Spirillum litorale and microecological preparation and application thereof

Abstract

The application belongs to the technical field of aquatic micro-ecological preparations, and particularly relates to a salt-tolerant and alkali-tolerant Ectothiorhodospira shaposhnikovii. The application obtains a high-salt-tolerant and high-alkali-tolerant Ectothiorhodospira shaposhnikovii (Ectothiorhodospira shaposhnikovii Cherni) through screening, and the preservation number of the strain is CGMCC No. 40095. The strain has a good effect of degrading nitrite in a water environment, and has a good application value for popularization.

Description

Technical Field

[0001] This invention belongs to the field of microecological preparation technology, specifically relating to a strain of *Exothromyces salsa*, its microecological preparations, and their applications. Background Technology

[0002] Photosynthetic bacteria (PSB) possess unique physiological functions such as carbon fixation, nitrogen fixation, desulfurization, denitrification, and hydrogen production, as well as rich metabolic diversity. They have great application prospects in biohydrogen production, environmental remediation, and agricultural production, and have been widely used in environmental management, aquaculture, and poultry farming.

[0003] Currently, there are few reports on research on *Exosulfuron-Saxon*. *Exosulfuron-Saxon* can utilize some small-molecule organic compounds such as sodium acetate and sodium propionate as carbon sources and hydrogen donors for growth, belonging to photosynthetic bacteria capable of photoautotrophic growth. *Exosulfuron-Saxon* can grow in media using Na₂S and Na₂S₂O₃ as the sole photosynthetic hydrogen donors, possesses catalase activity, can hydrolyze starch, cannot liquefy gelatin, and can grow using nitrite as a nitrogen source.

[0004] Carbon source utilization analysis using a fully automated microbial identification instrument indicates that this bacterium can utilize both small-molecule organic acid salts and glucose, demonstrating its autotrophic and heterotrophic metabolic capabilities. During liquid culture, the culture is deep red. The bacterial cells are mostly short rod-shaped.

[0005] The strain exhibits high tolerance to sulfides, grows well under high sulfide concentrations, and continuously utilizes sulfides during cultivation, demonstrating a high removal rate. This indicates that the bacterium shows promising potential for purifying eutrophic aquaculture water.

[0006] Market research indicates that the number of commercially available photosynthetic bacteria microecological preparations is currently limited. Many of these are substandard, lacking live bacteria and effective ingredients, are artificially colored, or lack salt and alkali tolerance. While some biotechnology companies are working on developing such products, the supply is far from meeting market demand. Existing *Exothromyces salicylates* exhibits varying abilities to remove ammonia and nitrite from water, poor environmental adaptability, unstable strain characteristics, susceptibility to contamination, and weak sulfide utilization. Therefore, there is an urgent need to develop *Exothromyces salicylates* with high nitrite degradation capabilities, strong tolerance to high-salt and high-alkali water bodies, the ability to utilize environmental sulfides, and stable characteristics. Summary of the Invention

[0007] To address the aforementioned problems, this invention provides a strain of *Ectothiorhodospira shaposhnikovii* Cherni, with accession number CGMCC No. 40095.

[0008] The present invention also provides a microecological preparation of the aforementioned *Exothromyces sarcoptes*.

[0009] The present invention also provides a method for preparing the aforementioned microecological preparation, characterized by comprising the following steps: inoculating the seed liquid of *Exothromyces salviae* CGMCC No. 40095 into the fermentation medium at an inoculation rate of 1-10% by volume, and culturing under light for 2-5 days to obtain the preparation.

[0010] The present invention also provides the application of the aforementioned *Exothiospirobacter salicylates* in degrading nitrite in water.

[0011] This invention also provides the application of the aforementioned microecological preparation in the degradation of nitrite in water.

[0012] The advantages of this invention compared to the prior art are as follows:

[0013] This patented strain has a salinity tolerance range of 10‰-70‰, exhibiting a wide salt tolerance range and adapting to the salinity conditions of most freshwater and seawater aquaculture bodies. pH is a crucial water quality indicator for aquaculture; a pH < 5.0 or pH > 9.0 in aquaculture water will cause abnormal blood pH in farmed fish, disrupting their blood buffering system, affecting respiration, and even leading to death. my country's fisheries water quality standards stipulate that the suitable pH range for freshwater aquaculture is 6.5-8.5, and for seawater, it is 7.0-8.5. This patented strain can grow within a pH range of 6.0-9.5, making it suitable for use in aquaculture water. After 48 hours, the strain achieves a nitrite degradation rate of over 90% in the water, demonstrating excellent potential for removing nitrogen sources from the water. It is a suitable strain of *Exosulfuron-Saxon* for purifying aquaculture water environments.

