A novel culture medium and culture method for bacterial diatom co-culture

Abstract

This invention discloses a novel culture medium and method for co-culturing bacteria and diatoms, comprising the following raw materials: calcium nitrate tetrahydrate Ca(NO3)2·4H2O: 300 mg / L; potassium nitrate KNO3: 200 mg / L; magnesium sulfate heptahydrate MgSO4·7H2O: 40 mg / L; sodium β-glycerophosphate pentahydrate C3H7Na2O6P·5H2O: 12 mg / L; vitamin B... 12 Vitamin B 12 The following are the ingredients of this novel bacterial-diatom co-culture medium: 0.1 ug / L; Vitamin B: 0.1 ug / L; Thiamine HCl: 10 ug / L; 4-Hydroxyethylpiperazine ethanesulfonic acid (HEPES) buffer: 0.5 g / L; Sodium silicate nonahydrate (Na2SiO3·9H2O): 400 mg / L; Yeast extract: 0.1 g / L; Maltose: 0.5 g / L. This novel bacterial-diatom co-culture medium formulation, through single-variable experimental comparison, optimized the medium to improve the growth rate of bacteria and diatoms during bacterial-algal symbiosis, increase their biomass, and reduce the cultivation cost of the algal-bacterial symbiosis. Specifically, when yeast extract and maltose were added simultaneously, and the total nitrogen, total phosphorus, and silicate concentrations were set to 500 mg / L, 12 mg / L, and 400 mg / L, respectively, the growth rate and biomass of bacteria and diatoms reached their maximum values.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a novel culture medium and method for co-culturing bacteria and diatoms. Background Technology

[0002] The co-culture system of bacteria and algae has significant effects on degrading organic matter, reducing pH value, stabilizing the physicochemical indicators of water bodies, and increasing the concentration of dissolved oxygen in water bodies. It is the key to developing new water quality remediation technologies. However, there are complex nutrient release and remineralization processes between bacteria and algae. The structure and concentration of nutrients (total nitrogen, total phosphorus, and silicates) in the culture medium are the key to achieving high-quality culture results.

[0003] Currently, microalgae cultivation technology has matured. However, this technology cannot be fully replicated in algal-microbe symbiotic systems. High production costs and low efficiency due to immature cultivation technology are the main factors limiting the cultivation of algal-microbe symbiotic systems. There are two ways to reduce the cost of algal-microbe symbiotic system cultivation: one is to reduce the cost of raw materials for the culture medium, and the other is to increase the yield of each nutrient component. Therefore, it is necessary to find the optimal ratio of different culture media for different nutrients in the algal-microbe symbiotic system.

[0004] Diatoms are one of the most important dominant microalgae in natural water bodies. They have huge biomass, rich species, unique siliceous structure and ecological functions. Bacterial-diatom symbiotic systems have been widely used in water treatment plants, aquaculture industry, bioremediation and other technical fields. However, there is little research on the optimal conditions for co-culturing bacteria and diatoms, and the optimal culture medium formula has not yet been found. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the existing defects and provide a novel culture medium formula for bacterial-diatom co-culture. The culture medium obtained by optimizing the formula through single-variable experimental comparison can improve the growth rate of bacteria and diatoms during bacterial-algal symbiosis, increase the biomass of both, and reduce the culture cost of algal-bacterial symbiosis.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a novel culture medium for co-culturing bacteria and diatoms, wherein the composition of the novel bacterial-diatom co-culturing culture medium is as follows:

[0007] Calcium nitrate tetrahydrate Ca(NO3)2·4H2O: 250-350 mg / L;

[0008] Potassium nitrate (KNO3): 150-250 mg / L;

[0009] Magnesium sulfate heptahydrate MgSO4·7H2O: 35-45 mg / L;

[0010] Sodium β-glycerophosphate pentahydrate C3H7Na2O6P·5H2O: 10-15 mg / L;

[0011] Vitamin B12: 0.05-0.15 ug / L;

[0012] Vitamin B6 iotin: 0.05-0.15 ug / L;

[0013] Thiamine HCl hydrochloride: 8-14 ug / L;

[0014] 4-Hydroxyethylpiperazine ethanesulfonic acid (HEPES) buffer: 0.3-0.7 g / L;

