Luminescent bacterium culture medium

Abstract

The invention relates to a culture medium for improving the luminescent property of luminescent bacteria. The culture medium comprises 5.43-26.48 w / v% of a carbon source; 2.02 to 16.16 w / v% of a nitrogen source; 0.53 to 2.64 w / v% of potassium ions; 0.0047 to 1.89 w / v% of sodium ions; 0.024 to 1.46 w / v% of magnesium ions; 0.0028 to 23.43 w / v% of chloride ions; 0.87 to 4.35 w / v% of phosphate ions; and 0.064 to 3.83 w / v% of sulfate ions. The improved culture medium effectively enhances the luminous property of marine luminous bacteria, and has wide application prospects in the fields of marine ecological research, environmental monitoring and the like.

Description

Technical Field

[0001] This invention relates to the field of microbial culture technology, and in particular to a luminescent bacterial culture medium. Background Technology

[0002] Marine bioluminescent bacteria are a class of microorganisms capable of producing bioluminescence, and they have significant applications in marine ecosystems, environmental monitoring, and biotechnology. Currently, commonly used culture media for marine bioluminescent bacteria, such as 2216E medium, while meeting the basic growth requirements of these bacteria, have limitations in promoting bioluminescence; the brightness and duration of the luminescence need improvement. Therefore, optimizing the culture medium formulation to enhance the bioluminescent performance of marine bioluminescent bacteria is a pressing issue that needs to be addressed. Summary of the Invention

[0003] To address the technical problems existing in the prior art, this invention proposes a culture medium for improving the luminescence performance of luminescent bacteria, comprising: 5.43 - 26.48 w / v% carbon source; 2.02 - 16.16 w / v% nitrogen source; 0.53 - 2.64 w / v% potassium ions; 0.0047 - 1.89 w / v% sodium ions; 0.024 - 1.46 w / v% magnesium ions; 0.0028 - 23.43 w / v% chloride ions; 0.87 - 4.35 w / v% phosphate ions; and 0.064 - 3.83 w / v% sulfate ions.

[0004] In the culture medium described above, the carbon source is selected from one or more of the following group: sugars, protein derivatives, alcohols, organic acids, peptones, yeast extracts, and beef extracts.

[0005] In the culture medium described above, the nitrogen source is selected from one or more of the following group: peptone, tryptone, casein peptone, beef extract, yeast extract, fish meal, soybean meal, amino acids, urea, gelatin, serum, ammonium ions, nitrate ions, and ammonia.

[0006] The culture medium as described above further comprises: 0.37 - 2.94 w / v% ammonium ions.

[0007] The culture medium as described above further comprises: 0.000056 - 0.0056 w / v% iron ions; 0.000055 - 0.0055 w / v% manganese ions; 0.000048 - 0.0077 w / v% zinc ions; and 0.000039 - 0.0062 w / v% copper ions.

[0008] The culture medium described above comprises: 11-15 w / v% peptone; 1-8 w / v% yeast extract; 5-25 v / v% glycerol; 2-10 w / v% potassium dihydrogen phosphate; 0.01-0.5 w / v% sodium dihydrogen phosphate; 1-8 w / v% ammonium chloride; 0.1-6 w / v% magnesium sulfate; and 10-40 w / v% sodium chloride.

[0009] The culture medium as described above further comprises: 0.1-2 v / v% trace element solution.

[0010] The culture medium as described above, wherein the trace element solution comprises: 0.1-0.5 w / v% ferric chloride; 0.1-0.5 w / v% manganese chloride; 0.1-0.8 w / v% zinc chloride; and 0.1-0.8 w / v% copper sulfate.

[0011] Application of any of the above-described culture media in enhancing the luminescence properties of luminescent bacteria.

[0012] In the applications described above, the luminescent bacteria are selected from the genera *Isobacterium*, *Luminobacterium*, *Shewanella*, and *Vibrio*.

[0013] In the application described above, the luminescent bacteria are marine luminescent bacteria.

