Production method of amino acid fermentation coupling membrane separation technology

Abstract

The invention discloses a process and a device for producing amino acid by an amino acid fermentation coupling membrane separation technology. The process comprises the following specific steps: step 1, inoculating a fermentation seed of an amino acid producing strain into a sterilized fermentation tank for culturing; 2, after initial fermentation is performed to a later stage of a logarithmic phase or a mature stage, separating amino acid by using a membrane separation technology, returning thalli intercepted by a membrane and other components into the tank for continuous fermentation, and meanwhile, feeding a fresh culture medium to keep the volume of fermentation liquid in the tank constant; and 3, recycling the unconsumed substrate in the amino acid clear liquid separated in the previous step through a small-aperture nanofiltration membrane, and returning to the front end to be continuously used for fermentation. The method has the advantages that semi-continuous or continuous fermentation is realized through a fermentation coupling membrane separation technology, the utilization rate of equipment and the yield of products are improved, and the method is mild in process condition, small in environmental pollution and easy to automatically control.

Description

Technical Field

[0001] The invention relates to a method and a device for fermenting and purifying amino acids, and belongs to the field of fermentation separation. Background Art

[0002] Amino acids are a general term for a class of organic compounds containing amino and carboxyl groups, and are the basic substances that constitute protein macromolecules. There are 20 basic amino acids in organisms, including 12 non-essential amino acids and 8 essential amino acids. A wide variety of powerful protein molecules are formed by the condensation of these amino acids in a certain order. Therefore, amino acids are extremely important biological small molecules. They can be used as a component unit of important macromolecular proteins in living organisms, determining the various biological functions of proteins, and can also be used as independent molecules to directly participate in the process of life activities.

[0003] At present, amino acids are mainly produced by biological fermentation, but a common bottleneck in fermentation is the inhibitory effect of products or by-products. Using engineering means to timely separate products or by-products can effectively slow down the inhibition of metabolites in the fermentation process, thereby carrying out continuous fermentation, which greatly reduces the time of repeated operations such as cleaning, feeding, sterilization, inoculation, and tank placement during the fermentation process, thereby shortening the total fermentation time. Moreover, since microorganisms grow at a high speed under a constant state, the number of newly added live bacteria is greatly increased, and the productivity of fermentation is improved. In addition, the use of continuous fermentation is also conducive to reducing labor intensity, saving power, and rationally utilizing equipment. It is also convenient for automatic control and the product quality is relatively stable.

[0004] However, there are certain problems in removing products during continuous fermentation. The main problem is that the separation process needs to maintain the controllability of the separation effect. When the stability of the separation process decreases, the fermentation process needs to be slowed down in time to avoid excessive product production. Summary of the invention

[0005] The present invention provides a continuous or semi-continuous production process for amino acids, which uses membrane separation technology to timely separate products or by-products in the fermentation liquid that have inhibitory or toxic effects on biological reactions, eliminate their inhibitory effects, extend the fermentation cycle, and increase cell activity, thereby improving the efficiency of fermentation.

[0006] A process and device for producing amino acids by amino acid fermentation coupled with membrane separation technology, comprising the following steps:

[0007] Step 1: Inoculate the fermentation seeds of the amino acid-producing strain into a sterilized fermentation tank for cultivation;

[0008] Step 2: When the initial fermentation reaches the late logarithmic growth phase or the mature phase, the amino acids are separated using membrane separation technology, and the bacteria trapped by the membrane and other components are returned to the tank for continued fermentation;

[0009] In the third step, the unconsumed substrate in the amino acid clear liquid is recovered by a nanofiltration membrane and further used for fermentation; at the same time, fresh culture medium is added to the fermentation tank to keep the volume of the fermentation liquid in the tank constant.

[0010] In one embodiment, the amino acid is selected from the group consisting of glutamic acid, lysine, threonine, leucine, isoleucine, serine, tryptophan, methionine, phenylalanine, and the like.

[0011] In one embodiment, the membrane separation technology can be an inorganic membrane or an organic membrane. Among inorganic membranes, a ceramic membrane is preferred, with a pore size of 4-500nm, a pressure of 0.1-0.5Mpa, a membrane surface flow rate of 2-5m / s, and a concentration multiple of 1 to 10 times.

[0012] In one embodiment, the nanofiltration uses a ceramic nanofiltration membrane with a pore size range of 4-10 nm and a filtration pressure of 0.4-4.0 MPa.

[0013] In one embodiment, 1. The process and device for producing amino acids by amino acid fermentation coupled with membrane separation technology is characterized in that it also includes regularly discharging a portion of the ceramic membrane concentrate, which can be used to prepare feed or fertilizer.

[0014] The present invention also provides use of the above device in producing amino acids.