[0015] Sodium silicate nonahydrate Na2SiO3·9H2O: 350-450 mg / L;

[0016] Yeast extract: 0.05-0.15 g / L;

[0017] Maltose: 0.4-0.7 g / L;

[0018] Disodium ethylenediaminetetraacetate (Na2EDTA): 4-5 mg / L;

[0019] Manganese chloride tetrahydrate MnCl2·4H2O: 0.2-0.4 mg / L;

[0020] Zinc chloride heptahydrate (ZnCl2·7H2O): 25-35 ug / L;

[0021] Sodium molybdate dihydrate Na2MoO4·2H2O: 22-26 ug / L;

[0022] Ferric chloride hexahydrate FeCl3·6H2O: 0.5-1.5 mg / L;

[0023] Cobalt chloride hexahydrate CoCl2·6H2O: 10-14 ug / L.

[0024] Preferably, a novel bacterial-diatom co-culture medium is provided, wherein the composition of the novel bacterial-diatom co-culture medium is as follows:

[0025] Calcium nitrate tetrahydrate Ca(NO3)2·4H2O: 300 mg / L;

[0026] Potassium nitrate (KNO3): 200 mg / L;

[0027] Magnesium sulfate heptahydrate MgSO4·7H2O: 40 mg / L;

[0028] Sodium β-glycerophosphate pentahydrate C3H7Na2O6P·5H2O: 12 mg / L;

[0029] Vitamin B 12 Vitamin B 12 0.1 ug / L;

[0030] Vitamin B1O2: 0.1 ug / L;

[0031] Thiamine HCl hydrochloride: 10 ug / L;

[0032] 4-Hydroxyethylpiperazine ethanesulfonic acid HEPES buffer: 0.5 g / L;

[0033] Sodium silicate nonahydrate Na2SiO3·9H2O: 400 mg / L;

[0034] Yeast extract: 0.1g / L;

[0035] Maltose: 0.5g / L;

[0036] Disodium ethylenediaminetetraacetate (Na2EDTA): 4.5 mg / L;

[0037] Manganese chloride tetrahydrate MnCl2·4H2O: 0.25 mg / L;

[0038] Zinc chloride heptahydrate ZnCl2·7H2O: 30ug / L;

[0039] Sodium molybdate dihydrate Na2MoO4·2H2O: 24ug / L;

[0040] Ferric chloride hexahydrate FeCl3·6H2O: 1.0 mg / L;

[0041] Cobalt chloride hexahydrate CoCl2·6H2O: 12ug / L.

[0042] A novel method for co-culturing bacteria and diatoms includes the following steps:

[0043] 1): Prepare the co-culture medium according to the above formula, adjust the pH to 7.0 with HEPES buffer, sterilize in an autoclave at 120℃ for 20 minutes, and store the sterilized medium in a refrigerator at 4℃ for later use.

[0044] 2): The sterile freshwater diatom (taking *Strombocys granulosus* as an example) pure algal strain obtained by the antibiotic method was cultured in CSI medium to the logarithmic growth phase for later use;

[0045] 3): Place the above-mentioned *Cyclocarya granulosa* in a nitrogen-, phosphorus-, silicon-, and iron-free CSI medium for starvation culture for 48 hours (the culture medium is a nitrogen-, phosphorus-, and silicon-free CSI medium) for later use;

[0046] 4): After starvation culture, *Streptomyces granulosus* was centrifuged at 4000 rpm for 4 min. The supernatant was then poured off and inoculated into Erlenmeyer flasks containing novel culture media (experimental group) and CSI culture medium (control group) with different formulations. The pure algae inoculation amount was 0.5 × 10⁻⁶. 5 ind. / mL, seal with sealing film;

[0047] 5): Transfer the conical flask into the light incubator, set the temperature to 25℃, the light intensity to 3000 lx, and the light-dark ratio to 12h:12h;

[0048] 6): Shake the conical flask once every 3 hours and culture continuously for 10 days. All experimental operations were carried out in a clean bench and all instruments were sterilized.