[0014] In the applications described above, the marine bioluminescent bacteria are selected from Vibrio fischeri or luminescent bacillus.

[0015] A method for increasing the luminescence performance of luminescent bacteria includes: obtaining a culture medium as described above; and inoculating the luminescent bacteria into the culture medium.

[0016] The improved culture medium described in this application effectively enhances the luminescence performance of marine bioluminescent bacteria, and has broad application prospects in marine ecological research, environmental monitoring and other fields. Detailed Implementation

[0017] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0018] In the following detailed description, reference can be made to specific embodiments that form part of this application. These specific embodiments are described in sufficient detail below to enable those skilled in the art to implement the technical solutions of this application. It should be understood that other embodiments or modifications to the embodiments of this application may also be used.

[0019] The "luminescent bacteria" mentioned in this article refer to bacteria capable of bioluminescence. They are a type of non-pathogenic, Gram-negative facultative anaerobic bacteria with rod-shaped or arc-shaped cells, polar flagella, and most are marine. Under aerobic conditions, luminescent bacteria can emit visible blue-green fluorescence with wavelengths between 474 and 505 nm.

[0020] Currently, several bioluminescent bacteria have been named, including multiple species within genera such as *Isobacterium*, *Bacillus*, *Shewanella*, and *Vibrio*. Among them, *Isobacterium* and *Vibrio qinghaiensis* are freshwater bioluminescent bacteria, while most others are marine bioluminescent bacteria.

[0021] The "marine bioluminescent bacteria" discussed in this article are a type of bioluminescent bacteria that primarily inhabit marine environments. Their most prominent characteristics are the need for high osmotic pressure and high salinity environments, as well as their dependence on sodium ions.

[0022] The "Vibrio fischeri" mentioned in this article is a marine bioluminescent bacterium belonging to the genus Vibrio. It is Gram-negative, facultatively anaerobic, with short rod-shaped or arc-shaped cells possessing polar flagella that enable motility. It relies on a high-salt environment and sodium ions for growth and luminescence, emitting a blue-green fluorescence at a wavelength of approximately 480 nm. In its natural state, it mostly inhabits marine waters, sediments, or the surface / digestive tract of marine animals. Some strains can form symbiotic relationships with cephalopods, fish, and other marine organisms, and it can also serve as a model strain for environmental toxicity testing.

[0023] In some embodiments, Vibrio fischeri includes multiple subtypes. The ATCC7744, ATCC14048 and ATCC33715 mentioned in this application are only representative subtypes selected to illustrate the performance of the culture medium of this application, and are not a limitation on the Vibrio fischeri subtypes that can be cultured in the culture medium of this application.

[0024] The "Bacillus luminescenceus" discussed in this article refers to a marine bioluminescent bacterium belonging to the genus *Bacillus*. It is Gram-negative, facultatively anaerobic, with straight rod-shaped cells and well-developed polar flagella. It also relies on high salt and sodium ions, exhibits stable luminescence intensity, and has a fluorescence wavelength of approximately 490 nm, making it a classic strain for studying bioluminescence mechanisms. Its habitats include marine waters, sediments, and the bodies of marine invertebrates. It lives freely or attaches to other organisms and is a commonly used indicator species in acute toxicity testing of environmental pollutants (such as bioluminescent bacterium toxicity tests).

[0025] In some embodiments, *Bacillus luminifera* includes multiple subtypes. The ATCC11040, ATCC35080, and ATCC35081 mentioned in this application are only representative subtypes selected to illustrate the performance of the culture medium in this application, and are not a limitation on the subtypes of *Bacillus luminifera* that can be cultured in the culture medium of this application.

[0026] According to one embodiment of this application, the Vibrio fischeri ATCC7744, ATCC14048, ATCC33715 and Bacillus luminifera ATCC11040, ATCC35080, ATCC35081 recorded in this application are bacterial strains retained by the laboratory of Hefei Shenbi Company.

