Method for biosynthesizing ectoine by using sequencing batch anaerobic membrane bioreactor

Abstract

The invention discloses a method for biosynthesizing ectoine by using a sequencing batch anaerobic membrane bioreactor. The method comprises the following steps: adding an acidic trace element solution, an alkaline trace element solution, KHCO3, KH2PO4, CaCl2 and MgSO4. 7H2O into deionized water to prepare an inorganic culture medium; monosodium glutamate and sodium chloride are added into the inorganic culture medium and then introduced from a water inlet in the top of the sequencing batch anaerobic membrane bioreactor; inoculating a proper amount of halomonas flora in the sequencing batch anaerobic membrane bioreactor; taking an inorganic culture medium, and adjusting the pH value of the inorganic culture medium to 7.2-8.0 by using NaOH; introducing the adjusted inorganic culture medium into a sequencing batch anaerobic membrane bioreactor inoculated with a halomonas flora; finally, tetrahydropyrimidine is synthesized. According to the method disclosed by the invention, the enrichment amount of halomonas strains is remarkably increased, the operation process of a bacterial milking method is simplified, and the synthesis efficiency of ectoine is effectively improved.

Description

Technical Field

[0001] The invention relates to the technical field of microbial application, and in particular to a method for biosynthesizing ectoine by utilizing a sequencing batch anaerobic membrane bioreactor. Background Art

[0002] Ectinoline, also known as methylpyrimidine carboxylic acid, is a compatible solute originally found in halophilic bacteria and salt-tolerant microorganisms. Ectinoline can not only regulate the osmotic pressure between microorganisms and the external environment, but also help stabilize enzymes, DNA, cytoplasmic membranes and even the entire cell in the cell. Ectinoline plays an important role in the adaptation of microorganisms to high-salt environments. In addition to being an osmotic pressure protectant, ectoine can also be used as a biological stabilizer and skin protectant; it is also widely used in the biomedical field, such as Alzheimer's disease, allergic rhinitis and conjunctivitis, lung infection, gastrointestinal inflammation, skin trauma, etc. In the field of new cosmetics research and development, ectoine can protect and stabilize cell membranes from damage by surfactants. It can also be used as a moisturizer that is more effective than glycerin to prevent dehydration of human skin and improve the skin's regeneration ability.

[0003] At present, ectoine production mainly uses halophilic Halomonas and Chromohalobacter bacteria. The biological preparation processes of ectoine include "bacterial milking" method, batch fermentation method, batch fed fermentation method, etc. The "bacterial milking" method cleverly utilizes the characteristics of halophilic bacteria synthesizing ectoine under high salt conditions and releasing ectoine under low salt conditions, and has been successfully applied to the production of ectoine.

[0004] In summary, the defects of the prior art are that in the "bacterial milking" production process, the repeated impact of hypertonicity and hypotonicity has the problems of cumbersome operation and low efficiency of synthesis of ectoine. Summary of the invention

[0005] In order to overcome the above technical problems, the purpose of the present invention is to provide a method for biosynthesizing ectoine using a sequencing batch anaerobic membrane bioreactor, which significantly increases the enrichment of Halomonas species, simplifies the operating procedures of the "bacterial milking" method, and effectively improves the synthesis efficiency of ectoine.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] A method for biosynthesizing ectoine using a sequencing batch anaerobic membrane bioreactor comprises the following steps: Step (1): preparing an inorganic culture medium

[0008] Acidic trace element solution, alkaline trace element solution, KHCO3, KH2PO4, CaCl2, MgSO4·7H2O are added to deionized water to prepare an inorganic culture medium;

[0009] (2) Initial inoculum source enrichment

[0010] adding monosodium glutamate and sodium chloride to the inorganic culture medium, and then introducing them from the water inlet at the top of the sequencing batch anaerobic membrane bioreactor;

[0011] Inoculate an appropriate amount of Halomonas bacteria in a sequencing batch anaerobic membrane bioreactor;

[0012] (3) Reaction stage

[0013] A. Take the inorganic culture medium prepared in step (1) and adjust the pH to 7.2-8.0 with NaOH;

[0014] B. Introducing the inorganic culture medium adjusted in step A into a sequencing batch anaerobic membrane bioreactor inoculated with a Halomonas colony, and internally circulating and enriching the Halomonas colony: controlling the temperature of the reactor to be medium during continuous operation, and regularly monitoring the dissolved oxygen in the reactor;

[0015] C. After the internal circulation of the reactor is stable and the monitored OD600 value is maintained at a constant value, the growth of Halomonas enters the stable period from the logarithmic growth period, and the density of the microbial population is stably maintained at a high level; at this time, a sufficient amount of Halomonas population is enriched in the sequencing batch anaerobic membrane bioreactor; and finally tetrahydropyrimidine is synthesized.