[0049] 7): In this experiment, the beneficial bacteria *Roseobacterium pomeroyi*, which has a symbiotic relationship with diatoms, was selected. The pure culture was inoculated into sterile granular *Chaetoceros* algal solution that had undergone starvation culture. The inoculum was then transferred to Erlenmeyer flasks containing different formulations of novel culture medium (experimental group) and CSI medium (control group). The diatom inoculum size was 0.5 × 10⁻⁶. 5 ind. / mL, bacterial inoculum size was 1.0 × 10⁻⁶. 6 cells / mL;

[0050] 8): Transfer the conical flask into the light incubator, set the temperature to 25℃, the light intensity to 3000 lx, and the light-dark ratio to 12h:12h;

[0051] 9): Shake the conical flask once every 3 hours and culture continuously for 10 days. All experimental operations were carried out in a clean bench and all instruments were sterilized.

[0052] 10): Samples of bacterial and diatom culture media were taken every 2 days. Bacterial abundance was determined by flow cytometry, and diatom density was counted under a microscope. The optimal culture medium formulation was determined based on the growth curves of diatoms and bacteria.

[0053] The physicochemical properties of each component of the above-mentioned culture medium and their functions in this invention are as follows:

[0054] (1) Calcium nitrate tetrahydrate Ca(NO3)2·4H2O: colorless crystals. It is a commonly used fertilizer in production, using calcium carbonate-rich stones and nitric acid as raw materials to provide nitrogen and calcium sources required for the growth of diatoms and bacteria; it is hygroscopic and has a melting point of 45℃; it is soluble in water, ethanol, methanol, and acetone, but almost insoluble in nitric acid; its 5% aqueous solution (based on anhydrous matter) has a pH of 6.0; it is a strong oxidizing agent and decomposes upon heating, releasing oxygen; it contains more water of crystallization than calcium nitrate, so its oxidizing hazard is lower than that of calcium nitrate.

[0055] (2) Potassium nitrate (KNO3): colorless transparent orthorhombic or trigonal prism crystals or white crystalline powder, soluble in glycerol, insoluble in anhydrous ethanol; when dissolved in water, it can lower the temperature, the pH of the aqueous solution is about 7, and it decomposes at 400℃ to release oxygen; it has strong oxidizing properties and provides a nitrogen source for the growth of diatoms and bacteria.

[0056] (3) Magnesium sulfate heptahydrate (MgSO4·7H2O) is a colorless, fine needle-like or oblique columnar crystal; odorless, bitter taste, relatively stable below 47℃; easily soluble in water, the solution is neutral, pH 6-7; slightly soluble in ethanol. It is easily efflorescent in dry air; if exposed to humid air, it quickly absorbs water; it becomes anhydrous at about 250℃; it provides trace amounts of metallic magnesium for the growth of diatoms and bacteria.

[0057] (4) Sodium β-glycerophosphate pentahydrate (C3H7Na2O6P·5H2O): colorless crystals, soluble in water and alcohol, slightly soluble in ether; stable under normal conditions, it is a stable inorganic compound and an important coenzyme, which plays an important catalytic role in many organisms; it provides phosphorus source for the growth of diatoms and bacteria.

[0058] (5-7) Vitamin B 12 Biotin and Thiamine HCl: provide trace elements and B vitamins for the growth of diatoms and bacteria.

[0059] (8) 4-Hydroxyethylpiperazine ethanesulfonic acid (HEPES): a buffer solution, an amphoteric organic chemical buffer, the best buffer for biological research, soluble in water and alcohol; insoluble in nonpolar solvents; has the strongest buffering capacity in the pH range of 6.0-8.5; used to adjust the pH of the culture medium to 7.0.

[0060] (9) Sodium silicate nonahydrate (Na2SiO3·9H2O): colorless crystals with a tasteless and salty taste; soluble in water and dilute alkaline solutions, but insoluble in alcohols and acids; its aqueous solution is alkaline, and its solubility increases with increasing temperature; relative density 0.7-0.9; melting point 40-48℃; easily exposed to air and easily absorbs moisture and deliquesces; has the ability to clean, emulsify, disperse, wet, penetrate, and buffer pH; provides silicon for the growth of diatoms and bacteria.

[0061] (10) The function of yeast extract is to provide B vitamins, nucleotides, trace elements, etc. The amount added is very small and insufficient to maintain bacterial growth, and does not meet the definition of carbon source; maltose and organic carbon released by diatoms are the carbon source of bacteria.

[0062] (11) Maltose provides trace elements for the growth of diatoms and bacteria, as well as carbon source for bacterial growth.