[0027] The “2216E medium” mentioned in this article is a solid or liquid culture medium formulated based on the characteristics of the high-salt and mineral-rich marine environment. It can simulate the natural nutrient conditions of the ocean and provide marine heterotrophic bacteria with the carbon source, nitrogen source, minerals and growth factors required for growth, supporting their normal proliferation and metabolism. It is a classic synthetic culture medium designed specifically for culturing marine heterotrophic bacteria, and is especially suitable for the isolation, purification and culture of marine luminescent bacteria such as Vibrio fischeri and luminescent bacillus.

[0028] The "luminescence performance" mentioned in this article refers to the detectable luminescence intensity, luminescence duration, and other properties of luminescent bacteria after cultivation. The stronger the detectable luminescence intensity and / or the longer the luminescence duration, the better the luminescence performance of the luminescent bacteria.

[0029] The term "luminescence intensity" as used herein refers to the intensity of fluorescence or light emitted by luminescent bacteria, as detected by a specific instrument; that is, the luminescence brightness of the luminescent bacteria, which can be directly observed by the naked eye. In some embodiments, the luminescence intensity is the average luminescence intensity value of the luminescent bacteria obtained by detecting and recording the luminescence intensity of the luminescent bacteria over a certain period of time. For example, after culturing luminescent bacteria for 36 hours, the average luminescence intensity is continuously detected for half an hour. In other embodiments, the luminescence intensity can also be the luminescence intensity of the luminescent bacteria at a specified culturing time, such as the luminescence intensity detected after culturing luminescent bacteria for 36 hours. In still other embodiments, the luminescence intensity can also be the highest luminescence intensity value obtained after detecting and recording the luminescence intensity of the luminescent bacteria at regular time intervals.

[0030] In some embodiments, the method for detecting the luminescence intensity of luminescent bacteria is as follows: Dynamic detection method of relative luminescence intensity in a dark environment fermentation system using a LightScout full-spectrum quantum meter (Shanghai Zequan Technology): The detection port is aligned with the culture medium after inoculation and fermentation in a dark environment, and the luminescence intensity is detected at different times. In some embodiments, the time interval for detecting luminescence intensity is 5-10 minutes. In some embodiments, the luminescence intensity recorded in this application is the strongest luminescence intensity detected.

[0031] In some embodiments, the duration of luminescence is recorded by reviving luminescent bacteria and inoculating them into a designated culture medium. When the detected luminescence intensity is 1 × 10⁻⁶... 12 photns / mm 2 Recording begins at a rate of 1 / min, with the luminescence intensity gradually increasing and then decreasing until it drops to 1×10⁻⁶. 12 photns / mm 2 When the time reaches / min, stop recording to obtain the luminescence duration of the luminescent bacteria.

[0032] The "carbon source" mentioned in this article refers to carbon-containing organic or inorganic matter that can be absorbed and utilized by marine bioluminescent bacteria.

[0033] In some embodiments, the organic carbon source is selected from sugars, protein derivatives, alcohols, organic acids, etc. Sugars are selected from monosaccharides such as glucose and fructose, disaccharides such as sucrose and lactose, and polysaccharides such as starch and dextrin. Protein derivatives are selected from peptone, tryptone, casein, etc. Alcohols are selected from ethanol, glycerol, etc. Organic acids are selected from citric acid, lactic acid, etc. In some embodiments, the organic carbon source may also be selected from yeast extract, peptone, beef extract, etc.

[0034] In some embodiments, the inorganic carbon source is selected from carbonates, bicarbonates, carbon dioxide, etc.

[0035] The "nitrogen source" mentioned in this article refers to nitrogen-containing organic or inorganic substances that can be absorbed and utilized by marine bioluminescent bacteria.

[0036] In some embodiments, the organic nitrogen source is selected from peptone, tryptone, casein peptone, beef extract, yeast extract, fish meal, soybean meal, pure amino acids such as glutamic acid and lysine, urea, gelatin, serum, etc.

[0037] In some embodiments, the inorganic nitrogen source is selected from ammonium ions, nitrate ions, ammonia, etc.

