Photoaerobic organism complete mixing reactor for synthesizing purple membrane protein by enriching halophilic microorganisms
By using a photo-aerobic biological fully mixed reactor, combined with the decoupled control and temporal regulation of light, oxygen, and heat, the problem of high synthesis cost of purple membrane protein was solved, and the efficient enrichment of halophilic microorganisms and stable synthesis of purple membrane protein were achieved, thus improving yield and quality.
Patent Information
- Application Number
- CN202511603361.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-04
- Publication Date
- 2026-03-06
AI Technical Summary
The high cost of purple membrane protein synthesis makes it impossible to meet the market's mass production needs. The high cost of synthetic culture media required for the growth of halophilic microorganisms and their sterilization specificity make them unsuitable for large-scale use. Furthermore, the synthesis of purple membrane protein is affected by environmental factors such as light and aeration, and there is a lack of efficient reactor operation modes.
Design a photo-aerobic biological fully mixed reactor, including a biological reaction zone and a bottom light management zone, to provide an environment with constant temperature, controllable dissolved oxygen, uniform nutrition, and sufficient light. Complete mixing is achieved through a stirring paddle and a central aeration disc. By combining the decoupled control and time-sequential regulation strategy of light, oxygen, and heat, the reactor can achieve efficient enrichment of halophilic microorganisms and efficient synthesis of purple membrane proteins.
It has achieved efficient enrichment of halophilic microorganisms and stable synthesis of purple membrane proteins at low cost, breaking through the bottleneck of synergistic optimization of cell growth and product synthesis, improving yield and quality uniformity, and enhancing the energy efficiency and system versatility of the production process.
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Figure CN121610334A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of microbial application technology, specifically to a photoaerobic biological fully mixed reactor for enriching halophilic microorganisms to synthesize purple membrane proteins. Background Technology
[0002] In actual production, the high cost of purple membrane protein synthesis makes it impossible to meet market demand for mass production. Large-scale production of purple membrane protein has become a hot topic of widespread concern in the energy revolution field.
[0003] Purple membrane protein synthesis depends on the growth and metabolism of halophilic microorganisms and is influenced by nutrients in a high-salt environment. Currently, the synthetic culture media required for the growth of halophilic microorganisms not only need glycerol and amino acid complex sources (tryptone, casein amino acids), but also various growth factors (yeast extract) and the trace element Mn. 2+ Zn 2+ Fe 2+ and Cu 2+ As cofactors in enzyme systems, nutrient culture media (amino acids and vitamins) require high costs on a laboratory scale, and their sterilization specificity is unlikely to promote large-scale use of synthetic media. Furthermore, the synthesis of purple membrane proteins is affected by environmental factors such as light and aeration, and currently, an efficient reactor operation mode has not been established to achieve efficient enrichment of halophilic microbial communities.
[0004] How to achieve efficient enrichment of halophilic microorganisms and production of purple membrane proteins under low-cost conditions is an urgent problem to be solved in the practical engineering applications of the field of microbial application technology. Summary of the Invention
[0005] To overcome the above technical problems, the present invention aims to provide a photo-aerobic biological hybrid reactor for enriching halophilic microorganisms to synthesize purple membrane proteins. This hybrid reactor can obtain halophilic microbial communities that efficiently synthesize purple membrane proteins, provide the aerobic / photosynthetic conditions required for the synthesis process of purple membrane proteins, provide sufficient salt and nutrients to the halophilic microbial communities, and realize a reactor operation mode for low-cost, stable and efficient synthesis of purple membrane proteins.
[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A photoaerobic biomixed reactor for enriching purple membrane proteins synthesized by halophilic microorganisms includes a bioreactor zone and a bottom light management zone; The bioreactor zone is the core chamber for completing microbial cell proliferation, metabolic regulation, and the synthesis of target products (purple membrane protein). It provides a constant temperature, controllable dissolved oxygen, uniform nutrition, and sufficient light environment for halophilic microbial communities, so as to achieve precise control of high-density culture and efficient induction of bacteria. The bottom light management area is used to efficiently, uniformly and controllably provide light energy of a specific wavelength (200-700nm) and an intensity of >1000 lux to the bioreaction zone, serving as a key environmental signal to induce halophilic microorganisms to synthesize purple membrane proteins, while solving the problem of heat dissipation of the light source and avoiding heat transfer affecting the temperature stability of the bioreaction zone.