[0016] In step (1), 0.5 mL of an acidic trace element solution is prepared, wherein Fe 2+ :Cu 2+ :Mn 2+ The ratio is 22:6:5;

[0017] Prepare 0.2 mL of alkaline trace element solution, 1.0 g of KHCO3, 0.05 g of KH2PO4, 0.226 g of CaCl2, and 0.2 g of MgSO4 7H2O; wherein the alkaline trace element solution is Na2MoO4·2H2O;

[0018] The above solution was added to 1 liter of deionized water to prepare an inorganic culture medium.

[0019] In the step (2), the concentration of monosodium glutamate is 5 to 40 g / L, and the concentration of sodium chloride is 50 to 200 g / L.

[0020] The method for obtaining the Halomonas bacteria group is as follows: activating the freeze-dried powder of the Halomonas bacteria, using tryptone as its nitrogen source, adopting medium-temperature enrichment culture in a shaking flask medium with a salt concentration of 5%, regularly monitoring the OD600 value in the shaking flask medium to reach a stable period, and then inoculating it into a sequencing batch anaerobic membrane bioreactor until the operation is stable, that is, obtaining the Halomonas bacteria group with stable bacterial content;

[0021] The salt mononas group contains the following bacteria calculated by the relative abundance of dominant species at the genus level: more than 80% of the salt mononas genus, and the remainder is miscellaneous bacteria that are not enriched objects; the internal circulation is 7 to 14 days for the biological bacteria group to attach to the surface of the industrial membrane.

[0022] The shake flask medium was cultured at a constant temperature of 34° C. for 7 days, and the OD600 in the shake flask medium was measured to be 1.0-1.2.

[0023] In step B of the reaction stage (3), the volume ratio of the Halomonas bacteria to the inorganic culture medium is 1:10.

[0024] In step C of the reaction stage (3), a high-salt culture solution and a low-salt water solution are circulated in the reaction device; the procedure of each cycle includes: water inlet, reaction, precipitation and water outlet; an operation cycle is 6 days, and a high-salt (20%) culture solution is introduced into the reactor for hypertonic shock in the first 48 hours, and the membrane intercepts the biological flora and then all the high-salt culture solution is discharged, and a low-salt (1%) water solution is introduced for hypotonic shock for 30 minutes, and the membrane intercepts the biological flora and then all the low-salt water solution is discharged, and then a high-salt (20%) culture solution is subjected to hypertonic shock for 24 hours, and this is repeated six times.

[0025] The structure of the sequencing batch anaerobic membrane bioreactor is as follows:

[0026] The reactor body includes a 2L plexiglass rectangular reactor, a rubber hose and a biofilm frame. The upper water inlet of the reactor is connected to the rubber hose and is connected to the feed trough 1 through a circulating peristaltic pump 2; the middle water outlet of the reactor is connected to the internal industrial membrane 4 and is connected to the water outlet trough 6 through a circulating peristaltic pump 5; the internal circulation direction is that the middle water outlet of the reactor is connected to the upper water inlet of the reactor through a rubber hose; considering the aerobic characteristics of the salt monocomonas group, a vent 7 is arranged on the upper part of the reactor.

[0027] Furthermore, considering that the low-salt solution must be introduced into the reactor after the high-salt culture solution is completely discharged, the reactor industrial membrane 4 is adjusted downward to the bottom of the membrane chamber to ensure that the low-salt solution can completely replace the original high-salt culture solution;

[0028] A membrane chamber is arranged inside the reactor, and an industrial membrane 4 is placed in the membrane chamber. The industrial membrane 4 is a PVDF (polyvinylidene fluoride) membrane.

[0029] Beneficial effects of the present invention:

[0030] The novel sequencing batch anaerobic membrane bioreactor adopted in the present invention significantly increases the enrichment of Halomonas species, simplifies the operation process of the "bacterial milking" method, and effectively improves the synthesis efficiency of ectoine.

