Method for improving fermentation performance of halophilic bacteria under salt stress and application
By adding citric acid and betaine to the fermentation broth of senophora in a high-salt environment, the problem of inhibition of fermentation performance of senophora in a high-salt environment was solved, and its growth rate and product under high-salt conditions were significantly improved. The synthesis rate reduces production costs and achieves simplified operation and environmental protection effects.
Patent Information
- Application Number
- CN202411158826.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-08-22
- Publication Date
- 2025-01-03
AI Technical Summary
In high-salt environments, the fermentation performance of prolonged salmonas is inhibited, resulting in slower growth, extended fermentation cycles and increased production costs, limiting its prospects in industrial applications.
Under the condition that the salt concentration is 13%, a mixture of citric acid and betaine is added to the basal culture medium, with a concentration of 0 to 2 g/L, preferably 0.5 and 1 g/L, respectively. The specific addition time is the growth delay period to increase the bacterial rate. body growth rate and product synthesis rate.
It significantly shortens the fermentation cycle, improves the maximum specific growth rate of bacteria and product synthesis rate, reduces production costs, and citric acid and betaine are both natural, safe and environmentally friendly substances, making it easy to operate.
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Figure CN119242553A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the field of fermentation, and more specifically to a method for improving the fermentation performance of halophilic bacteria under salt stress and its application. Background Art
[0002] As one of the representatives of extreme microorganisms, halophilic microorganisms have the advantages of being less susceptible to bacterial contamination and saving freshwater resources. They can effectively deal with the energy consumption, pollution, cost and other problems existing in current industrial biotechnology. They have been widely used in the production of PHA, ectoine, hydrolase and carotenoids. Therefore, halophilic microorganisms have important industrial application value.
[0003] Halomonas elongata ATCC 33173 is a moderately halophilic bacterium that is widely found in high-salt environments such as salt lakes, salt ponds, and saltwater lakes. It can tolerate sodium chloride concentrations higher than 10% (w / v), and can accumulate a large amount of compatible solute ectoine in the cell to resist the high-salt environment outside. H.elongata can meet the requirements of the next generation of industrial microorganisms, namely, open continuous intelligent processing based on low-cost mixed substrates, reduced freshwater consumption, energy saving, and long-term, so the industry uses this bacterium to produce tons of ectoine, which has important application value.
[0004] Although Halomonas extensa has shown significant application potential in industrial biotechnology, in a high-salt environment, excessive environmental pressure significantly inhibits its fermentation performance, which inhibits the growth of bacteria and increases the fermentation cycle, which not only reduces production efficiency but also increases fermentation costs. This limits the application prospects of Halomonas extensa in other fields, such as high-salt wastewater treatment. Therefore, there is an urgent need to develop a method that can promote the growth of Halomonas extensa under high-salt conditions. This method not only needs to be easy to operate, but also must ensure that the synthesis process of the target product is not inhibited, so as to achieve high efficiency and economy in practical applications.
[0005] Therefore, there is an urgent need in the art for a method that can improve the fermentation performance of Halomonas elongatus under salt stress and has green, natural and safe characteristics. Summary of the invention
[0006] In order to solve the above problems, the present invention provides a method and application of improving the fermentation performance of Halomonas elongata under salt stress by using citric acid and betaine. The present invention will provide a theoretical basis for studying the improvement of the fermentation performance of Halomonas elongata under salt stress by using a mixture of citric acid and betaine.
[0007] In order to achieve the above technical objectives, the technical solution adopted by the present invention is as follows:
[0008] The present invention first provides an application of a mixture of citric acid and betaine to improve the fermentation performance of Halomonas prolonga under salt stress; further, the application is to improve the fermentation performance of Halomonas prolonga under a salt concentration of 13% (w / v) stress; the concentrations of citric acid and betaine in the mixture of citric acid and betaine are 0-2 g / L and 0-2 g / L, respectively, preferably 0.5 and 1 g / L.
[0009] The invention also provides a method for improving the fermentation performance of Halomonas under salt stress, which comprises adding a mixture of citric acid and betaine to a basic culture medium at a suitable time.
