High-density fermentation technology

High-density culture technology (High-cell-density cultivation, HCDC), or high-density fermentation technology, is the result of increasing the ratio of the product (the yield of the product per unit of volume in time) by increasing the fermentation density of the bacteriaThis method can not only reduce the cultivation volume, strengthen the downstream separation extraction, but also shorten the production cycle, reduce equipment investment to reduce production costs, can greatly improve the competitiveness of products in the marketIn addition to the expressive nature of the recombinant bacteria itself, the optimal environmental conditions for the growth and product expression of recombinant bacteria must be given, including suitable medium composition, suitable culture temperature, pH, stable growth rate, suitable dissolved oxygen and reasonable flow of nutrients"style"text-align: center;"img src""alt""high-density fermentation technology" "high-density fermentation technology" "High-density fermentation technology"is often encountered in a variety of problems, resulting in a large gap between actual yield and expected estimates, because the fermentation process is too many variable factors, only through the improvement of the process and more perfect human-machine dialogue to shortenWith regard to the inevitable loss of plasmids, the pressure of antibiotic selection can be increased appropriately to reduce plasmid lossAs for maintaining batch consistency, or easy to achieve, according to the seed preparation protocol to prepare seeds, seed transmission proliferation, to ensure the consistency of fermentation conditions can be1, high-density fermentation factors (ecoli for example)high-density fermentation on the fermentation equipment has high requirements, but also has very high requirements for fermentation conditionsIn the production of fermentation tank will pursue high yield, but high density is not equal to high yield, fermentation has its instability, in the shaker expression of successful strains, after the tank is not necessarily successful, may not be expressed, or express the product characteristics of changeThere are many factors affecting high-density fermentation, such as the nutrients needed for bacterial growth, the accumulation of growth inhibitors in fermentation process, the concentration of dissolved oxygen, the temperature of culture, the pH of the fermentation fluid, the method of replenishment and the rheological characteristics of fermentation fluid"text-align: center;"img src""alt""high-density fermentation technology" title" "high-density fermentation technology", nutrient sourcehigh-density fermentation to obtain high biomass and high concentration expression products, need to be invested several times the biomass of the matrix, to meet the needs of rapid growth of bacteria and a large number of expression of gene productsHigh-density fermentation also requires more varieties and quantities of nutrient sources in the matrix than the general fermentation methodHowever, the concentration of nutrients in the matrix is too high, beyond a certain limit can make the growth inhibition of a large amount of accumulation, so that the growth and reproduction of bacteria and the target gene expression is inhibited, so that the production efficiency is reduced1.2, growth inhibitors In the process of high-density fermentation, the accumulation of bacterial metabolites, such as organic acids, CO2 and other harmful substances can inhibit the growth of bacteria, especially acetic acid, will inhibit the expression of exogenous protein E coli in fermentation, carbon source supply is higher than the rate of cell assimilation, than the growth rate is too large, insufficient oxygen supply, will lead to the production of metabolic by-products such as acetic acid, the growth of bacteria and the expression of recombinant genes inhibited 1.3, dissolved oxygen concentration dissolved oxygen concentration is one of the important factors affecting the growth of bacteria in the process of high density fermentation The growth and metabolism of E coli requires the participation of oxygen, dissolved oxygen concentration on the growth of bacteria and product production has a great influence, the concentration of dissolved oxygen is too high or too low will affect the metabolism of bacteria, so that the later growth becomes extremely slow In the process of amassing a large number of bacteria, oxygen consumption is oxidized and decomposed, so the timely supply of saturated oxygen is very important With the extension of fermentation, the density of the bacteria increases rapidly, the dissolved oxygen concentration decreases and cell growth slows down In the late stage of high-density fermentation, due to the increase of bacterial density and the high oxygen consumption, the physical parameters of the fermentation tank can not meet the supply of oxygen, resulting in a very low expression of external protein Therefore, maintaining a higher dissolved oxygen concentration can improve the growth of E coli with recombinant plasmids, which is beneficial to the formation of exogenous protein products 1.4, temperature culture temperature are important factors affecting bacterial growth and regulating cell metabolism Higher temperatures are beneficial to the growth of bacteria and improve the biomass of bacteria, while low temperature culture can improve the expression of recombinant products Studies have shown that the use of different culture temperatures at different stages is beneficial to improve the growth density of bacteria and the expression of recombinant products, and can shorten the incubation cycle For the engineering bacteria of temperature control induced expression, the growth state of bacteria and the duration of induction have a great influence on the expression and productivity of recombinant proteins 1.5, pH