[0063] (12) Disodium ethylenediaminetetraacetate (Na2EDTA); white or off-white crystalline powder; odorless; soluble in water, practically insoluble in methanol, ethanol or chloroform; pH 4.0~5.0; provides trace amounts of metallic sodium for the growth of diatoms and bacteria.

[0064] (13) Manganese chloride tetrahydrate (MnCl2·4H2O): pale pink monoclinic translucent crystals; there are two forms: the α form is more stable and belongs to the monoclinic columnar crystal system; the β form is unstable and belongs to the monoclinic plate crystal system; slightly deliquescent; soluble in ethanol, insoluble in ether; hygroscopic and deliquescent; provides trace amounts of metallic manganese for the growth of diatoms and bacteria.

[0065] (14) Zinc chloride heptahydrate (ZnCl2·7H2O): soluble in methanol, ethanol, glycerol, acetone and ether, but insoluble in liquid ammonia; highly hygroscopic, it can absorb moisture from the air and deliquesce; it has the property of dissolving metal oxides and cellulose; it provides trace amounts of zinc for the growth of diatoms and bacteria.

[0066] (15) Sodium molybdate dihydrate (Na2MoO4·2H2O): A lustrous, colorless, orthorhombic plate-like crystal; stable at room temperature and pressure. Avoid light, open flame, and high temperature. Slightly soluble in water, insoluble in acetone; melting point 687ºC; provides trace amounts of metallic sodium for the growth of diatoms and bacteria.

[0067] (16) Ferric chloride hexahydrate (FeCl3·6H2O): The solid product is a brown crystal; melting point is about 37℃; relative density is 1.82; it readily absorbs moisture from the air and deliquesces; the liquid product is a reddish-brown solution; it is readily soluble in water, ethanol, glycerol, ether and acetone, but sparingly soluble in benzene; it has good solubility and excellent flocculation effect; it can be used for activated sludge dewatering; the pH range for use is 6.0 to 11.0; it provides trace amounts of metallic iron for the growth of diatoms and bacteria.

[0068] (17) Cobalt chloride hexahydrate (CoCl2·6H2O): Red or purplish-red monoclinic crystals; a low-toxicity substance with deliquescent properties; soluble in water, glycerol, ethanol, acetone, and ether; melting point 87℃; its aqueous solution turns blue when heated or with concentrated hydrochloric acid, chloride, or organic solvent; provides trace amounts of metallic cobalt for the growth of diatoms and bacteria.

[0069] Compared with existing technologies, the beneficial effects of this invention are as follows: The novel bacterial-diatom co-culture culture medium formulation has the following advantages: The culture medium optimized by this invention can improve the growth rate of bacteria and diatoms during bacterial-algal symbiosis, increase the biomass of both, and reduce the cultivation cost of the algal-bacterial symbiosis. Specifically, when yeast extract and maltose are added simultaneously, and the concentrations of total nitrogen, total phosphorus, and silicate are set to 500 mg / L, 12 mg / L, and 400 mg / L respectively, the growth rate and biomass of bacteria and diatoms reach their maximum values. Attached Figure Description

[0070] Figure 1 This is a schematic diagram illustrating the effect of total nitrogen concentration in the culture medium of this invention on diatom cell density (A) and bacterial abundance (B).

[0071] Figure 2 This is a schematic diagram illustrating the effect of total phosphorus concentration in the culture medium of this invention on diatom cell density (A) and bacterial abundance (B).

[0072] Figure 3 This is a schematic diagram illustrating the effect of silicate concentration in the culture medium of the present invention on diatom cell density (A) and bacterial abundance (B). Detailed Implementation

[0073] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and 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.