[0010] Furthermore, when the mixture of citric acid and betaine is added in the present invention, the concentrations of citric acid and betaine are 0-2 g / L and 0-2 g / L, respectively, preferably 0.5 and 1 g / L.
[0011] Furthermore, the citric acid and betaine mixture of the present invention is added during the growth lag phase, the growth logarithmic phase and the growth stable phase, preferably during the growth lag phase.
[0012] Furthermore, the ingredients of the basal culture medium and their concentration ranges are: glucose 17-23 g / L, peptone 18-22 g / L, yeast powder 9-11 g / L, sodium chloride 1% to 21% (w / v), preferably glucose 20 g / L, peptone 20 g / L, yeast powder 10 g / L, sodium chloride 13% (w / v).
[0013] Furthermore, the improved performance may include increasing bacterial growth rate, increasing specific growth rate, increasing product synthesis rate, shortening fermentation cycle, reducing fermentation cost, etc.
[0014] Furthermore, the number of the extended Halomonas is Halomonas elongata ATCC 33173.
[0015] Beneficial effects of the present invention:
[0016] (1) The present invention provides an application of a mixture of citric acid and betaine in improving the fermentation performance of Halomonas elongata under salt stress. The present invention has been verified through experiments that, under the condition of a salt concentration of 13%, compared with a treatment without addition (control), after adding 2.6mmol / L citric acid and 8.5mmol / L betaine during the growth lag phase, the fermentation period of Halomonas elongata is shortened by 36.4%, the highest specific growth rate of the bacteria is increased by 62.5%, and the product synthesis rate is increased by 111.1%, thereby significantly reducing the production cost.
[0017] (2) The citric acid and betaine used in the present invention are both safe and healthy substances. Citric acid is a natural organic acid widely found in citrus fruits and is commonly used in food and beverages as an acidity regulator. Betaine is an amino acid derivative naturally found in plants such as beets, which is environmentally friendly, non-toxic, safe and reliable.
[0018] (3) The present invention is easy to operate and can be realized by simply adding the substance to the fermentation broth during a fixed growth phase without the need for complex control conditions or additional equipment support. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1 The effect of different additives on the growth of Halomonas prolongans.
[0020] Figure 2 The effect of different addition times of citric acid and betaine mixture on the growth of Halomonas cells.
[0021] Figure 3 This is a comparison curve of the biomass of Halomonas, ectoine content and residual sugar with and without the addition of a mixture of citric acid and betaine.
[0022] Figure 4 The graph is a comparison curve of oxygen uptake rate (OUR) and carbon dioxide release rate (CER) under the conditions of adding and not adding a mixture of citric acid and betaine.
[0023] Figure 5 It is a comparison curve of the oxygen consumption rate per unit cell (qo2) and the carbon dioxide release rate per unit cell (qco2) with and without the addition of a mixture of citric acid and betaine. DETAILED DESCRIPTION
[0025] The inventors have conducted in-depth research and have revealed a method for improving the fermentation performance of Halomonas elongata under salt stress. The inventors have found that adding a mixture of citric acid and betaine at a specific point during the fermentation process can effectively improve the fermentation performance of Halomonas elongata. The present invention provides a method that is easy to operate and has simple process control.
[0026] As used in the present invention, the improvement of "fermentation performance" includes the improvement of bacterial cell specific growth rate and the improvement of product synthesis rate.
[0027] As used in the present invention, the "specific node" includes the growth lag period, the logarithmic period, the stable period and the decay period; preferably, it refers to the growth lag period.
[0028] As used in the present invention, the "mixture of citric acid and betaine" includes 0-2 g / L citric acid and 0-2 g / L betaine; preferably, it includes 0.5 g / L citric acid and 1 g / L betaine.
[0029] As used in the present invention, the term “containing”, “having” or “includes” includes “comprising”, “mainly consisting of”, “substantially consisting of”, and “consisting of”; “mainly consisting of”, “substantially consisting of” and “consisting of” are subordinate concepts of “containing”, “having” or “including”.