[0038] In this application, the applicant adjusts the ratio between the components to regulate the synergistic effect of each component ratio on the luminescent bacteria, thereby increasing the luminescence intensity of the luminescent bacteria and further extending the luminescence time of the luminescent bacteria.

[0039] In this application, the ratio of glycerol, peptone, and ammonium chloride is adjusted to regulate the synergistic effect of carbon and nitrogen sources on luminescent bacteria. Glycerol can directly function as a carbon source for luminescent bacteria; peptone provides organic nitrogen for the synthesis of proteins such as luciferase; and ammonium chloride provides inorganic nitrogen for rapid nitrogen supplementation. This application optimizes the ratio of these three components. For example, in experimental culture medium 10, 20 mL of glycerol + 15 g of peptone + 6 g of ammonium chloride avoids the problems of "too much carbon and too little nitrogen (glycogen accumulation inhibits luminescence)" or "too much nitrogen and too little carbon (insufficient energy)". According to Examples 3-4 of this application, the luminescence stability period of the six bacterial strains was extended from 40 h in 2216E to 90 h in experimental culture medium 10, avoiding the sudden drop in luminescence caused by "late-stage carbon and nitrogen deficiency" in 2216E culture medium.

[0040] In this application, the ion concentration ratio is adjusted to regulate the synergistic effect of each ion on luminescent bacteria. For example, in experimental culture medium 10, the sodium ion concentration is adjusted to 25 g / L to maintain the cell osmotic pressure of luminescent bacteria and activate membrane transport, while 6 g / L ammonium ions supplement nitrogen and assist in pH buffering. These three factors synergistically construct a physiological environment of "high extracellular sodium ions - high intracellular potassium ions - sufficient nitrogen source". The luminescence duration of *Bacillus luminifera* ATCC35080, which is sensitive to ion concentration, increased from 42.1 h at 2216E to 92.8 h in experimental culture medium 9, an increase of 1.2 times, far exceeding the effect of adjusting a single ion. For instance, in experimental culture medium 4, increasing the ion concentration alone only increased the luminescence duration of *Bacillus luminifera* ATCC35080 to 70.2 h.

[0041] In this application, the composition of trace elements in the culture medium is regulated. Among them, iron ions participate in the formation of the active site of luciferase in luminescent bacteria; manganese ions promote the activation of glycerol metabolism and glycolytic enzymes in luminescent bacteria; zinc ions stabilize the cell membrane of luminescent bacteria; and copper ions regulate the redox balance of luminescent bacteria.

[0042] In this application, the proportions of each group in the culture medium are further adjusted to suit luminescent bacteria with different ion and nutrient requirements. For example: Regarding Vibrio fischeri ATCC14048: When 19.45g of NaCl was added to test medium 6, its luminescence performance was lower than that of test medium 7 with 25g of NaCl added, indicating that the strain has strong salt tolerance. Therefore, in test medium 11, the sodium ion concentration was increased to 30g / L compared to test medium 10. Regarding the luminescent bacterium ATCC11040: When 1g of ammonium chloride was added to the culture medium of test case 5, its luminescence performance was lower than that of the culture medium of test case 6 with 3g of ammonium chloride added, indicating that this strain requires high levels of ammonium chloride. Therefore, the ammonium chloride in group 10 was increased to 6g.

[0043] Gain: The luminescence performance of the two strains in the dedicated optimization group (experimental culture medium 10-12) is improved by 5%-7% compared with the "general optimal group experimental culture medium 9", achieving the upgrade from "universal optimization to precise adaptation".

[0044] The terms "one embodiment," "some embodiments," "example," "specific example," or "some examples" used in this application refer to a specific feature, structure, material, or characteristic described in connection with that embodiment or example, which is included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0045] This application relates to a culture medium for improving the luminescence performance of luminescent bacteria, comprising: 5.43 - 26.48 w / v% carbon source; 2.02 - 16.16 w / v% nitrogen source; 0.53 - 2.64 w / v% potassium ions; 0.0047 - 1.89 w / v% sodium ions; 0.024 - 1.46 w / v% magnesium ions; 0.0028 - 23.43 w / v% chloride ions; 0.87 - 4.35 w / v% phosphate ions; and 0.064 - 3.83 w / v% sulfate ions.