[0074] Experimental Example

[0075] The novel bacterial-diatom co-culture medium was formulated based on a combination of CSI medium (a commonly used freshwater diatom medium), yeast extract, and maltose, with the pH adjusted to 7.0. The concentrations of total nitrogen, total phosphorus, and silicate were set at 1 / 2, 1, and 2 times the original CSI formulation, respectively. A single-factor controlled variable experiment was conducted, and the specific concentration settings for each group are shown in Table 1. Except for the nitrogen, phosphorus, and silicate concentrations, all other components in each treatment group were the original CSI formulation. Three replicates were set for each treatment group. During the experiment, test tubes containing *Gnaphalium granulosum* algal solution were shaken well and aseptically transferred directly into 50 ml glass Erlenmeyer flasks. The flasks were sealed and incubated in a light incubator at 25°C and 3000 Lux for 12 h / 12 ​​h. After completing the light culture of the freshwater diatoms, 5 ml of the algal solution was added to 10 ml of fresh medium for activation and then incubated in a sterile Erlenmeyer flask. After approximately 15 days of cultivation, the OD value of the algal solution was measured daily until the diatoms reached the exponential growth phase. Then, a large-scale culture of freshwater diatoms was conducted at a transfer ratio of 1:5 (algal solution: culture medium). During cultivation, the conical flask was shaken twice daily. Subsequently, a sufficient concentration of *Streptomyces granulosus* and *Roseobacterium rubrum* strain *Ruegeria pomeroyi* were co-cultured in novel culture media and CSI media with different formulations. The culture was placed in a constant-temperature shaker for 2 hours daily at 160 rpm and 25°C to complete the symbiotic culture. The culture was then placed in a light incubator for symbiotic light cultivation for 10 consecutive days. Bacterial and diatom culture samples were collected every 2 days. Bacterial abundance was determined using flow cytometry, and diatom density was counted under a microscope. The OD values, growth rates, and biomass of the symbiotic culture media in different formulations were analyzed and compared to screen for the optimal culture medium formulation ratio.

[0076] Table 1. Optimal Concentrations of Nitrogen, Phosphorus, and Silicon Nutrients in Experimental Groups

[0077]

[0078] The method for determining bacterial abundance is as follows: 1 ml of bacterial suspension is injected into a 2 ml cryovial, and glutaraldehyde solution (final concentration 0.5%) is immediately added for fixation. The solution is mixed well, placed in the dark for 15 min, and then rapidly frozen in liquid nitrogen for storage. Two parallel samples are collected for bacterial abundance testing. During the test, the thawed water sample at room temperature is stained with SYBR Green I fluorescent dye in the dark for 15 min, and 10 μL of 1 μm diameter standard fluorescent beads (concentration 10) are added. 7 cellml -1 After mixing, samples were analyzed using a flow cytometer. High-abundance samples were diluted with TE buffer before analysis. Heterotrophic bacteria were counted using a flow cytometer at an injection rate of 10-20 μL / min. -1Bacterial cell collection speed is 100-300 events. -1 During sample injection, after all parameters stabilize for 30 seconds, begin collecting bacterial abundance data for 1-2 minutes, ensuring the total number of events obtained is greater than 10,000. Measure each sample twice, and take the average of the two parallel sample measurements as the final abundance. (This is done at the Becton-Dickinson Accurial Center.) TM The C6 system software identifies and counts bacterial abundance based on lateral scattered light and green fluorescent patterns.

[0079] The method for determining the density of diatom thallus is as follows: 1 mL of uniform algal solution is injected into the Sedgewick-Rafte phytoplankton counting frame, and the diatom thallus are counted under a Nikon TS-100 inverted microscope.

[0080] The method for determining the OD value of the bacterial-algae co-culture solution is as follows: take an appropriate amount of culture solution and measure the absorbance value under a UV-Vis spectrophotometer with the wavelength set at 680 nm.

[0081] Experimental results are as follows Figure 1 , 2 As shown in Figure 3.

[0082] Figure 1 In this study, the total nitrogen concentration in the novel culture medium was set to 1 / 2, 1, and 2 times that of the traditional CSI medium, respectively, while the contents of other components remained unchanged. Experimental results showed that during the culture period, the algal density in the low-nitrogen group (total nitrogen concentration of 125 mg / L) ranged from 0.5 to 6.5 × 10⁻⁶. 5 Ind. / mL, the algal density in the medium nitrogen group (total nitrogen concentration of 250 mg / L) ranged from 0.5 to 72.3 × 10⁻⁶. 5 The algal density in the high-nitrogen group (total nitrogen concentration of 500 mg / L) ranged from 0.5 to 156.0 × 10⁻⁶. 5 ind. / mL ( Figure 1 A). During the culture period, the bacterial abundance in the low-nitrogen group ranged from 1.0 to 2.3 × 10⁻⁶. 6 The algal density in the medium nitrogen group ranged from 1.0 to 5.1 × 10⁻⁶ cells / mL. 6 The algal density in the high-nitrogen group ranged from 1.0 to 6.8 × 10⁻⁶ cells / mL. 6 cells / mL ( Figure 1(B) Throughout the culture period, the algal density and bacterial abundance in the high-nitrogen group were significantly higher than those in the low-nitrogen and medium-nitrogen groups, indicating that increasing nitrogen concentration is beneficial to promoting the growth rate and biomass of the algal-bacterial co-culture system. This is because nitrogen, as an important nutrient, is an essential element for both bacteria and diatoms; increasing its concentration can enhance the metabolic rate of bacterial and diatom cells and increase extracellular enzyme activity, thus promoting the growth rate and biomass of both.