[0030] The specific embodiments of the technical solution of the present invention will be described in detail below in conjunction with the accompanying drawings. These embodiments are only used to more clearly illustrate the technical solution of the present invention, and are therefore only used as examples and cannot be used to limit the scope of protection of the present invention. It should be noted that, unless otherwise specified, the technical terms or scientific terms used in this application should be based on the usual meanings understood by those skilled in the art.
[0031] In the method of the present invention, the initial fermentation medium includes: 17-23 g / L glucose, 18-22 g / L peptone, 9-11 g / L yeast powder, 1% to 21% sodium chloride (w / v); preferably, the medium includes: 20 g / L glucose, 20 g / L peptone, 10 g / L yeast powder, 13% sodium chloride (w / v). This medium formula, combined with the fermentation operation process of the present invention, provides a good nutritional environment foundation for bacterial growth and product synthesis. Nevertheless, it should be understood that the medium obtained by certain changes on the basis of the fermentation medium (such as the carbon source / nitrogen source is replaced by other carbon sources / nitrogen sources that can be utilized by the bacteria of the present invention) or some medium known in the art can also be applied to the present invention, in combination with the method of adding a mixture of citric acid and betaine.
[0032] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.
[0033] Materials and Methods
[0034] 1. Starting strain
[0035] Halomonas elongata ATCC 33173, a moderately halophilic and Gram-negative bacterium, was provided by the Institute of Microbiology, Chinese Academy of Sciences.
[0036] 2. Culture medium and culture conditions
[0037] Plate medium (g / L): peptone 10, yeast powder 5, sodium chloride 80, agar 16, pH 7.0±0.1. The culture temperature is 37°C and the culture is carried out for 24 hours.
[0038] Seed culture medium (g / L): peptone 10, yeast extract 5, sodium chloride 80, pH 7.0±0.1, culture temperature was 37°C, and culture was carried out at 220 rpm for 20 h.
[0039] 5L tank fermentation medium (g / L): peptone 20, yeast extract 10, glucose 20, sodium chloride (initial medium final concentration is 10g / L; added according to the experimental conditions specified later), defoamer 0.5mL / L, pH 7.0. Inoculation ratio is 10% (v / v), and the culture temperature is 37°C.
[0040] 3. Fermentation broth OD 600 and determination of dry weight of bacteria
[0041] Dilute the fermentation broth to a certain multiple and measure the absorbance using a microplate reader in low oscillation and absorbance 600 mode. 600 =600nm absorbance value × n (n is the dilution multiple). Take a certain amount of fermentation liquid and centrifuge it at 4°C, 12000rpm for 15min. Discard the supernatant and wash it twice with isotonic sodium chloride solution and centrifuge it. Discard the supernatant and take the precipitate and dry it in an oven at 90°C to constant weight and weigh it.
[0042] 4. Determination of Ecdoin in Products
[0043] Take 0.5mL of fermentation liquid, put it into the EP tube with grinding beads, and add 1mL of ultrapure water. The EP tube with fermentation liquid and grinding beads is placed in a freezer grinder at 75Hz for 30min. After the EP tube is taken out, it is centrifuged at 12000rpm / min for 5min. The supernatant is taken and diluted through the membrane and then detected by Shimadzu HPLC. The mobile phase is water: acetonitrile (98:2) solution, the flow rate is 0.5L / min, the column temperature is 30℃, the detector is SPD-20AV, and the detection wavelength is 210nm.
[0044] The supernatant of the fermentation broth was taken and diluted, and the glucose concentration was determined by enzyme membrane method using a biochemical analyzer (Shandong Academy of Sciences).
[0045] 5. Plate culture
[0046] The bacteria in the seed glycerol tube were inoculated into LB medium containing 8% NaCl and cultured at 220 rpm and 37°C for 24 hours for activation. -7 , and spread on a plate pre-inverted with 30 mL of activation medium, and cultured at 37°C for 24 hours. Subsequently, single colonies with good growth were selected and evenly spread on the plate, and cultured again at 37°C for 24 hours.
