Microbial fermentation technology is widely applied, and seed culture serves as a critical preliminary step in fermentation processes, with its quality directly influencing fermentation efficiency and yield. Key factors regulating seed quality include culture medium, temperature, seed age and inoculum size, aeration and agitation, as well as the balance between dissolved oxygen (DO) and biological oxygen demand (BOD). This article reviews the mechanisms and control strategies of these factors, providing insights for optimizing seed culture processes and enhancing fermentation efficiency.
The culture medium (nutritional conditions) is related to nutrient acquisition and has a direct impact on the growth, reproduction, enzyme activity, and yield of microorganisms. The medium can be categorized into: C/N basic growth nutrients, inorganic salts for osmotic pressure control, trace elements for structural maintenance, and a pH enzyme activity regulation system.
The seed medium is the fundamental environment for the growth and reproduction of the inoculum, providing the necessary nutrients, energy sources, and appropriate physicochemical conditions for the microorganism.
Firstly, sufficient and balanced nutritional components are key to ensuring the vitality and quantity of the seeds. For example, the carbon source is the energy basis for microbial growth, while the nitrogen source is an important component of cellular material. If the supply of carbon or nitrogen sources is insufficient, the growth of the seeds will be inhibited, and they will not be able to achieve the expected quantity and quality.
Inorganic salts in the medium, such as phosphorus, sulfur, magnesium, and potassium, play an indispensable role in maintaining the osmotic pressure balance of cells, enzyme activity, and the structural integrity of cells. Taking phosphorus as an example, it participates in the synthesis of nucleic acids and phospholipids. If phosphorus is deficient, the replication of genetic material and the formation of cell membranes will be hindered, thereby affecting the growth and reproduction of the seeds.
The purity and impurity content of the raw materials in the medium can affect the growth of microorganisms. If the raw materials contain toxic substances or inhibitory factors, they may have a toxic effect on the growth of the seeds, or even lead to the death of the seeds.
A. The medium in the final stage of the seed tank should be consistent with the production tank. At this point, the enzyme system required for fermentation has already been established in the previous tank, and there is no need to rebuild it under the new environmental conditions.
B. The proportion of nitrogen source in the seed tank medium should be increased, and the increment ratio of inorganic nitrogen should be relatively larger.
C. After the strain is transferred to the next stage, due to differences in equipment (such as tank type, agitation form, rotation speed, etc.), the medium formulation needs to be readjusted based on experiments.
In the field of biology, as one of the key environmental factors for microbial growth, temperature has a significant impact on the growth rate and metabolic activities of microorganisms. According to the general rule, the growth rate of microorganisms usually doubles for every 10℃ increase, mainly because temperature can directly affect the metabolic enzyme systems in cells.
Enzymes are proteins that catalyze chemical reactions in organisms, and almost all growth and metabolic processes of microorganisms are catalyzed by enzymes. The reaction rate of enzymes is greatly affected by temperature, and there is an optimal temperature range within which the enzyme activity is the highest, and the growth and metabolic rate of microorganisms are also the fastest.
When the temperature is lower than the optimal temperature, the enzyme activity 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, leading to hindered microbial growth or even death.
The dissolved oxygen state of the fermentation system is jointly determined by aeration rate, tank pressure and stirring conditions. The seed bacteria can grow normally only when the dissolved oxygen (DO) content in the fermentation broth is higher than the current biological oxygen demand (BOD) of the system. After inoculation, the bacteria rapidly enter the logarithmic growth phase, and their division and reproduction rates increase exponentially. At this stage, the aeration rate and stirring power are usually adjusted alternately to make the dissolved oxygen level meet the growth requirements of bacteria. If the oxygen demand of bacteria cannot be met only by adjusting aeration and stirring, the culture temperature can be appropriately reduced to slow down the oxygen consumption rate of bacteria. This operation will slightly reduce the division and growth efficiency of bacteria, but it can effectively avoid bacterial deformation and autolysis caused by oxygen deficiency, and ensure the normal metabolic survival of bacteria.
The aeration rate directly supplements the source of dissolved oxygen in the fermentation system by continuously injecting fresh air into the fermenter. Stirring can promote the thorough mixing of gas, liquid and solid particles in the fermentation broth, and effectively improve the distribution uniformity of dissolved oxygen and the gas-liquid mass transfer efficiency. Precise regulation of dissolved oxygen in the fermentation system can be realized by alternately adjusting aeration rate and stirring power. During the vigorous growth period of bacteria, synchronously increasing aeration rate and stirring power can significantly raise the dissolved oxygen level and fully meet the oxygen demand for rapid bacterial proliferation. When the system has excessive or insufficient dissolved oxygen, the two parameters can be flexibly adjusted to maintain the stability of the dissolved oxygen environment and ensure the continuous and stable growth and metabolism of bacteria.
Biological Oxygen Demand (BOD) is an important index to characterize the degree of organic pollution in water, which can directly reflect the total oxygen required by microorganisms to decompose organic substances in the system. During the fermentation and culture process, the balance between dissolved oxygen and biological oxygen demand is the core condition for the normal growth of microorganisms. Microorganisms can maintain normal growth and metabolic activities only when the dissolved oxygen (DO) content in the system is higher than the current biological oxygen demand (BOD). If the dissolved oxygen content is lower than BOD, the system will suffer oxygen deficiency stress, which inhibits the physiological metabolism of microorganisms, reduces the growth rate of bacteria and the synthesis and accumulation of metabolites. In severe cases, it will cause bacterial cell autolysis and lead to fermentation failure.
Young seeds, due to their insufficient growth and maturity, have not yet fully established various enzyme systems and metabolic mechanisms in the cells, resulting in weak ability to adapt to the new environment after tank transfer and slow start-up speed. Old seeds, on the other hand, may also perform poorly in the new environment due to decreased cell viability and declining metabolic capacity. Both situations will prolong the fermentation cycle, increase production costs, and ultimately lead to reduced yield.
When the inoculum size 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, enabling cells to rapidly enter the logarithmic growth phase and shorten the time to adapt to the new environment.
On the contrary, if the inoculum size is too small, the content of metabolites in the cells is insufficient, and the cells need more time to synthesize the required substances, thereby prolonging the lag phase. Taking yeast fermentation for ethanol production as an example, when the inoculum size is insufficient, the ethanol production rate in the early stage of fermentation will be significantly slowed down, leading to the extension of the entire fermentation cycle and the reduction of production efficiency.