[0007] The bioreactor zone includes a reactor body, a stirring paddle, a central aeration disc, a water bath insulation layer, an inlet, an outlet 1, an outlet 2, and an exhaust port. A dissolved oxygen meter and an air vent are located at the top of the reactor body. The stirring paddle and the central aeration disc work together to achieve "complete mixing" and efficient gas-liquid mass transfer. The stirring paddle is mounted on the shaft of the reactor drive motor and suspended in the center of the reactor body. The central aeration disc surrounds and is fixed to the bottom central shaft of the stirring paddle or is placed directly in the center of the bottom of the reactor body and is connected to an external air pump or gas mixing system through a pipe. The water bath insulation layer is wrapped around the outside of the reactor body. The water bath insulation layer is equipped with an independent circulating water inlet and outlet to form an independent circulating water system. The temperature is controlled by circulating water through an external constant temperature water bath. The probe of the dissolved oxygen meter penetrates through the top cover of the reactor body and is sealed and fixed with a threaded structure, deeply immersed in the culture medium, to monitor the dissolved oxygen concentration in the culture medium in real time. The inlet, outlet 1, outlet 2, and vent are used to feed, sample, harvest, and clean the reactor body; the inlet is located at the top of the reactor body and is connected to the culture medium.
[0008] Outlet 1 and outlet 2 are located at different heights, which facilitates sampling or transferring cultures at different liquid levels; The vent is located at the bottom of the reactor body and is connected to a pipe. It is used to completely empty the contents of the reactor body after the batch is finished.
[0009] The bottom light management area includes a heat dissipation component and a bottom light source; The heat dissipation component is an integrated active cooling system made of highly thermally conductive aluminum alloy or copper material. It is in direct contact with the substrate of the bottom light source to increase the contact area with the air. A forced air cooling fan is installed at the bottom of the heat dissipation component to accelerate airflow and remove the heat generated on the board surface.
[0010] A method for using a photoaerobic biological fully mixed reactor for enriching halophilic microorganisms to synthesize purple membrane proteins includes the following steps; Halophilic microorganisms, successfully domesticated and cultured from salt lake sediment, were inoculated into a photoaerobic biomixed reactor. While maintaining the inorganic nutrient composition of the synthetic medium, carbon and nitrogen sources, supplied through the reactor's side inlet, replaced high-cost amino acids and vitamins. To achieve efficient enrichment of the halophilic microorganisms and high-yield synthesis of their purple membrane protein, a sequencing batch reactor (SBR) culture mode was employed. A time-sequential control strategy was used to regulate the light intensity and dissolved oxygen levels at the bottom of the reactor, enabling efficient enrichment of the halophilic microorganisms and efficient, stable synthesis of the purple membrane protein. This strategy provided suitable light intensity and high dissolved oxygen during the exponential growth phase to maximize cell biomass, and then transitioned to low dissolved oxygen conditions in the later stationary phase to precisely induce and enhance purple membrane protein synthesis. This two-stage decoupled regulation successfully overcame the challenge of metabolic resource competition between microbial proliferation and target product synthesis.
[0011] The method for culturing halophilic microorganisms in salt lake sediment involves taking 3-8g of salt lake sediment and using casein amino acids, yeast extract, and sodium citrate as nutrient sources. The sediment is then acclimated at a mesophilic temperature of 37-39℃ in an inorganic nutrient environment. The inoculum size of the halophilic microorganisms is 5%-15%, and the total volume is 100-150ml (250ml Erlenmeyer flask). The bacterial density (OD600) in the shake flask culture medium is monitored periodically until it reaches a stable period, at which point the successfully acclimated halophilic microbial community is obtained.
[0012] The inorganic nutrients are MgSO4·7H2O at a concentration of 15-25 g / L, KCl at a concentration of 1.0-3.0 g / L, FeSO4 at a concentration of 5-20 ppm, and NaCl at a concentration of 150-250 g / L. The supplementary carbon and nitrogen sources are 1.0–10 g / L sodium acetate and 0.2–8.0 g / L ammonium chloride, used to prepare the synthetic culture medium.