[0047] 6. Shake flask culture
[0048] Use 5 mL of sterile 8% sodium chloride solution to rinse the bacteria on the cultured seed plate into a pre-sterilized 50 mL centrifuge tube (containing glass beads), and shake on a vortex shaker for 30 seconds to obtain a bacterial suspension. Subsequently, 5 mL of the bacterial suspension was inoculated into a 500 mL flask containing 95 mL of seed culture medium and cultured at 37 ° C, 220 rpm for 20 hours.
[0049] 7. Calculation formula for specific growth rate: Specific growth rate (μ) = (lnN2-lnN1) / (t2-t1), N2 and N1 are the bacterial counts at time t2 and t1 respectively.
[0050] 8. Product synthesis efficiency calculation formula: Product synthesis efficiency (Productivity) = (P1-P2) / (t2-t1), P1 and P2 are the product contents at time t2 and t1 respectively.
[0051] The calculation formula of unit bacterial oxygen consumption rate (qo2) and unit bacterial carbon dioxide release rate (qco2) is: qo2 = OUR / OD 600 ,qco2=CER / OD 600
[0052] Example 1. Effects of different additives on the growth of Halomonas spp.
[0053] In order to explore the ability to alleviate the inhibitory effect of high-salt environment (13% sodium chloride) on the growth of Halomonas elongata, screening experiments on a variety of common substances were carried out. These substances include amino acids, product synthesis precursors, organic acids and vitamins, specifically betaine, sorbitol, ectoine, mannitol, trehalose, glutamine, aspartic acid, asparagine, cysteine, proline, glutamic acid, citric acid, malic acid, ketoglutaric acid, thiamine, riboflavin, pantothenic acid, biotin, folic acid and cobalamin, etc. 20 substances, all added at a concentration of 0.5g / L. In the experiment, each substance was added to the fermentation system separately, and a control group without any substance was set up. All experimental conditions were kept consistent, and the OD of the fermentation broth was measured after the fermentation lasted for 20 hours. 600 The values were used to evaluate the effects of various additives on the growth of Halomonas prolongans.
[0054] like Figure 1 As shown in the figure, compared with the control group, 9 additives have a promoting effect on bacterial growth, namely betaine, ectoine, glutamine, aspartic acid, asparagine, cysteine, glutamic acid, citric acid and malic acid. It is worth noting that among all additives, citric acid and betaine have the most significant effect on promoting bacterial growth. The OD value of the fermentation liquid under the conditions of citric acid and betaine addition group is 600The values reached 40.5 and 41.4 respectively. Compared with the control group, the OD of the fermentation broth increased after adding citric acid and betaine. 600 The values increased by 16% and 18% respectively (see Table 1). These results show that the addition of citric acid and betaine can significantly improve the growth of Halomonas under 13% salt stress.
[0055] Table 1. Prolongation of OD of Halomonas fermentation broth under different additive conditions 600 Results
[0056] In order to further explore whether the co-addition of citric acid and betaine can produce better results, this experiment was based on the addition of 0.5g / L. According to the principle of full factorial experimental design, three levels of each of the two factors (0.5, 1 and 2g / L) were set, with a total of 9 experimental combinations. The results are shown in Table 2. The effects of citric acid and betaine on the response variables show that: the average response value of citric acid at 0.5g / L is the highest, while further increasing the concentration reduces the response value; the increase of betaine has a positive effect on the response value, especially when it increases from 0.5g / L to 1g / L, the response value is significantly improved. In the combination of experimental number 1, when citric acid and betaine were 0.5g / L and 1g / L respectively, the OD of the fermentation broth 600 The highest value was 48. Based on this result, the subsequent experiments will select the combination of 0.5 g / L citric acid and 1 g / L betaine to obtain the best effect.
[0057] Table 2. Effect of citric acid and betaine on response variables (OD 600 ) Experimental results of the influence of
[0058] Next, the effect of addition time on fermentation effect was explored. The growth cycle of microorganisms can be divided into lag phase, logarithmic phase, stable phase and decay phase. Different addition times may lead to different fermentation effects. Therefore, in this experiment, citric acid and betaine were added at the four stages under the condition that the addition concentrations were fixed at 0.5g / L and 1g / L respectively. The results are shown in Figure 2. Figure 2 As shown. The fermentation broth OD of the delayed addition group 600 The value was significantly higher than that in the other three stages, reaching 48.9. Compared with the addition in the stable phase and the decay phase, the OD 600 The values were 25% and 27% higher, respectively. This indicates that the addition of citric acid and betaine during the lag phase can more effectively promote the growth and fermentation of Halomonas.