[0031] Specifically, the salt mononas are effectively separated from the culture medium by a polyvinylidene fluoride (PVDF) industrial membrane, so that the salt mononas colony is attached and retained on the surface of the industrial membrane. This process ensures the physical isolation of the microorganisms from the culture medium, and provides a stable attachment matrix for the salt mononas, which is conducive to the growth and metabolic activities of the salt mononas. Furthermore, the hypertonic and hypotonic shock methods have the advantage of being able to be reused, thereby improving the utilization rate of the bacteria. This invention provides a new strategy for the biosynthesis of tetrahydropyrimidines, and provides a new perspective for the potential development of microorganisms in industrial applications. By further optimizing the internal structure of the traditional anaerobic membrane bioreactor, it is expected to achieve a more efficient and stable tetrahydropyrimidine production process. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a schematic diagram of the device structure for the application of a new sequencing batch anaerobic membrane bioreactor.

[0033] Figure 2 Schematic diagram of the intracellular ectoine content of Halomonas after high and low osmotic shock.

[0034] Description of the accompanying drawings: 1. Feed trough, 2. Water inlet pump, 3. Water bath, 4. Industrial membrane, 5. Water outlet pump, 6. Water outlet trough, 7. Vent. DETAILED DESCRIPTION

[0035] The present invention will be further described in detail below in conjunction with the accompanying drawings.

[0036] The structure of the anaerobic membrane bioreactor of the present invention is as follows Figure 1 As shown;

[0037] The reactor body includes a 2L organic glass rectangular reactor, a rubber hose and a biofilm frame. The upper water inlet of the reactor is connected to the rubber hose and connected to the feed tank 1 through a circulating peristaltic pump 2; the middle water outlet of the reactor is connected to the internal industrial membrane 4 and connected to the water outlet tank 6 through a circulating peristaltic pump 5; the internal circulation direction is that the middle water outlet of the reactor is connected to the upper water inlet of the reactor through a rubber hose; considering the aerobic characteristics of the salt monosaccharide group, a vent 7 is set on the upper part of the reactor;

[0038] Furthermore, considering the enrichment of the Halomonas bacteria, the original aeration chamber inside the reactor was removed so that the Halomonas bacteria could be enriched and retained on the industrial membrane;

[0039] Furthermore, considering that the low-salt solution must be introduced into the reactor after the high-salt culture solution is completely discharged, the reactor membrane frame is adjusted downward to the bottom of the membrane chamber to ensure that the low-salt solution can completely replace the original high-salt culture solution;

[0040] Furthermore, considering the temperature change during the operation of the reactor, the water bath chamber 3 is designed; the industrial membrane adopts PVDF (polyvinylidene fluoride) membrane.

[0041] In the present invention, the sequencing batch anaerobic membrane bioreactor is placed in a constant temperature room, and the temperature of the reactor during operation is 30°C.

[0042] The following is an introduction to the method of enriching Halomonas bacteria using an anaerobic membrane bioreactor through a specific application example.

[0043] (1) Preparation of inorganic culture medium

[0044] Add 0.5 mL of acidic trace element solution of FeSO4, CuSO4, and MnCl, 0.2 mL of alkaline trace element solution of Na2MoO4·2H2O, 1.0 g of KHCO3, 0.05 g of KH2PO4, 0.226 g of CaCl2, and 0.2 g of MgSO4·7H2O to 1 liter of deionized water to prepare the culture medium;

[0045] (2) Initial inoculum source enrichment

[0046] Adding monosodium glutamate and sodium chloride to the inorganic culture medium obtained in step (1), wherein the concentration of monosodium glutamate is 5 g / L and the concentration of sodium chloride is 200 g / L, and then introducing the monosodium glutamate and sodium chloride into the reactor from the top water inlet;

[0047] 100 mL of Halomonas colony is inoculated in a sequencing batch anaerobic membrane bioreactor, and the method for obtaining the Halomonas colony is as follows: activating freeze-dried powder of Halomonas species, using tryptone as its nitrogen source, and culturing it at a constant temperature of 34°C in a shaking flask medium with a salt concentration of 5% for 7 days, determining that the OD600 in the shaking flask medium is 1.0-1.2, and then inoculating it into the sequencing batch anaerobic membrane bioreactor until the operation is stable, so as to obtain a Halomonas colony with stable bacterial species content; the Halomonas colony contains the following bacterial species calculated by the relative abundance of dominant species at the genus level: Halomonas 79.42%, Pseudomonas 3.3%, Alcaligenes 2.28%, Corynebacterium 1.4%, Brevundimonas 1%, and the remainder is miscellaneous bacteria that are not enrichment objects; the internal circulation lasts for 7 days for the biological flora to attach to the surface of the biofilm.