[0013] The photoaerobic biological complete mixed reactor sequential batch culture mode includes the following program for each stage: water inlet, reaction, stabilization, and effluent, with continuous light supply. In specific operation, water enters the biological reaction zone through the inlet on the side of the reactor. After the reaction is stabilized, water is discharged through the outlet on the side of the reactor. The outlet height can be selected according to the liquid level. Each stage has a cycle of 6 days. The first 3 days provide high aeration to rapidly consume the substrate and enrich the amount of halophilic microorganisms. The last 3 days provide low aeration to reduce agitation and maintain the aerobic mode to induce the rapid maturation and synthesis of purple membrane proteins.
[0014] The nutrient source components and concentrations are as follows: casein amino acid concentration is 5-9 g / L, yeast extract concentration is 7.5-12 g / L, and sodium citrate concentration is 1-5 g / L; the shake flask culture medium is used for enrichment culture at 37℃ for 7 days, and the OD600 in the shake flask culture medium is measured to be 1.0-1.5.
[0015] The enriched halophilic microbial community was calculated based on the relative abundance of dominant species at the genus level: Halomonas accounted for more than 80%, with the remainder being non-enriched miscellaneous bacteria.
[0016] The inorganic nutrients are: MgSO4·7H2O at a concentration of 15-25 g / L, KCl at a concentration of 1.0-3.0 g / L, FeSO4 at a concentration of 5-20 ppm, and NaCl at a concentration of 150-250 g / L in 1 L of culture medium; the supplemented carbon and nitrogen sources are 1.0-10 g / L sodium acetate and 0.2-8.0 g / L ammonium chloride, and the pH is adjusted to 7.0-7.2 to prepare the synthetic culture medium.
[0017] The bottom light source provides continuous illumination, and the heat dissipation component ensures rapid heat dissipation. The continuous illumination level is 40W-100W white light, providing the light conditions required for the synthesis of purple membrane proteins. The high aeration strategy has an aeration rate of 1.0-1.2 vvm and a rotation speed of 150-200 r, while the low aeration strategy has an aeration rate of 0.6-1.0 vvm and a rotation speed of 80-100 r. The high aeration strategy achieves rapid proliferation of microorganisms in the exponential phase, while the low aeration strategy aims to further induce the synthesis of purple membrane proteins at the end of the stationary phase based on the increased cell biomass, thereby improving the synthesis level of purple membrane proteins. The rotation speed and aeration rate are jointly controlled by the agitator and the central aeration disc, respectively.
[0018] The beneficial effects of this invention are: This invention utilizes a photo-aerobic biological fully mixed reactor to obtain a halophilic microbial community that efficiently synthesizes purple membrane proteins. It provides the aerobic / photosynthetic conditions required for the synthesis of purple membrane proteins, and supplies the halophilic microbial community with sufficient salt and nutrients, thereby achieving a reactor operation mode for low-cost, stable, and efficient synthesis of purple membrane proteins.
[0019] The reactor system and method provided by this invention represent a significant advancement by overcoming the technical bottleneck of the difficulty in co-optimizing cell growth and product synthesis in traditional culture methods. Through the organic combination of decoupled control of light, oxygen, and heat, along with a time-sequential regulation strategy, not only is an order-of-magnitude increase in purple membrane protein yield achieved, but also unexpectedly superior results are achieved in product quality uniformity, production process energy efficiency, and system versatility. This is not a simple improvement on existing technologies, but rather provides a synergistically regulated production mode and platform equipment for industrial biotechnology based on halophilic microorganisms and their photodriven metabolism. Attached Figure Description
[0020] Figure 1 This is a diagram of a photoaerobic biological complete mixed reactor.
[0021] Figure 2 This diagram illustrates the enrichment of halophilic microorganisms and the content of purple membrane proteins.
[0022] Figure descriptions: 1. Biological reaction zone, 2. Bottom light management zone, 3. Stirrer, 4. Central aeration disc, 5. Water bath insulation layer, 6. Heat dissipation components, 7. Bottom light source, 8. Inlet, 9. Outlet 1, 10. Outlet 2, 11. Dissolved oxygen meter, 12. Vent, 13. Drain. Detailed Implementation
[0023] The present invention will be further described in detail below with reference to the embodiments.