[0015] Beneficial Effects

[0016] The present invention provides a production process of amino acid fermentation coupled with membrane separation technology, which uses membrane separation technology to timely separate products or by-products that have inhibitory or toxic effects on biological reactions in the fermentation liquid, eliminate their inhibitory effects, promote product conversion, and reduce the repeated operation time of each cleaning, feeding, sterilization, inoculation, tank placement, etc. during the fermentation process, thereby improving the efficiency of fermentation. In addition, the use of continuous or semi-continuous fermentation is also conducive to reducing labor intensity, saving power, and rationally utilizing equipment, and is convenient for automatic control, and the product quality is relatively stable. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is a production process flow chart of the present invention. DETAILED DESCRIPTION

[0018] The present invention is further described in detail below by specific embodiments. However, it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. If specific techniques or conditions are not specified in the examples, they are carried out according to the techniques or conditions described in the literature in this area or according to the product instructions. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be obtained commercially.

[0019] Values ​​expressed in range format should be understood in a flexible manner to include not only the values ​​explicitly listed as range limits, but also all individual values ​​or subranges encompassed within the range, as if each value and subrange were explicitly listed. For example, a concentration range of "about 0.1% to about 5%" should be understood to include not only the explicitly listed concentrations of about 0.1% to about 5%, but also individual concentrations (e.g., 1%, 2%, 3% and 4%) and subranges (e.g., 0.1% to 0.5%, 1% to 2.2%, 3.3% to 4.4%) within the indicated range.

[0020] The "one embodiment", "another embodiment", "implementation method" and the like mentioned in this specification refer to the specific features, structures described in conjunction with the embodiment or included in at least one embodiment generally described in this application. The same expression appearing in multiple places in the specification does not necessarily refer to the same embodiment. Furthermore, when a specific feature, structure or characteristic is described in conjunction with any embodiment, it is claimed that the realization of such feature, structure or characteristic in conjunction with other embodiments also falls within the scope of protection of this application.

[0021] It should be understood that when an element is referred to as being "connected" to another element, it can be directly connected to the other element or indirectly connected to the other element with elements interposed therebetween. Unless expressly stated to the contrary, the terms "including" and "having" should be understood to convey the inclusion of the listed elements but not the exclusion of any other elements.

[0022] As used herein, the words "comprises," "comprising," "having," or any other variations thereof, are intended to cover a non-exclusive inclusion. For example, a process, method, article, or apparatus that includes listed elements is not necessarily limited to those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.

[0023] The amino acid fermentation liquid to be treated by the present invention includes, but is not limited to, glutamic acid, lysine, threonine, leucine, isoleucine, serine, tryptophan, methionine, phenylalanine, etc. In the culture medium here, the fermentation raw material used can be a raw material that promotes the growth of the cultured strain and enables good production of the target fermentation product. For example, a liquid culture medium containing organic micronutrients such as carbon source, nitrogen source, inorganic salts, and amino acids and vitamins added as needed can be preferably used. As the above-mentioned carbon source, sugars such as glucose, sucrose, fructose, galactose, lactose and maltose, starch saccharification liquid containing these sugars, sweet potato molasses, beet molasses, high-grade molasses (High Test Molasses), cane juice, cane juice extract or concentrate, raw sugar purified or crystallized from cane juice, purified sugar purified or crystallized from cane juice, and organic acids such as acetic acid and fumaric acid, alcohols such as ethanol, and glycerol can be preferably used. The sugars described here refer to the initial oxidation products of polyols, which have an aldehyde group or a ketone group, and the sugars with aldehyde groups are classified as aldoses, and the sugars with ketone groups are classified as ketose carbohydrates, preferably glucose, sucrose, fructose, galactose, lactose or maltose. The above-mentioned carbon source can be added together at the beginning of the culture, and can also be added in batches or continuously during the culture. In addition, as the above-mentioned nitrogen source, for example, ammonia, ammonia water, ammonium salts, urea, nitrates, other auxiliary organic nitrogen sources such as oil cakes, soybean hydrolysis liquid, casein decomposition products, other amino acids, vitamins, corn steep liquor, yeast or yeast extract, meat extract, peptides such as peptone, various fermented bacteria and their hydrolysis products, etc. can be used. In addition, as the above-mentioned inorganic salts, for example, phosphates, magnesium salts, calcium salts, iron salts, and manganese salts can be appropriately added. Moreover, defoamers can also be added as needed. There are no particular restrictions on the fermentation culture conditions as long as the culture can be carried out, but it is preferably carried out at a pH of 4 to 8 and a temperature of 20 to 40° C. The pH of the fermentation culture solution is adjusted to a predetermined value within the above range using an inorganic acid or an organic acid, an alkaline substance, urea, calcium carbonate, ammonia, or the like.