[0059] Example 2.5 Validation experiment of citric acid and betaine addition in a 5L fermenter
[0060] Furthermore, the effect of adding citric acid and betaine was verified in a 5L fermenter. According to the results of the previous experiment, 0.5g / L citric acid and 1g / L betaine were added during the lag phase. During the fermentation process, samples were taken every hour to measure the OD600 and product content of the fermentation liquid, and OUR and CER were measured by the laboratory online fermentation parameter acquisition system. Figure 3 As shown, the addition group and the non-addition group showed significant differences in the OD600 value of the bacterial liquid, the residual sugar concentration and the product ectoine content. The increase rate of OD600 and ectoine content in the addition group was significantly faster than that in the control group. The highest OD600 and ectoine content in the addition group were 50.1 and 4.6 g / L, respectively, which were 24.9% and 17.9% higher than those in the control group (40.1 and 3.9 g / L). At the same time, the specific growth rate μ of the addition group was as high as 0.39 h -1 , compared with 0.24h in the control group -1 The average synthesis rate of ectoine in the addition group was 0.38 g / L / h, which was 111.1% higher than that of the control group (0.18 g / L / h). In addition, the residual sugar consumption rate in the addition group was also faster, and the fermentation cycle was shortened to 14 hours, while the control group was 22 hours, a shortening of 36.4%. In the addition group, OUR and CER were significantly higher than those in the control group, with the highest value reaching 50 mmol / L / h, while the control group was only 26 mmol / L / h (see Figure 4 ). Further analysis showed that the respiratory intensity (Qo2 and Qco2) per unit cell in the supplemented group was also significantly higher than that in the control group (see Figure 5 The above results showed that the addition of citric acid and betaine in a 5L fermenter significantly promoted the cell growth, product synthesis and metabolic activity of Halomonas elongatus.
[0061] Table 3. Changes in specific growth rate with and without addition of citric acid and betaine mixture
[0062] The present invention proposes a method and application for improving the fermentation performance of Halomonas prolongans under high salt stress conditions by adding citric acid and betaine. The method of the present invention is simple and easy to implement. It only needs to add 0.5g / L citric acid and 1g / L betaine to the fermentation liquid during the lag phase to significantly improve the growth of bacteria under salt stress, improve the product synthesis efficiency, and shorten the fermentation cycle.
[0063] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.
Claims
1. A method for improving the fermentation performance of halophilic bacteria under salt stress, characterized in that: The method comprises adding a mixture of citric acid and betaine to a basal medium.
2. The method according to claim 1, characterized in that The added concentrations of citric acid and betaine in the citric acid and betaine mixed additive are 0-2 g / L and 0-2 g / L respectively.
3. The method according to claim 2, characterized in that The added concentrations of citric acid and betaine in the citric acid and betaine mixed additive are 0.5 and 1 g / L respectively.
4. The method according to claim 2, characterized in that: The citric acid and betaine are mixed and added during the lag growth period of the halophilic bacteria.
5. The method according to claim 1, characterized in that The basic culture medium comprises a carbon source, a nitrogen source and a salt, wherein the carbon source is glucose, the nitrogen source is peptone and yeast powder, and the salt is sodium chloride.
6. The method according to claim 5, characterized in that The basic culture medium comprises: 20±3 g / L glucose, 20±2 g / L peptone, 10±1 g / L yeast powder, and 1% to 21% (w / v) sodium chloride.
7. The method according to claim 6, characterized in that The sodium chloride concentration in the basic culture medium is 13±1% (w / v).
8. The method according to claim 1, characterized in that The improved fermentation performance includes increasing the specific growth rate of halophilic bacteria, increasing the product synthesis rate, shortening the fermentation cycle, reducing the fermentation cost, etc.
9. The method according to any one of claims 1 to 8, characterized in that: The halophilic bacterium is Halomonas elongata ATCC 33173.