[0024] like Figure 1 As shown, a photo-aerobic bio-mixed reactor is used to enrich halophilic microorganisms that synthesize purple membrane proteins; the photo-aerobic bio-mixed reactor includes a bioreactor zone 1 and a bottom light management zone 2; The bioreactor 1 includes a stirrer 3, a central aeration disc 4, a water bath insulation layer 5, an inlet 8, an outlet 9 and 10, an exhaust port 13, and a dissolved oxygen meter 11 and an air vent 12 are installed at the top of the bioreactor 1. The bottom light management area 2 includes a heat dissipation component 6 and a bottom light source 7; Furthermore, considering the temperature changes during reactor operation, a water bath insulation layer 5 is designed to provide a constant temperature of 37℃; Furthermore, to ensure thorough mixing of halophilic microorganisms with the substrate, a stirrer 3 and a central aeration disc 4 are installed. Furthermore, considering the sensitivity of halophilic microorganisms to dissolved oxygen during the synthesis of purple membrane proteins, a dissolved oxygen meter 11 was set up to monitor changes in dissolved oxygen in real time. Furthermore, considering the dependence of halophilic microorganisms on photoinduced conditions during the synthesis of purple membrane proteins, a bottom light management area 2 is set up, and a bottom light source 7 provides white light illumination and supports multi-spectral adjustment to optimize photonutrient culture conditions; the heat dissipation component 6 ensures rapid heat dissipation.
[0025] The following example of a photoaerobic biological fully mixed reactor further illustrates the method for enriching halophilic microorganisms to synthesize purple membrane proteins.
[0026] (1) Acclimation of halophilic microorganisms in salt lake sediment: Take an appropriate amount of salt lake sediment and use 5-9 g / L casein amino acids, 7.5-12 g / L yeast extract, and 1-5 g / L sodium citrate as nutrient sources. Acclimate the halophilic microbial community at 37℃ for 7 days in 1L inorganic culture medium. After the bacterial density OD600 in the shake flask culture medium reaches the stable period, the halophilic microbial community is obtained. The relative abundance of dominant species in the halophilic microorganisms at the genus level is as follows: Halomonas 80.93%, Bacillus halophilus 4.35%, halophilic mineral bacteria 4.23%, and the remainder are non-enriched miscellaneous bacteria. (2) Preparation of synthetic culture medium: In 1L of culture medium, the inorganic culture components are 15-25g / L MgSO4·7H2O, 1.0-3.0g / L KCl, 5-20ppm FeSO4, and 150-250g / L NaCl; the carbon source and nitrogen source are 1.0~10 g / L sodium acetate and 0.2~8.0 g / L ammonium chloride, respectively, and the pH is adjusted to 7.0-7.2; (3) Sequencing batch culture in a photo-aerobic biological complete mixing reactor: 100 ml of halophilic microorganisms were inoculated into the photo-aerobic biological complete mixing reactor, and 900 ml of synthetic culture medium was introduced from the inlet on the side of the photo-aerobic biological complete mixing reactor. Sequencing batch culture was carried out in the reaction device. The procedure of each stage included: water inlet, reaction, stabilization and water outlet. The cycle of each stage was 6 days. The first 3 days provided high aeration to rapidly consume the substrate and enrich the halophilic microorganisms. The last 3 days provided low aeration to reduce stirring disturbance and maintain the oxygen consumption mode to induce the rapid maturation and synthesis of purple membrane protein. Finally, the well-mixed bacterial solution was collected.
[0027] (4) Determination of purple membrane protein: The OD600 of the enriched bacterial solution was determined. The harvested bacterial cells were centrifuged and then mixed with ultrapure water to break the cells and form a bacterial suspension, releasing purple membrane protein fragments. The bacterial suspension and alkaline fixative were mixed evenly in the dark at a volume ratio of 9:1. The concentration of purple membrane protein was determined using a UV-Vis spectrophotometer.
[0028] The ultraviolet-visible spectrophotometer measurement method in step (4) is as follows: The absorbance of the solution at 568 nm was measured under dark conditions. This indicates that the absorbance at 568 nm was measured again after 48 hours of illumination. This indicates that, based on the purple membrane protein's molecular weight of 26 kDa and molar extinction coefficient of 63000 M, -1 cm -1 The concentration of purple membrane protein can be calculated using the following formula: Purple membrane protein (g / L) = 26000 × .