[0024] Through membrane separation technology, the membrane permeate that passes through the fermentation liquid also contains fermentation products, culture medium, and some other inorganic salts, etc. The fermentation products are separated from the culture medium, inorganic salts, etc. by nanofiltration. As the material of the nanofiltration membrane used in the present invention, polymer materials such as cellulose acetate polymers, polyamides, sulfonated polysulfones, polyacrylonitrile, polyesters, polyimides and vinyl polymers can be used. The membrane is not limited to a membrane composed of only one material, and can be a membrane containing multiple materials. Regarding the membrane structure, the membrane can be an asymmetric membrane, which has a dense layer on at least one side of the membrane, and has micropores with gradually increasing pore diameters from the dense layer to the inside of the membrane or the other side; or a composite membrane, which has a very thin functional layer formed by other materials on the dense layer of the asymmetric membrane. The nanofiltration membrane has a molecular weight cutoff of 150 to 1000 Da and a filtration pressure of 0.4-4.0 Mpa.

[0025] Example 1

[0026] Take lysine fermentation broth as an example: First, add 65% of the volume of the fermentation tank culture medium to the fermentation tank. After sterilization, the mature seed liquid is connected to the culture medium of the fermentation tank for fermentation culture. The inoculation amount of the seed liquid is 10% of the volume of the fermentation tank culture medium. After inoculation, control the appropriate temperature, pH and ventilation volume for fermentation culture. After 48 hours of fermentation culture, the fermentation broth is sent to the ceramic membrane for filtration, the filtration temperature is controlled below 35°C, the filtration pressure is 0.25Mpa, and it is concentrated to 4 times. The obtained ceramic membrane clear liquid enters the next process, the remaining concentrated liquid is returned to the fermentation tank, and fresh culture medium and mature seed liquid are added to the fermentation tank for continued fermentation. After continuing fermentation culture for 36 hours, the fermentation broth is sent to the ceramic membrane for filtration again, the filtration temperature is controlled below 35°C, the filtration pressure is 0.25Mpa, and it is concentrated to 4 times. The obtained ceramic membrane clear liquid enters the next process, the remaining concentrated liquid is returned to the fermentation tank, and fresh culture medium and mature seed liquid are added to the fermentation tank for continued fermentation. After another 60 hours of fermentation, the fermentation liquid is sent to the ceramic membrane for filtration. The filtration temperature is controlled below 35°C, the filtration pressure is 0.25Mpa, and the concentration is 4 times. Softened water is added for filtration, and the amount of water added is three times that of the remaining concentrated solution. Finally, the ceramic membrane permeate and the ceramic membrane concentrated solution are obtained respectively. The obtained ceramic membrane clear solution enters the next process, and the remaining ceramic membrane concentrated solution is used to prepare feed, in which the unconsumed substrate in the amino acid clear solution is recovered by a small pore size nanofiltration membrane with a pore size of 4-5nm, and then it is further used for fermentation.

[0027] Example 2

[0028] Take lysine fermentation broth as an example: First, add 65% of the volume of the fermentation tank culture medium to the fermentation tank. After sterilization, the mature seed liquid is connected to the culture medium of the fermentation tank for fermentation culture. The inoculation amount of the seed liquid is 12% of the volume of the fermentation tank culture medium. After inoculation, control the appropriate temperature, pH and ventilation volume for fermentation culture. After 48 hours of fermentation culture, the fermentation broth is sent to the ceramic membrane for filtration, the filtration temperature is controlled below 35°C, the filtration pressure is 0.3Mpa, and it is concentrated to 4.5 times. The obtained ceramic membrane clear liquid enters the next process, the remaining concentrated liquid is returned to the fermentation tank, and fresh culture medium and mature seed liquid are added to the fermentation tank for continued fermentation. After continuing fermentation culture for 36 hours, the fermentation broth is sent to the ceramic membrane for filtration again, the filtration temperature is controlled below 35°C, the filtration pressure is 0.3Mpa, and it is concentrated to 4.5 times. The obtained ceramic membrane clear liquid enters the next process, the remaining concentrated liquid is returned to the fermentation tank, and fresh culture medium and mature seed liquid are added to the fermentation tank for continued fermentation. After running like this for 9 consecutive days, no fresh culture medium is added to the fermentation tank. After the last batch of fermentation matures, the fermentation liquid is sent to the ceramic membrane for filtration. The filtration temperature is controlled below 35°C, the filtration pressure is 0.3Mpa, and the concentration is 4.5 times. Softened water is added for filtration, and the amount of water added is three times that of the remaining concentrated liquid. Finally, ceramic membrane permeate and ceramic membrane concentrated liquid are obtained respectively. The obtained ceramic membrane clear liquid enters the next process, and the remaining ceramic membrane concentrated liquid is used to prepare feed, in which a small-pore nanofiltration membrane with a pore size of 4-5nm is used to recover the unconsumed substrate in the amino acid clear liquid and continue to be used for fermentation.