[0083] Figure 2 In this study, the total phosphorus concentration in the novel culture medium was set to 1 / 2, 1, and 2 times that of the traditional CSI medium, respectively, while the contents of other components remained unchanged. The experimental results showed that during the culture period, the algal density in the low-phosphorus group (total phosphorus concentration of 12 mg / L) ranged from 0.5 to 112.3 × 10⁻⁶. 5 Ind. / mL, the algal density in the medium phosphorus group (total phosphorus concentration of 25 mg / L) ranged from 0.5 to 80.9 × 10⁻⁶. 5 The algal density in the high-phosphorus group (total phosphorus concentration of 50 mg / L) ranged from 0.5 to 100.0 × 10⁻⁶. 5 ind. / mL ( Figure 2 A). During the culture period, the bacterial abundance in the low-phosphorus group ranged from 1.0 to 5.2 × 10⁻⁶. 6 The abundance of bacteria in the medium phosphorus group ranged from 1.0 to 3.2 × 10⁻⁶ cells / mL. 6 The bacterial abundance in the high-phosphorus group ranged from 1.0 to 1.8 × 10⁻⁶ cells / mL. 6 cells / mL ( Figure 2 (B) During the first four days of cultivation, the algal density in the low-phosphorus group was lower than that in the high-phosphorus and medium-phosphorus groups. However, in the later stages of cultivation, the algal density in the low-phosphorus group increased significantly and was significantly higher than that in the high-phosphorus and medium-phosphorus groups. Throughout the cultivation period, the bacterial abundance in the low-phosphorus group was significantly higher than that in the high-phosphorus and medium-phosphorus groups. This is because, in the early stages of cultivation, bacteria compete with diatoms for phosphorus. The bacteria in the low-phosphorus group, due to their larger specific surface area, have a higher capacity and rate of phosphorus absorption and utilization than diatoms, resulting in a slower growth rate of diatoms.

[0084] Figure 3 In this study, the silicate concentration in the novel culture medium was set to 1 / 2, 1, and 2 times that of the traditional CSI medium, while the contents of other components remained unchanged. Experimental results showed that the algal density in the low-silica group (silicate concentration of 100 mg / L) varied from 0.5 to 6.5 × 10⁻⁶ during the culture period. 5 Ind. / mL, the algal density of the medium silicate group (silicate concentration of 200 mg / L) ranged from 0.5 to 17.6 × 10⁻⁶. 5 In the high-silica group (silicate concentration 400 mg / L), the algal density ranged from 0.5 to 20.1 × 10⁻⁶.5 ind. / mL ( Figure 3 A). During the culture period, the bacterial abundance in the low-silica group varied from 1.0 to 1.6 × 10⁻⁶. 6 The abundance of bacteria in the mid-silica group ranged from 1.0 to 2.3 × 10⁻⁶ cells / mL. 6 The bacterial abundance in the high-silica group ranged from 3.1 to 1.8 × 10⁻⁶ cells / mL. 6 cells / mL ( Figure 3 B). During the culture period, the growth rate and biomass of diatoms and bacteria were higher in high-concentration silicate media. This is because silicate, as the main raw material for diatom cell wall synthesis, plays a significant role in promoting the growth and reproduction of diatoms. In turn, the release of organic carbon by diatoms promotes bacterial growth. Therefore, in the later stages of culture, the bacterial biomass in high-concentration silicate media was significantly higher than that in medium- and low-concentration silicate media.