[0029] The enrichment of halophilic microorganisms and the content of purple membrane proteins were determined by ultraviolet-visible spectrophotometry, as shown below. Figure 2 As shown; On day 8 under light conditions, 249.06 mg / L of purple membrane protein was synthesized. After enrichment culture in a photoaerobic biological complete mixed reactor, the diversity and abundance of halophilic microbial community were significantly improved. The dominant community changed from 80.93% of Halomonas to a multifunctional community enriched mainly by 36.74% of Halomonas.
Claims
1. A photo-aerobic bioreactor for enrichment of halophilic microorganisms synthesizing purple membrane proteins, characterized in that, It comprises a biological reaction zone (1) and a bottom light management zone (2); The biological reaction zone (1) is used for completing microbial cell proliferation, metabolic regulation and purple membrane protein synthesis, providing a constant temperature, controllable dissolved oxygen, uniform nutrition and sufficient light environment for halophilic microbial flora to realize precise control of high-density cultivation and high-efficiency induction of bacteria; The bottom light management zone (2) is used for efficiently, uniformly and controllably providing 200-700nm wavelength and >1000lux intensity light energy to the biological reaction zone (1) as an environmental signal for inducing halophilic microorganisms to synthesize purple membrane proteins.
2. The photo-aerobic bioreactor for enrichment of halophilic microorganisms for synthesis of purple membrane proteins according to claim 1, characterized in that, The biological reaction zone (1) comprises a reactor body, a stirring paddle (3), a central aeration disc (4), a water bath insulation layer (5), a water inlet (8), a water outlet one (9), a water outlet two (10) and a emptying port (13), and a dissolved oxygen meter (11) and a vent (12) are arranged at the top of the reactor body; the stirring paddle (3) and the central aeration disc (4) work cooperatively to realize "complete mixing" and efficient gas-liquid mass transfer, the stirring paddle (3) is installed on the rotating shaft of a reactor driving motor and suspended in the center of the reactor body, and the central aeration disc (4) is surrounded and fixed to the top central shaft of the stirring paddle (3) or directly arranged at the center of the top of the reactor body and connected to an external air pump or gas mixing system through a pipeline; The water bath insulation layer (5) is wrapped around the periphery of the reactor body, and independent circulating water is arranged on the water bath insulation layer (5) to realize circulating temperature control through an external constant temperature water bath pot; the probe of the dissolved oxygen meter (11) penetrates the reactor body top cover and is fixed and immersed in the culture solution in the reactor body through a threaded structure to realize real-time online monitoring of the dissolved oxygen concentration in the culture solution; The water inlet (8), the water outlet one (9), the water outlet two (10) and the emptying port (13) realize feeding, sampling, harvesting and cleaning of the reactor body; the water inlet (8) is located at the upper part of the reactor body and connected to a culture medium; The water outlet one (9) and the water outlet two (10) are located at different heights to facilitate sampling or transferring the culture at different liquid levels; The water inlet (8) is located above the water outlet one (9) and the water outlet two (10), and the water inlet (8), the water outlet one (9) and the water outlet two (10) penetrate the water bath insulation layer (5) and communicate with the reactor body; The emptying port (13) is located at the bottom of the reactor body and connected to a pipeline to completely empty the contents in the reactor body after batch completion.
3. A photo-aerobic bioreactor for enrichment of halophilic microorganisms synthesizing purple membrane proteins according to claim 2, characterized in that, The bottom light management zone (2) comprises a heat dissipation assembly (6) and a bottom light source (7); The heat dissipation assembly (6) is made of high-thermal-conductivity aluminum alloy or copper material and directly contacts with the substrate of the bottom light source (7) to increase the contact area with air; a forced air cooling fan is installed at the bottom of the heat dissipation assembly (6) to accelerate air flow and take away the heat generated on the panel surface.