[0014] This strain has a wide range of salinity and pH tolerance and can be used to effectively remove nitrite nitrogen in both freshwater and marine aquaculture environments, showing broad application prospects in the purification of aquaculture water environments. Attached Figure Description

[0015] Figure 1 This is a colony diagram of the plate culture of the *Exothiophora salicylates* strain of the present invention.

[0016] Figure 2 This is a microscopic image of *Exothiospira salicylides* from the present invention.

[0017] Figure 3 This is a standard curve of sodium nitrite content.

[0018] Figure 4 The degradation effect of *Exothiospermum sarcodactylus* on nitrite under light conditions is shown in this invention.

[0019] Figure 5The invention demonstrates the degradation effect of *Exothiophora salicylates* on nitrite under dark conditions.

[0020] Figure 6 The effect of different salinities on the growth of the *Exothiophora salicylates* strain of this invention.

[0021] Figure 7 The effect of different pH values ​​on the growth of the *Exothiophora salicylata* strain of this invention. Detailed Implementation

[0022] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention.

[0023] Example 1: Isolation and identification of *Ectothiorhodospira shaposhnikovii* Cherni

[0024] (1) Prepare photosynthetic bacteria culture medium

[0025] Enrichment medium ( / L): Sodium acetate trihydrate 1.5g, ammonium oxalate 2.5g, sodium chloride 1.0g, potassium dihydrogen phosphate 0.5g, yeast extract 1.5g, sodium bicarbonate 3g, pH 7.0, autoclaved at 121℃ for 20min.

[0026] The isolation medium was prepared by adding agar to the enrichment medium, with a final mass fraction of 1.5%, pH 7.0, and autoclaved at 121°C for 20 min.

[0027] (2) Enrichment

[0028] The collected mud-water samples were placed in screw-top anaerobic tubes containing enrichment medium. Each tube contained 15 mL of medium and 10 mL of pond water and bottom mud, and each was covered with 2-3 cm of paraffin oil. The tubes were anaerobically cultured at 30°C under a light intensity of 1000-2000 lx.

[0029] (3) Separation and purification

[0030] After one week of enrichment, 1 mL of liquid was taken from the anaerobic tube where the culture medium had turned red, and the solution was diluted to 10⁻⁶. -3 10 -4 10 -5 Spread 100 μL of the liquid onto a plate, and observe the growth of red colonies after about 10 days; pick out the red colonies and streak them onto the plate to obtain a pure strain.

[0031] (4) Identification

[0032] Single colonies were selected for 16S rRNA colony PCR and sent to Invitrogen for sequencing. The sequence is shown in SEQ ID No. 1. The sequence was then compared with NCBI using BLAST and preliminarily identified as *Ectothiorhodospira* sp. Rh35, with 100% similarity to the 16S rRNA gene of *Ectothiorhodospira* sp. B7-7. This bacterium grows slowly; single colonies appear after 5 days of anaerobic culture, ranging in size from 0.3 mm to 1.2 mm. Under anaerobic conditions, the colonies are dark red with intact edges. It is a Gram-negative bacterium, and the strain morphologically consists of short rods. Solid plate culture yields... Figure 1 As shown, the morphology under the microscope is as follows Figure 2 As shown.

[0033] The isolated *Ectothiorhodospira shaposhnikovii* Cherni is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The deposit date is June 23, 2022, and the accession number is CGMCCNo. 40095.

[0034] Example 2: Preparation of *Exothromyces salsa* bacterial suspension

[0035] (1) Preparation of seed culture of *Exothromyces salviae*

[0036] Seed culture medium formula: ammonium chloride 1.0 g / L, potassium dihydrogen phosphate 0.6 g / L, calcium chloride dihydrate 0.1 g / L, magnesium chloride 0.3 g / L, sodium acetate 4 g / L, sodium bicarbonate 3 g / L, sodium chloride 25 g / L.

[0037] Culture steps: Take out 100 μL of *S. salvage* frozen at -80℃, inoculate it into a 10 ml anaerobic tube, and culture it under light at 30℃ for 5 days; inoculate the well-grown bacterial solution into a 100 ml anaerobic bottle at a ratio of 5%, and culture it under light at 30℃ for 5 days to obtain *S. salvage* seed culture.