[0048] (3) Reaction stage

[0049] A. Take the inorganic culture medium prepared in step (1) and adjust the pH to 7.2-8.0 with NaOH;

[0050] B. Introducing the inorganic culture medium in step A into a sequencing batch anaerobic membrane bioreactor inoculated with the Halomonas bacteria to enrich the Halomonas bacteria in an internal circulation: controlling the temperature of the reactor during continuous operation to be 30° C. and monitoring the dissolved oxygen in the reactor to be above 2 mg / L;

[0051] C. After the internal circulation of the reactor is stable and the monitored OD600 value is maintained at a constant value, the anaerobic batch reactor is enriched with halomonas bacteria capable of synthesizing tetrahydropyrimidine; high-salt culture solution and low-salt solution are circulated in the reaction device; the main procedures of each cycle include: water inlet, reaction, precipitation and water outlet; one operation cycle is 6 days, and high-salt (20%) culture solution is passed into the reactor for hypertonic shock for the first 48 hours, and all high-salt culture solution is discharged after the membrane intercepts the biological flora, and low-salt (1%) aqueous solution is passed for hypotonic shock for 30 minutes, and all low-salt solution is discharged after the membrane intercepts the biological flora, and then hypertonic shock is carried out in high-salt (20%) culture solution for 24 hours, and this is repeated six times.

[0052] In the present invention, the sequencing batch anaerobic membrane bioreactor is placed in a constant temperature room, and the temperature of the reactor during operation is 30°C.

[0053] (4) Measurement phase

[0054] The enriched bacterial liquid in step (1) is collected for OD600 determination, and the bacterial cells after shock in step (3) are collected for extraction of intracellular ectoine, and then ectoine is quantitatively detected using high performance liquid chromatography (HPLC).

[0055] The quantitative detection method of ectoine in the step (4) is: HPLC method, using the external standard method to calculate the peak area of ​​the spectrum, detection conditions: UV detector, detection wavelength 210nm, mobile phase using methanol / pure water (V / V, 70:30), Hypersil BDS-C18 column, 150×4.6mm, column temperature 30°C, pressure 96bar, injection 5μL, flow rate 1.0mL·min -1 , running time 5min.

[0056] After quantitative analysis by high performance liquid chromatography, the results of the synthesis of tetrahydropyrimidine by Halomonas under osmotic pressure shock are shown in Figure 2 First, the cells were cultured in high-salt culture medium (20%) for 48 hours to synthesize 33.40 mg / L of ectoine, and then subjected to hypotonic shock for 30 minutes in low-salt solution (1%), and then subjected to hypertonic shock for 24 hours to resynthesize ectoine. After 96 hours, the cells synthesized 124.67 mg / L of ectoine. After six shocks, 195.8 mg / L of ectoine could be produced in the cells.

Claims

1. A method for biosynthesizing ectoine using a sequencing batch anaerobic membrane bioreactor, characterized in that: The steps include: Step (1): Preparation of inorganic culture medium Acidic trace element solution, alkaline trace element solution, KHCO3, KH2PO4, CaCl2, MgSO4·7H2O are added to deionized water to prepare an inorganic culture medium; (2) Initial inoculum source enrichment adding monosodium glutamate and sodium chloride to the inorganic culture medium, and then introducing them from the water inlet at the top of the sequencing batch anaerobic membrane bioreactor; Inoculate an appropriate amount of Halomonas bacteria in a sequencing batch anaerobic membrane bioreactor; (3) Reaction stage A. Take the inorganic culture medium prepared in step (1) and adjust the pH to 7.2-8.0 with NaOH; B. Introducing the inorganic culture medium adjusted in step A into a sequencing batch anaerobic membrane bioreactor inoculated with a Halomonas colony, and internally circulating and enriching the Halomonas colony: controlling the temperature of the reactor to be medium during continuous operation, and regularly monitoring the dissolved oxygen in the reactor; C. After the internal circulation of the reactor is stable and the monitored OD600 value is maintained at a constant value, the growth of Halomonas enters the stable period from the logarithmic growth period, and the density of the microbial population is stably maintained at a high level; at this time, a sufficient amount of Halomonas population is enriched in the sequencing batch anaerobic membrane bioreactor; and finally tetrahydropyrimidine is synthesized.