4. Use of a photo-aerobic bioreactor for the enrichment of halophilic microorganisms for the synthesis of purple membrane proteins according to any one of claims 1 to 3, characterized in that, It comprises the following steps: The salt lake sediment is inoculated with the successfully domesticated halophilic microorganism in a photo-oxygenic biological complete mixing reactor; the inorganic nutrient components in the synthetic culture medium are maintained unchanged, and the carbon source and nitrogen source are supplemented through the water inlet (8) on the side of the reactor; in order to realize the efficient enrichment of the halophilic microorganism and the high-yield synthesis of the purple membrane protein, the reactor is operated in a sequencing batch culture mode, the light intensity of the bottom light source (7) and the dissolved oxygen level are controlled through a time sequence regulation strategy, so as to precisely induce and strengthen the synthesis of the purple membrane protein.
5. The method according to claim 4, wherein the method is characterized in that, The salt lake sediment halophilic microorganism culture method takes 3-8 g of salt lake sediment, uses casein amino acid, yeast extract and sodium citrate as the nutrient source, adopts medium temperature domestication of 37-39℃ in the inorganic nutrient components, the inoculation amount of the salt lake halophilic microorganism is 5%-15%, the total volume is 100-150 ml, and after the bacterial density OD600 in the shake flask culture medium reaches the stable period, the successfully domesticated halophilic microorganism flora is obtained.
6. The method according to claim 4, wherein the method is characterized by, The inorganic nutrient components include MgSO4·7H2O, KCl, FeSO4 and NaCl; The concentration of MgSO4·7H2O is 15-25 g / L, the concentration of KCl is 1.0-3.0 g / L, the concentration of FeSO4 is 5-20 ppm, and the concentration of NaCl is 150-250 g / L; The supplemented carbon source and nitrogen source are 1.0-10 g / L of sodium acetate and 0.2-8.0 g / L of ammonium chloride, and the synthetic culture medium is configured.
7. The method according to claim 4, wherein the method is characterized by, The sequencing batch culture mode of the photo-oxygenic biological complete mixing reactor includes the procedures of water feeding, reaction, stabilization and water discharge in each stage, and light is continuously provided. In the specific operation process, the water feeding enters the biological reaction zone (1) through the water inlet (8) on the side of the reactor, and after the stable operation and reaction are completed, the water is selectively discharged from the water outlet one (9) and the water outlet two (10) at different heights on the same side according to the liquid level height, and each stage cycle is 6 days, the high aeration amount is provided in the first 3 days to rapidly consume the substrate and enrich the halophilic microorganism amount, and the low aeration amount is provided in the last 3 days to reduce the stirring disturbance and maintain the oxygen consumption mode to induce the rapid mature synthesis of the purple membrane protein.
8. The method according to claim 4, wherein the method is characterized by, The nutrient source components and concentrations are as follows: the concentration of casein amino acid is 5-9 g / L, the concentration of yeast extract is 7.5-12 g / L, and the concentration of sodium citrate is 1-5 g / L; the shake flask culture medium is enriched and cultured at 37℃ for 7 days, and the OD600 in the shake flask culture medium is 1.0-1.
5.
9. The method according to claim 4, wherein the method is characterized by, The relative abundance of the dominant species of the halophilic microorganism flora at the genus level is calculated as follows: the Halomonas is more than 80%, and the rest is miscellaneous bacteria which are not the enrichment objects; The inorganic nutrient components in 1 L of the culture medium are as follows: the concentration of MgSO4·7H2O is 15-25 g / L, the concentration of KCl is 1.0-3.0 g / L, the concentration of FeSO4 is 5-20 ppm, and the concentration of NaCl is 150-250 g / L; the supplemented carbon source and nitrogen source are 1.0-10 g / L of sodium acetate and 0.2-8.0 g / L of ammonium chloride, the pH is adjusted to 7.0-7.2, and the synthetic culture medium is configured.
10. The method according to claim 4, wherein the method is characterized by, The bottom light source (7) provides continuous light, and the heat dissipation assembly (6) ensures rapid heat dissipation. The continuous light level is 40W-100W white light, which provides the light conditions required for purple membrane protein synthesis. The high aeration strategy is that the aeration volume is 1.0-1.2vvm, and the rotating speed is 150-200r. The low aeration strategy is that the aeration volume is 0.6-1.0vvm, and the rotating speed is 80-100r. The rotating speed and the aeration volume are jointly controlled by the stirring paddle (3) and the central aeration disc (4) respectively.