[0046] In some embodiments, the carbon source is selected from one or more of the group consisting of: sugars, protein derivatives, alcohols, organic acids, peptone, yeast extract, and beef extract. For example, the organic carbon source is selected from sugars, protein derivatives, alcohols, organic acids, etc. Sugars are selected from monosaccharides such as glucose and fructose, disaccharides such as sucrose and lactose, and polysaccharides such as starch and dextrin. Protein derivatives are selected from peptone, tryptone, casein, etc. Alcohols are selected from ethanol, glycerol, etc. Organic acids are selected from citric acid, lactic acid, etc. In some embodiments, the organic carbon source may also be selected from yeast extract, peptone, beef extract, etc. In some embodiments, the inorganic carbon source is selected from carbonates, bicarbonates, carbon dioxide, etc.

[0047] In some embodiments, the nitrogen source is selected from one or more of the following group: peptone, tryptone, casein peptone, beef extract, yeast extract, fish meal, soybean meal, amino acids, urea, gelatin, serum, ammonium ions, nitrate ions, and ammonia.

[0048] In some embodiments, the culture medium further comprises 0.37 - 2.94 w / v% ammonium ions.

[0049] In some embodiments, the culture medium further comprises: 0.000056 - 0.0056 w / v% iron ions; 0.000055 - 0.0055 w / v% manganese ions; 0.000048 - 0.0077 w / v% zinc ions; and 0.000039 - 0.0062 w / v% copper ions.

[0050] In some embodiments, the improved culture medium of this application comprises: 11-15 w / v% peptone; 1-8 w / v% yeast extract; 5-25 v / v% glycerol; 2-10 w / v% potassium dihydrogen phosphate; 0.01-0.5 w / v% sodium dihydrogen phosphate; 1-8 w / v% ammonium chloride; 0.1-6 w / v% magnesium sulfate; and 10-40 w / v% sodium chloride.

[0051] In some embodiments, the culture medium further comprises: 0.1-2 v / v% trace element solution.

[0052] In some embodiments, the trace element solution comprises: 0.1-0.5 w / v% ferric chloride; 0.1-0.5 w / v% manganese chloride; 0.1-0.8 w / v% zinc chloride; and 0.1-0.8 w / v% copper sulfate.

[0053] The culture medium of this application can be used for applications that enhance the luminescence performance of luminescent bacteria. In some embodiments, the luminescent bacteria are selected from the genera *Isobacterium*, *Luminobacterium*, *Shewanella*, and *Vibrio*. In some embodiments, the luminescent bacteria are selected from marine luminescent bacteria. In some embodiments, the marine luminescent bacteria are selected from *Vibrio fischeri* or *Luminobacterium luminiferum*.

[0054] This application relates to a method for increasing the luminescence performance of luminescent bacteria, comprising: obtaining a culture medium as described in this application; and preparing a culture medium with a concentration of 1×10⁻⁶. 6 -1×10 7 CFU / mL of luminescent bacteria were inoculated into the culture medium and cultured at 25-30°C on a shaker at 180 r / min for 24-96 hours.

[0055] The technical solution of this application will be further illustrated through the following embodiments.

[0056] Example 1: Culture medium preparation In this embodiment, specified amounts of each component are measured into a container, and RO water is added to bring the volume to 1000 mL, adjusting the pH to 7.6-8.0. This application uses the commonly used 2216E medium as the basal medium, adjusting its components and the distribution ratio of each group. The components of the 2216E medium and the improved medium of this application are shown in Table 1.

[0057] Table 1 Components of each culture medium

[0058] The method for preparing the trace element solution includes: adding 0.01 g of ferric chloride, 0.01 g of manganese chloride, 0.01 g of zinc chloride, and 0.001 g of copper sulfate to RO water to a final volume of 100 mL.