2. The method for biosynthesizing ectoine using a sequencing batch anaerobic membrane bioreactor according to claim 1, characterized in that: In step (1), 0.5 mL of an acidic trace element solution is prepared, wherein Fe 2+ :Cu 2+ :Mn 2+ The ratio is 22:6:5; Prepare 0.2 mL of alkaline trace element solution, 1.0 g KHCO3, 0.05 g KH2PO4, 0.226 g CaCl2, and 0.2 g MgSO47H2O; wherein the alkaline trace element solution is Na2MoO4·2H2O; The above solution was added to 1 liter of deionized water to prepare an inorganic culture medium.

3. The method for biosynthesizing ectoine using a sequencing batch anaerobic membrane bioreactor according to claim 1, characterized in that: In the step (2), the concentration of monosodium glutamate is 5 to 40 g / L, and the concentration of sodium chloride is 50 to 200 g / L.

4. The method for biosynthesizing ectoine using a sequencing batch anaerobic membrane bioreactor according to claim 3, characterized in that: The method for obtaining the Halomonas bacteria group is as follows: activating the freeze-dried powder of the Halomonas bacteria, using tryptone as its nitrogen source, adopting medium-temperature enrichment culture in a shaking flask medium with a salt concentration of 5%, regularly monitoring the OD600 value in the shaking flask medium to reach a stable period, and then inoculating it into a sequencing batch anaerobic membrane bioreactor until the operation is stable, that is, obtaining the Halomonas bacteria group with stable bacterial content; The salt mononas group contains the following bacteria calculated by the relative abundance of dominant species at the genus level: more than 80% of the salt mononas genus, and the remainder is miscellaneous bacteria that are not enriched objects; the internal circulation is 7 to 14 days for the biological bacteria group to attach to the surface of the industrial membrane.

5. The method for biosynthesizing ectoine using a sequencing batch anaerobic membrane bioreactor according to claim 3, characterized in that: The shake flask medium was cultured at a constant temperature of 34° C. for 7 days, and the OD600 in the shake flask medium was measured to be 1.0-1.

2.

6. The method for biosynthesizing ectoine using a sequencing batch anaerobic membrane bioreactor according to claim 1, characterized in that: In step B of the reaction stage (3), the volume ratio between the Halomonas bacteria and the inorganic culture medium is 1:

10.

7. The method for biosynthesizing ectoine using a sequencing batch anaerobic membrane bioreactor according to claim 1, characterized in that: In step C of the reaction stage (3), a high-salt culture solution and a low-salt water solution are circulated in a reactor; the procedure of each cycle includes: water inlet, reaction, precipitation and water outlet; an operation cycle is 6 days, and a high-salt (20%) culture solution is introduced into the reactor for hypertonic shock in the first 48 hours, and the industrial membrane (4) intercepts the biological flora and then discharges all the high-salt culture solution, and then a low-salt (1%) water solution is introduced for hypotonic shock for 30 minutes, and the membrane intercepts the biological flora and then discharges all the low-salt water solution, and then a high-salt (20%) culture solution is subjected to hypertonic shock for 24 hours, and this is repeated six times.

8. The method for biosynthesizing ectoine using a sequencing batch anaerobic membrane bioreactor according to claim 1, characterized in that: The structure of the sequencing batch anaerobic membrane bioreactor is as follows: The reactor body comprises a 2L organic glass rectangular reactor, the upper water inlet of the reactor is connected to a rubber hose, and is connected to a feed tank (1) through a circulating peristaltic pump (2); the middle water outlet of the reactor is connected to an internal industrial membrane (4), and is connected to a water outlet tank (6) through a circulating peristaltic pump (5); the internal circulation direction is that the middle water outlet of the reactor is connected to the upper water inlet of the reactor through a rubber hose; and a vent (7) is arranged on the upper part of the reactor.

9. The method for biosynthesizing ectoine using a sequencing batch anaerobic membrane bioreactor according to claim 8, characterized in that: The reactor industrial membrane (4) is adjusted downward to the bottom of the membrane chamber to ensure that the low-salt solution can completely replace the original high-salt culture solution.

10. The method for biosynthesizing ectoine using a sequencing batch anaerobic membrane bioreactor according to claim 8, characterized in that: A membrane chamber is arranged inside the reactor, and an industrial membrane (4) is placed in the membrane chamber. The industrial membrane (4) adopts a PVDF (polyvinylidene fluoride) membrane.

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