[0029] Example 3

[0030] Take lysine fermentation broth as an example: First, add 65% of the volume of the fermentation tank culture medium to the fermentation tank. After sterilization, the mature seed liquid is connected to the culture medium of the fermentation tank for fermentation culture. The inoculation amount of the seed liquid is 14% of the volume of the fermentation tank culture medium. After inoculation, control the appropriate temperature, pH and ventilation volume for fermentation culture. After 48 hours of fermentation culture, the fermentation broth is sent to the ceramic membrane for filtration. The filtration temperature is controlled below 35°C, the filtration pressure is 0.35Mpa, and it is concentrated to 5 times. The obtained ceramic membrane clear liquid enters the next process, and the remaining concentrated liquid is returned to the fermentation tank, and fresh culture medium and mature seed liquid are added to the fermentation tank for continued fermentation. After continuing fermentation culture for 36 hours, the fermentation broth is sent to the ceramic membrane for filtration again. The filtration temperature is controlled below 35°C, the filtration pressure is 0.35Mpa, and it is concentrated to 5 times. The obtained ceramic membrane clear liquid enters the next process, and the remaining concentrated liquid is returned to the fermentation tank, and fresh culture medium and mature seed liquid are added to the fermentation tank for continued fermentation. After 12 days of continuous operation, no fresh culture medium is added to the fermentation tank. After the last batch of fermentation matures, the fermentation liquid is sent to the ceramic membrane for filtration. The filtration temperature is controlled below 35°C, the filtration pressure is 0.35Mpa, and the concentration is 5 times. Softened water is added for filtration, and the amount of water added is three times the remaining concentrated liquid. Finally, ceramic membrane permeate and ceramic membrane concentrated liquid are obtained respectively. The obtained ceramic membrane clear liquid enters the next process, and the remaining ceramic membrane concentrated liquid is used to prepare feed, in which a small-pore nanofiltration membrane with a pore size of 4-5nm is used to recover the unconsumed substrate in the amino acid clear liquid and continue to be used for fermentation.

[0031] Comparative Example 1

[0032] Lysine was prepared according to the method of Example 1, except that fresh culture medium was not added to the fermenter during the fermentation culture, the fermenter was discharged after 60 hours of fermentation culture, the lysine concentration of the discharged tank and the lysine concentration of the intermediate discharge were measured, and the single tank output, acid supply per unit time and equipment utilization were calculated.

[0033]

[0034]

[0035] It can be seen from Table 1 that the method of the present invention can prolong the fermentation cycle and significantly improve the acid supply per unit time and equipment utilization rate.

[0036] The method for continuous fermentation of lysine provided by the present invention greatly prolongs the fermentation culture time and improves the metabolic capacity of lysine bacteria. Under the condition that the equipment does not fail and the culture medium is not contaminated with bacteria, the fermentation can continue, thereby increasing the acid supply per unit time, improving the equipment utilization rate, and thus reducing the lysine fermentation cost.

Claims

1. A process and device for producing amino acids by amino acid fermentation coupled with membrane separation technology, characterized in that: The steps include: Step 1: Inoculate the fermentation seeds of the amino acid-producing strain into a sterilized fermentation tank for cultivation; Step 2: When the fermentation reaches the late logarithmic growth phase or the mature phase, the amino acids are separated using membrane separation technology, and the bacteria trapped by the membrane and other components are returned to the tank for further fermentation; In the third step, the unconsumed substrate in the amino acid clear liquid is recovered by a nanofiltration membrane and further used for fermentation; at the same time, fresh culture medium is added to the fermentation tank to keep the volume of the fermentation liquid in the tank constant.

2. The process and device for producing amino acids by amino acid fermentation coupled with membrane separation technology according to claim 1, characterized in that: The amino acids are selected from the group consisting of glutamic acid, lysine, threonine, leucine, isoleucine, serine, tryptophan, methionine, phenylalanine and the like.

3. The process and device for producing amino acids by amino acid fermentation coupled with membrane separation technology according to claim 1, characterized in that: The membrane separation technology can be an inorganic membrane or an organic membrane. Among inorganic membranes, a ceramic membrane is preferred, and the pore size of the ceramic membrane is preferably 4-500nm; the pressure is 0.1-0.5Mpa; the flow rate on the membrane surface is 2-5m / s; and the concentration multiple is 1 to 10 times.

4. The process and device for producing amino acids by amino acid fermentation coupled with membrane separation technology according to claim 1, characterized in that: The nanofiltration adopts a ceramic nanofiltration membrane with a pore size range of 4-10nm and a filtration pressure of 0.4-4.0Mpa.

5. The process and device for producing amino acids by amino acid fermentation coupled with membrane separation technology according to claim 1, characterized in that: The method also includes regularly discharging a portion of the membrane filtration concentrate, which can be used to prepare feed or fertilizer.

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