[0059] Example 2: Cultivation and Detection of Marine Bioluminescent Bacteria 1. Bacterial strains: Vibrio fischeri (ATCC7744, ATCC14048, ATCC33715) and luminescent bacillus (ATCC11040, ATCC35080, ATCC35081) are six luminescent bacterial strains.

[0060] 2. Inoculation: Inoculate each bacterial strain into 2216E medium and experimental culture media 1-8, respectively, with an inoculation concentration of approximately 1×10⁻⁶. 6 - 1×10 7 CFU / mL.

[0061] 3. Incubation: Incubate at 25-30 ℃ for 24-96 hours. In this example, the incubation temperature is 28 ℃ and the incubation time is 24 hours.

[0062] 4. Luminescence Intensity Detection: Using a LightScout full-spectrum quantum optical meter (Shanghai Zequan Technology), the detection port was aligned with the culture medium after inoculation and fermentation in a dark environment to detect the relative luminescence intensity at different times. In this embodiment, the luminescence intensity was detected and recorded every 10 minutes. Unless otherwise specified, the luminescence intensity listed in this application is the strongest recorded luminescence intensity.

[0063] Example 3: Comparison of luminescent bacteria culture results: As shown in Table 1 of the examples, 2216E medium is a commonly used medium for culturing luminescent bacteria in the art. Examples 1-4 represent adjustments to the component composition based on this application, while Examples 5-9 represent further adjustments to the component ratios. Tables 2 and 3 show the luminescence intensity and duration of the luminescent bacteria cultured in each medium, respectively.

[0064] Table 2. Luminescence intensity of various luminescent bacteria in different culture media (×10⁻¹⁰)12 photns / mm 2 / min)

[0065] Table 3. luminescence duration (h) of various luminescent bacteria in different culture media.

[0066] Table 4. OD values ​​(OD 600) of various luminescent bacteria in different culture media when the luminescence intensity was highest.

[0067] Table 5. OD values ​​(OD 600) of various luminescent bacteria in different culture media when the luminescence time was longest.

[0068] As shown in Tables 2 and 3, based on the statistical analysis of the luminescence intensity and duration of six species of all two types of bacteria, the bacteria in the 2216E medium had the lowest luminescence brightness and a luminescence duration of approximately 40.1–47.6 h.

[0069] In Experiment 1, an additional 10 mL of glycerol was added to the 2216E medium to provide a readily available carbon source for the bacteria and reduce cellular metabolic stress. The results showed that the luminescence intensity of all six bacterial species from both strains increased by 44.7%–65.6% compared to the 2216E medium. Notably, the luminescence duration of *Bacillus luminifera* ATCC35081 increased from 43.4 h to 52.6 h. This demonstrates that providing sufficient carbon source supply can enhance the luminescence of luminescent bacteria.

[0070] The percentage increase in luminescence intensity of the luminescent bacteria in the test medium compared to that in the 2216E medium was calculated as follows: the percentage of the ratio of the luminescence intensity of the luminescent bacteria in the test medium (minus its luminescence intensity in the 2216E medium) to the luminescence intensity of the luminescent bacteria in the 2216E medium. The calculation method for the percentage increase in luminescence intensity compared to other media is similar and will not be repeated below.

[0071] In Experiment 2, the culture medium was supplemented with 0.3 mL of a trace element solution, containing approximately 0.045 mg of iron ions, 0.048 mg of manganese ions, 0.046 mg of zinc ions, and 0.0025 mg of copper ions. The results showed that the luminescence intensity of all six bacterial species from both experiments increased by 20%-32.4% compared to the medium in Experiment 1. Specifically, the luminescence duration of *Vibrio fischeri* ATCC7744 increased from 48.5 h to 57.7 h. This demonstrates that compensating for the trace element deficiency of 2216E in the culture medium can enhance the luminescence intensity and prolong the luminescence time of luminescent bacteria.

