in the cell have not been fully established, resulting in weak ability to adapt to the new environment and slow start-up speed . The old seeds may also perform poorly in the new environment because of the decline in cell vitality and metabolic capacity. Both of these conditions will prolong the fermentation cycle, increase production costs, and ultimately lead to lower yields.
The amount of inoculation had a significant effect on the lag period under the new environment.
When the amount of inoculation is large, the content of metabolites such as RNA required for cell division and reproduction is abundant. These metabolites can quickly support the growth and reproduction of cells in the new environment, so that cells quickly enter the logarithmic growth phase and shorten the time to adapt to the new environment.
Conversely, if the amount of inoculation is too small, the metabolite content in the cell is insufficient, and the cell needs to spend more time to synthesize the required substance, thus prolonging the lag period. Taking yeast fermentation to produce alcohol as an example, when the amount of inoculation is insufficient, the alcohol generation rate in the early stage of fermentation will be significantly slowed down, resulting in the extension of the whole fermentation cycle and the reduction of production efficiency.
Look at temperature in the early stage, look at dissolved oxygen in the late stage
Any microorganism has its optimal growth temperature and production temperature, as follows:
| Species | thermal lethal conditions | growth temperature /℃ | |||
| temperature/℃ | time/min | Optimum | limit | ||
| Mycete | Mycelium | 60 | 5-10 | 25-30 | 15-37 |
| Spore | 65-70 | 5-10 | |||
| Yeast | Trophic cells | 55-65 | 2-3 | 20-28 | 10-35 |
| Spore | 60 | 10-15 | |||
| Bacteria | Trophic cells | 63 | 30 | 35-40 | 5-45 |
| Spore | Above 100 | ||||
In the field of biology, temperature as one of the key environmental factors of microbial growth, has a significant impact on the growth rate and metabolic activities of microorganisms. According to the universal rule, every 10℃ increase, the growth rate of microorganisms is usually doubled, this is mainly because the temperature can directly affect the metabolic enzyme system in the cell. Enzymes are proteins that catalyze chemical reactions in organisms, and the growth and metabolic processes of microorganisms are almost all catalyzed by enzymes. The reaction rate of enzyme is greatly affected by temperature, and there exists an optimal temperature range, in which the activity of enzyme is the highest, and the growth and metabolism of microorganism is also the fastest. When the temperature is lower than the optimal temperature, the activity of enzymes decreases and the growth rate of microorganisms slows down. When the temperature is higher than the optimal temperature, the enzyme may lose its activity due to denaturation, resulting in microbial growth retardation or even death.
4.1 Influence of pH value on enzyme activity
Enzyme activity regulation: Enzymes are key proteins that catalyze biochemical reactions in microorganisms, and their activity is significantly affected by pH value. Each enzyme has its optimal pH range, in which the enzyme activity is highest. When the pH value deviates from the optimal range, the enzyme activity will gradually decrease, or even completely deactivate. This causes the metabolic process of microorganisms to be hindered, affecting the absorption and utilization of nutrients, and thereby reducing the seed quality.
Affecting metabolic pathways: Changes in pH value may also cause changes in microbial metabolic pathways. For example, under acidic conditions, certain microorganisms may increase the lactic acid fermentation pathway to maintain pH homeostasis, which may change the variety and proportion of metabolites, which in turn affects seed quality.
4.2 Influence of pH value on cell membrane
Cell membrane permeability: The pH value can affect the state of charge carried by the microbial cell membrane, thus changing the permeability of the cell membrane. Cell membrane is the main channel for the exchange of substances between microorganisms and the external environment, and the change of its permeability will directly affect the absorption of nutrients and the excretion of metabolites by microorganisms.
Cell stability: Under extreme pH conditions, the structure of the cell membrane may be damaged, resulting in the leakage of substances within the cell or the entry of harmful substances from the outside into the cell, which in turn affects the normal physiological function of the cell.
4.3 Influence of pH value on nutrient absorption
Affecting the dissociation of nutrients: nutrients in the medium (such as amino acids, minerals, etc.) have different degrees of dissociation under different pH conditions, which will affect the absorption and utilization efficiency of these nutrients by microorganisms.
Influence the competition of nutrients: In the mixed culture system, the absorption and utilization of nutrients by different microorganisms are different. Changes in pH may alter this competitive relationship, which in turn affects the growth and reproduction of dominant strains.
Ventilation, tank pressure and agitation jointly determine the current dissolved oxygen state. Only when the dissolved oxygen is greater than the current BOD, the seeds can grow normally. After the seed transfer, the bacteria enter the logarithmic growth phase, and the rate of fission and reproduction increases exponentially. The oxygen consumption rate of the bacteria is reduced, and the cell division and growth efficiency is reduced, but it will not be hypoxic malformation or autolysis.
5.1 Synergistic effect of ventilation and agitation
Ventilation increases the source of dissolved oxygen directly by injecting fresh air into the fermenter. At the same time, stirring improves the distribution uniformity and mass transfer efficiency of dissolved oxygen in the culture medium by promoting the mixing of gas, liquid and solid particles. The fine control of dissolved oxygen level can be achieved by alternating adjustment of ventilation and stirring power. For example, in the flourishing period of cell growth, increasing ventilation and stirring power can improve the dissolved oxygen level and meet the demand of cell for oxygen. In excess or shortage of dissolved oxygen, the stability of dissolved oxygen can be maintained by adjusting the ventilation rate and stirring power.