[0085] The above experimental results show that the new culture medium, based on CSI medium, adds yeast extract to provide trace elements for bacteria and maltose to provide a carbon source. The optimal nitrogen, phosphorus, and silicon concentrations are 2 times, 1 / 2 times, and 2 times that of the original CSI medium, respectively, i.e., 500 mg / L, 12 mg / L, and 400 mg / L. After continuous cultivation in this new culture medium for 10 days, the highest diatom density reached 156.0 × 10⁻⁶. 5 ind. / mL, which is the maximum density in the original culture medium of 6.5 × 10⁻⁶. 5 The bacterial abundance was 24 times that of ind. / mL; the highest bacterial abundance reached 6.76 × 10⁻⁶. 6 The number of cells / mL is approximately 2.28 × 10⁻⁶, representing the maximum bacterial abundance in the original culture medium. 6 This novel bacterial-diatom symbiotic culture medium can significantly increase the growth rate and biomass of both bacteria and diatoms, resulting in cost savings in culture. (The cell / mL ratio is three times higher than that of other cultures.)

[0086] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made based on the content of the present invention specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A method for co-culturing bacteria and diatoms, characterized in that, The composition of the co-culture medium is as follows: Calcium nitrate tetrahydrate Ca(NO3)2·4H2O: 300 mg / L; Potassium nitrate (KNO3): 200 mg / L; Magnesium sulfate heptahydrate MgSO4·7H2O: 40 mg / L; Sodium β-glycerophosphate pentahydrate C3H7Na2O6P·5H2O: 12 mg / L; Vitamin B 12 Vitamin B 12 0.1 ug / L; Vitamin B1O2: 0.1 ug / L; Thiamine HCl hydrochloride: 10 ug / L; 4-Hydroxyethylpiperazine ethanesulfonic acid HEPES buffer: 0.5 g / L; Sodium silicate nonahydrate Na2SiO3·9H2O: 400 mg / L; Yeast extract: 0.1g / L; Maltose: 0.5g / L; Disodium ethylenediaminetetraacetate (Na2EDTA): 4.5 mg / L; Manganese chloride tetrahydrate MnCl2·4H2O: 0.25 mg / L; Zinc chloride heptahydrate ZnCl2·7H2O: 30ug / L; Sodium molybdate dihydrate Na2MoO4·2H2O: 24ug / L; Ferric chloride hexahydrate FeCl3·6H2O: 1.0 mg / L; Cobalt chloride hexahydrate (CoC) l2 ·6H2O: 12ug / L; The co-culture method includes the following steps: 1): Prepare the co-culture medium according to the above formula, adjust the pH to 7.0 with HEPES buffer, sterilize in an autoclave at 120℃ for 20 minutes, and store the sterilized medium in a refrigerator at 4℃ for later use. 2): The pure algal strain of *Streptomyces granulosus* obtained by the antibiotic method was cultured in CSI medium until the logarithmic growth phase for later use; 3): Starvation culture for 48 hours; 4): After starvation culture, the *Streptomyces granulosus* was centrifuged at 4000 r / min for 4 min. The supernatant was then poured off and inoculated into Erlenmeyer flasks containing the co-culture medium. The pure algae inoculation amount was 0.5 × 10⁻⁶. 5 ind. / mL, seal with sealing film; 5): Transfer the conical flask into the light incubator, set the temperature to 25℃, the light intensity to 3000 lx, and the light-dark ratio to 12h:12h; 6): Shake the conical flask once every 3 hours and culture continuously for 10 days. All experimental operations were carried out in a clean bench and all instruments were sterilized. 7): The selected bacterial strain is *Roseobacterium pomeroyi*, a beneficial bacterium that coexists with diatoms. The pure culture of this bacterium was inoculated into sterile granular *Chaetoceros oryzae* solution that had undergone starvation culture, and then transferred to an Erlenmeyer flask containing the co-culture medium. The inoculation amount was 1.0 × 10⁻⁶. 6 cells / mL; 8): Transfer the conical flask into the light incubator, set the temperature to 25℃, the light intensity to 3000 lx, and the light-dark ratio to 12h:12h; 9): Shake the conical flask once every 3 hours and culture continuously for 10 days. All experimental operations were carried out in a clean bench and all instruments were sterilized. 10): Bacterial and diatom culture medium samples were taken every 2 days. Bacterial abundance was determined by flow cytometry and diatom density was counted under a microscope.

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