[0072] In Experiment 3, the culture medium was supplemented with an additional 1g of ammonium chloride compared to Experiment 2. The results showed that the luminescence duration of all six bacterial species from both strains was prolonged compared to Experiment 2. Specifically, in the medium with added ammonium ions, the luminescence intensity of *Vibrio fischeri* ATCC14048, which prefers inorganic nitrogen, exhibited dynamic changes with culture time: luminescence rapidly initiated and reached its peak in the early stages of culture, with a significantly higher peak brightness than the untreated group; subsequently, throughout the entire culture period (0-65.4h), the luminescence intensity remained at a detectable level, without any rapid decline in brightness due to nitrogen limitation in the later stages, ultimately extending the luminescence duration from 57.7h to 65.4h. Monitoring the luminescence intensity at different time points clearly demonstrated the dual effect of readily available nitrogen sources on improving both the brightness and duration of bacterial luminescence. Adding additional ammonium ions as a readily available nitrogen source to the culture medium can shorten the nitrogen metabolism pathway of luminescent bacteria, eliminating "late-stage nitrogen limitation" and thus increasing the brightness of the luminescent bacteria.

[0073] In Experiment 4, the culture medium was supplemented with an additional 1g of sodium dihydrogen phosphate compared to that in Experiment 3. The results showed that the luminescence intensity of all six bacterial species from both strains increased by approximately 17%-33.3% compared to the medium in Experiment 3, with the luminescence duration of *Vibrio fischeri* ATCC7744 increasing to 72.3 hours. This demonstrates that increasing the sodium dihydrogen phosphate content can maintain pH stability, regulate osmotic pressure, and thus improve luminescence duration and intensity.

[0074] In Experiment 5, the carbon source concentration was increased compared to Experiment 4, thereby providing an energy source and promoting biomass growth. The results showed that the brightness of all six bacterial species from both strains was improved, and the luminescence duration of *Bacillus luminifera* ATCC11040 increased from 71.6 h to approximately 79.4 h.

[0075] Based on Experiment 5, the carbon source, nitrogen source, and the proportion of each ion added in Experiments 6-9 were further adjusted to obtain a universal culture medium that is more suitable for the culture of luminescent bacteria.

[0076] As shown in Table 2, all six species of Vibrio fischeri and luminescent bacillus showed the highest luminescence intensity in test medium 9, followed by test medium 8, test medium 7, and 2216E medium, which showed the lowest luminescence intensity.

[0077] Therefore, it is evident that, regardless of the luminescence intensity or duration of each bacterium, the culture medium of Example 9 is superior to that of 2216E and other culture media. Thus, culture medium 9 can be used as a universal culture medium for the cultivation of marine luminescent bacteria.

[0078] Example 4: Culture media for different luminescent bacteria In this embodiment, to adapt to luminescent bacteria with different ion requirements, the component ratios of the culture medium were specifically adjusted based on the experimental culture medium 9, as shown in Table 6.

[0079] Table 6 Components of Each Culture Medium

[0080] Tables 7 and 8 show the luminescence intensity and duration recorded for luminescent bacteria cultured in medium 9-12 according to the method of Example 2. As shown in Figures 7 and Tables 8, Table 7. Luminescence intensity of various luminescent bacteria in different culture media (×10⁻⁶) 12 photns / mm 2 / min)

[0081] Table 8. luminescence duration (h) of various luminescent bacteria in different culture media.

[0082] Table 9. OD values ​​(OD600) of various luminescent bacteria in different culture media when the luminescence intensity was highest.

[0083] Table 10. OD values ​​(OD600) of various luminescent bacteria in different culture media when the luminescence intensity was highest.

[0084] As shown in Tables 7 and 8, for the culture medium of Experiment Example 10, the luminescence duration of the six species of the two luminescent bacteria was similar to that of Experiment Example 9, but the luminescence brightness of each bacterium was increased.

[0085] For experimental culture medium 11, the sodium ion concentration was reduced and the glycerol content was increased compared to experimental medium 10. As shown in Tables 7 and 8, the luminescence duration of Vibrio fischeri ATCC14048 increased from 86.3 h to 92.8 h, and the luminescence brightness increased by approximately 8.9% compared to experimental medium 10. The luminescence intensity and duration of other bacterial species did not change significantly. Therefore, experimental culture medium 11 can be specifically adapted for bacterial species that prefer "low sodium ion, high carbon source" conditions; furthermore, this culture medium is suitable for the cultivation of Vibrio fischeri ATCC14048.