5.2 Relationship between dissolved oxygen and BOD
Biological oxygen demand (BOD) is a measure of the level of organic matter pollution in water, and it reflects the amount of oxygen required by microorganisms to break down these organic matter. During fermentation, microorganisms can grow normally only when the dissolved oxygen (DO) is greater than the current biological oxygen demand (BOD). If DO is lower than BOD, microorganisms will be inhibited by hypoxia, resulting in reduced growth rates, reduced metabolite accumulation, and even cellular autolysis.
The fewer the number of stages, the less likely it is to be infected with bacteria. The number of stages is determined by the properties of the bacteria, such as the growth rate, the concentration of bacteria in the logarithmic stage, the development level of spores, etc. After continuous optimization and debugging, the number of expanded seed pot should be reduced as much as possible, but the premise is that the increase of the lag period of the production pot and the growth rate in the logarithmic growth stage are not affected or less affected.
In the fermentation industry, the stage of seed tank is a key process parameter, which directly affects the risk of microbial contamination, growth efficiency and the stability and cost of the whole fermentation process. The determination of the stage is usually based on the characteristics of the strain, such as growth rate, logarithmic bacterial concentration, and spore development level, which together determine the needs and adaptability of microorganisms at different growth stages. What are the advantages of scaling fewer stages?
6.1 Reduce the risk of infection
Fewer stages means that microorganisms undergo fewer operational steps and environmental changes during the expansion process, reducing the risk of contamination. Bacterial contamination is a common problem in fermentation industry, which can lead to fermentation failure, product quality decline and even production interruption, so reducing the number of stages is of great significance to improve the stability and reliability of the fermentation process.
6.2 Increase growth efficiency
Under suitable conditions, the growth rate of microorganisms is constant, but after each transfer and expansion, there will be a certain growth loss and adaptation period. Therefore, reducing the number of stages can shorten the time from inoculation to the logarithmic growth phase of microorganisms and improve the growth efficiency.
6.3 Cost Reduction
Fewer stages means fewer resources such as equipment, manpower and materials are required, which helps to reduce production costs.
6.4 Precautions
In the process of reducing the series, it must be ensured that the increase in the lag period of the production tank and the growth rate of the logarithmic growth period are not significantly affected. This is because the lag period is the key period for microorganisms to adapt to the new environment and adjust the metabolic state, if the lag period is too long, it will lead to the extension of the entire fermentation cycle and the reduction of yield. At the same time, the growth rate of the logarithmic growth period directly determines the reproduction rate and final biomass of microorganisms. If the growth rate decreases, the fermentation effect and product quality will also be affected.
Contamination refers to the contamination of non-target microorganisms during culture. This can lead to culture failure or a decline in product quality. Therefore, strict aseptic operation measures must be taken, such as the use of aseptic equipment, media and inoculum, regular disinfection and sterilization.
7.1 Bacteria contamination will directly lead to degradation of seed quality
The viability of seeds was manifested in the ability to divide, the total number of viable bacteria and the start-up time. Normal seeds should have a pure, single microbial population, but after bacteria contamination, other bacteria can be mixed in, which will change the microbial composition and proportion of the seed. This will affect the growth characteristics and metabolic activity of the seeds, for example, hybrid bacteria may compete for nutrients, interfere with the normal growth and metabolism of the target microorganisms, resulting in insufficient seed vitality and increasing the time spent on expanding culture.
7.2 Bacteria contamination will affect the yield and quality of fermentation
Due to the presence of other bacteria in the seeds, forced inoculation or operation ignoring the risk of contamination, these bacteria may produce substances different from the target product after the tank, resulting in a decrease in the yield and purity of the fermentation product. For example, in the production of antibiotics, bacteria contamination may cause the titer of antibiotics to decrease, or even fail to meet medicinal standards.
7.3 SOP invalidation and the process is temporarily adjusted
After inoculating the seed liquid containing hybrid bacteria, the physical and chemical properties of the fermentation liquid in the production tank may be changed, such as pH value, dissolved oxygen, etc., thus destroying the appropriate fermentation environment, affecting the normal growth and metabolic regulation of microorganisms, resulting in the fermentation process out of order, unable to operate according to the fixed SOP, requiring the technical personnel to conduct temporary intervention and guidance.
7.4 Bacteria contamination will increase production costs
Once the bacteria contamination is found, it is often necessary to take a series of measures to deal with it, such as stopping fermentation, cleaning equipment, re-preparing seeds, etc., which requires a lot of manpower and material resources and increases the time cost. The contamination during the activation period can be avoided by increasing parallel operation and selecting high-quality strain shake bottles, even if the bacteria contamination is also an increase in time cost, and it will not lead to a large number of material costs and equipment maintenance losses; The loss of bacteria contamination in the expansion stage is greater than that in the activation stage, but it is still advisable to terminate the fermentation after the occurrence of bacteria contamination.