stable pH is a necessary condition to keep the bacteria in optimal growth Since the change of pH in the outside world changes the pH in the cell of the bacteria by the change of weak acid or weak alkali, thus affecting the metabolic response of the bacteria, the change of pH in the fermentation process affects the cell's biomass and gene product expression 2, improve the yield of the method 2.1, choose fermentation mode in order to solve the high concentration substrate inhibition, feed batch fermentation has been widely used in all kinds of microorganisms high-density fermentation The batch fermentation of supplements refers to the culture technique of adding certain materials continuously in some way in the batch fermentation of microorganisms This technique can not only make up for the deficiency of batch operation, but also some difficulties in the continuous operation of alkali light, especially to avoid the technical difficulties of maintaining a constant volume 2.2, reduce growth inhibitor accumulation In order to reduce the accumulation of growth inhibitors in the process of high-density fermentation, first of all, limit the concentration of nutrients in the fermentation solution can not be too high, because the concentration of too high nutrient source can easily lead to the production of growth inhibitors By using batch feed fermentation, the flow-plus high-concentration feed can not only meet the large demand for nutrient sources during high-density fermentation, but also reduce the production of growth inhibitors The sufficient supply of oxygen in the fermentation process can not only make the bacteria expand rapidly, but also reduce the production of harmful substances 2.3, improve oxygen supply In order to maintain a certain concentration of dissolved oxygen in the fermentation process, can appropriately increase the stirring speed, but too high speed will produce a large number of foam, but will lead to a decrease in the concentration of dissolved oxygen Now small fermentation tanks have the use of combined with pure oxygen ventilation to improve oxygen pressure, but because the use of pure oxygen is not safe, not economical, at the same time in the large fermentation tank may be locally uneven, easy to make bacteria produce oxygen poisoning, but inhibited the growth of the seedlings A number of methods have recently been developed to improve oxygen supply, such as increasing oxygen pressure by increasing the pressure of fermentation tanks, adding hydrogen chloride to the fermentation solution, releasing oxygen under the action of cell perchlorinase, for use by bacteria 2.4, fermentation fluid rheological general fermentation is also for the intended homogeneity, and in high-density fermentation, can not ignore the volume of the bacteria, at this time the performance of gas-liquid solid three-phase In addition, the viscosity of high-density fermentation slag will also increase significantly, as a non-Newtonian fluid, the transfer of oxygen and nutrients will have a greater impact 3, the application of supplements in the recombinant E coli high-density fermentation the key to the success of recombinant E coli high-density fermentation technology is the replenishment strategy, that is, according to the growth characteristics of recombinant bacteria and the expression of products to adopt a reasonable nutritional logistics plus method Carbon source and nitrogen source are two commonly used restrictive matrix, glucose has been widely used as a restrictive matrix for high-density fermentation of recombinant bacteria due to the fast and inexpensive use of bacteria E coli in excess glucose or hypoxia conditions will occur "glucose effect", accumulation of a large number of organic acids and affect the growth of recombinant bacteria and effective expression of exogenous protein Therefore, in the high-density fermentation of E coli, the reasonable flow and carbon source reduce the glucose effect, which is the key to success first of all, the feed rate and ratio growth rate directly affect the production rate and accumulation of acetic acid (mainly the feed rate and the ratio growth rate affect the amount of residual sugar in the fermentation liquid, and then affect the formation of acetic acid), so the appropriate control of the feed rate and the ratio growth rate, for the control of the amount of acetic acid has a very good effect secondly, it is necessary to ensure adequate dissolved oxygen, and strictly control the pH, and the rate of acid-to-alkali as far as possible to moderate, not too fast; third, choose a reasonable induction time is very important, the general induction time is selected in the exponential growth later, and the ratio growth rate of induction is best controlled within 0.2, selected at this time induction: (1) the rapid growth of the bacteria and protein synthesis period separate, so that the two stages do not affect each other, conducive to high protein expression; fourth, the carbon-nitrogen ratio in the replenishment process is also important If the nitrogen source is too high, it will make the bacteria grow too vigorous, pH is too high, not conducive to the accumulation of metabolites; Too much carbon source, it is easy to form a lower pH, inhibit the growth of bacteria, carbon source is insufficient, it is easy to cause the aging and self-soluble of bacteria In addition, improper carbon-nitrogen ratio will also cause the bacteria to absorb nutrients proportionally, which directly affects the growth of bacteria and the synthesis of products Experts with relevant experience have found that, in general, for a stable fermentation process, if always in a fixed fermentation period of the phenomenon of solubility, and can rule out the possibility of phage and bacteria, it may be due to the unreasonable carbon-nitrogen ratio caused by the appropriate adjustment of carbon-nitrogen ratio Source: , Sino-Nongwitt Biotech Co., Ltd.