[0086] For experimental culture medium 12, the potassium ion concentration was reduced and the amount of trace elements was increased compared to experimental medium 11. As shown in Tables 7 and 8, the luminescence duration of *Bacillus luminifera* ATCC11040 increased from 89.1 h to 93.7 h, and the luminescence brightness increased by approximately 9.6% compared to experimental medium 11. The luminescence intensity and duration of other bacterial species did not change significantly. Therefore, experimental culture medium 11 can be specifically adapted for bacterial species requiring "low potassium ion and high trace element" conditions; furthermore, this culture medium is suitable for the cultivation of *Bacillus luminifera* ATCC11040.

[0087] The above embodiments are for illustrative purposes only and are not intended to limit the invention. Those skilled in the art can make various changes and modifications without departing from the scope of the invention. Therefore, all equivalent technical solutions should also fall within the scope of the invention.

Claims

1. A culture medium for improving the luminescence performance of luminescent bacteria, comprising: 5.43-26.48 w / v% carbon source; 2.02-16.16 w / v% nitrogen source; 0.53 - 2.64 w / v% potassium ions; 0.0047 - 1.89 w / v% sodium ions; 0.024 - 1.46 w / v% magnesium ions; 0.0028 - 23.43 w / v% chloride ions; 0.87 - 4.35 w / v% phosphate ions; and 0.064 - 3.83 w / v% sulfate ions.

2. The culture medium according to claim 1, wherein the carbon source is selected from one or more of the following: sugars, protein derivatives, alcohols, organic acids, peptones, yeast extracts, and beef extract.

3. The culture medium according to claim 1, wherein the nitrogen source is selected from one or more of the following: peptone, tryptone, casein peptone, beef extract, yeast extract, fish meal, soybean meal, amino acids, urea, gelatin, serum, ammonium ions, nitrate ions, and ammonia.

4. The culture medium according to claim 1, wherein the carbon source further comprises: 0.37 - 2.94 w / v% ammonium ions.

5. The culture medium according to claim 1, further comprising: 0.000056 - 0.0056 w / v% iron ions; 0.000055 - 0.0055 w / v% manganese ions; 0.000048 - 0.0077 w / v% Zinc ions; as well as 0.000039 - 0.0062 w / v% copper ions.

6. The culture medium according to any one of claims 1-5, comprising: 11-15 w / v% peptone; 1-8 w / v% yeast extract; 5-25 v / v% glycerin; 2-10 w / v% potassium dihydrogen phosphate; 0.01-0.5 w / v% Sodium dihydrogen phosphate; 1-8 w / v% ammonium chloride; 0.1-6 w / v% magnesium sulfate; and 10-40 w / v% Sodium chloride.

7. The culture medium according to claim 6, further comprising: 0.1-2 v / v% trace element solution.

8. The culture medium according to claim 7, wherein, The trace element solution includes: 0.1-0.5 w / v% ferric chloride; 0.1-0.5 w / v% manganese chloride; 0.1-0.8 w / v% zinc chloride; and 0.1-0.8 w / v% copper sulfate.

9. The use of the culture medium as described in any one of claims 1-8 in increasing the luminescence performance of luminescent bacteria.

10. The application according to claim 9, wherein the luminescent bacteria are selected from: *Heterobacterium*, *Luminobacterium*, *Shewanella*, and *Vibrio*.

11. The application according to claim 9, wherein the luminescent bacteria are marine luminescent bacteria.

12. The application according to claim 11, wherein the marine bioluminescent bacteria are selected from Vibrio fischeri or luminescent bacillus.

13. A method for increasing the luminescence properties of luminescent bacteria, comprising: Obtain the culture medium as described in any one of claims 1-8; The luminescent bacteria were inoculated into the culture medium.

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