[0038] (2) Large-scale culture of *Exosulfuron-Saxon*

[0039] The formulation of the expanded culture medium for *Exothromyces sarcoptes* is as follows: ammonium chloride 1.0 g / L, potassium dihydrogen phosphate 0.6 g / L, calcium chloride dihydrate 0.1 g / L, magnesium chloride 0.3 g / L, sodium acetate 4 g / L, sodium bicarbonate 3 g / L, and sodium chloride 25 g / L.

[0040] Expanded culture steps:

[0041] 1) Weigh each component according to the culture medium formula, dissolve it in water, and dispense the culture medium into 5L PET plastic containers (4.5L each) that have been sterilized with alcohol. Inoculate each container with 500mL of *Salmonella exostreatus* CGMCC No. 40095 seed culture and seal the container. Place the container in a cardboard box and incubate under incandescent light for 4 days. This culture medium can then be used as the seed culture for the next large-scale culture.

[0042] 2) Weigh all the components required for 1 ton of *Rhodospirillum sarcopenia* expansion culture medium. First, completely dissolve them in a small amount of tap water, then thoroughly mix them with the remaining tap water in a large container. Inoculate 10 containers of seed culture (4.5L each), mix well, and then dispense into 5L transparent PET plastic containers and seal. Incubate under light for 5 days until the culture turns reddish-brown, at which point you will obtain high-purity *Rhodospirillum sarcopenia* bacterial culture.

[0043] Example 3: Determination of the salinity versatility of *Exothrostilus salina* CGMCC No. 40095

[0044] (1) Salinity culture medium: Prepare 100ml of liquid culture medium and place it in a 150ml anaerobic bottle. Adjust the salinity to 0‰, 10‰, 20‰, 30‰, 40‰, 50‰, 60‰, 70‰, 80‰, 90‰ and 100‰ respectively. Autoclave at 121℃ for 20min.

[0045] (2) Three replicates were set up for each salinity, and the inoculation amount of the culture medium was 5% seed liquid. The culture was placed at 30°C for seven days of anaerobic culture.

[0046] (3) According to the experimental results, such as Figure 6 As shown, the strains all grew normally at salinities of 10‰-70‰, and showed good growth performance in the salinity range of 20‰-30‰.

[0047] Example 4: pH adaptability determination of *Exothromyces salsa* CGMCC No. 40095

[0048] (1) pH culture medium: Prepare 100ml of liquid culture medium and place it in a 150ml anaerobic bottle. Adjust the pH to 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, and 12.0 respectively. Autoclave at 121℃ for 20min.

[0049] (2) Three replicates were set for each pH, ​​and the seed culture was inoculated with 5% seed culture at 30°C for seven days.

[0050] (3) According to the experimental results, such as Figure 7 As shown, the strains all grew normally in pH environments ranging from 6.0 to 9.5, and exhibited good growth within a pH range of 6.0 to 7.5.

[0051] Example 5: Test of the nitrite degradation ability of *Exothromyces sarcopentae* CGMCC No. 40095

[0052] 1. Experimental Methods

[0053] (1) Prepare a sodium nitrite standard curve: Prepare nitrite solutions of different concentrations and measure OD under an enzyme-linked immunosorbent assay (ELISA) reader. 550 Create a standard curve.

[0054] (2) Nitrite culture medium for the experiment: Sterile water was placed in 150mL anaerobic bottles, 50mL / bottle, and 0.05g of shrimp feed and 50ul of sodium nitrite stock solution were added to each bottle. The mixture was sterilized by autoclaving at 121℃ for 20min.

[0055] (3) Set up dark and light conditions, and set up experimental and control groups under each condition. Each strain of bacteria was replicated 3 times. 50 μL of photosynthetic bacteria seed liquid was added to the experimental group and placed under dark and light conditions respectively. The bacteria were then placed at 30°C for incubation.

[0056] (4) Measure the nitrite content every 24 hours. The specific method is as follows: Take 1 mL of liquid into a 1.5 mL centrifuge tube in a clean bench, centrifuge at 12,000 rpm for 2 minutes, and aspirate 200 μL of the supernatant into a 96-well plate. Add 20 μL each of Gliss reagent I and II successively, let stand for 5 minutes to allow the reaction to proceed completely, and then measure the OD under a microplate reader. 550 .

[0057] (5) Calculate the nitrite concentrations of the experimental group and the control group based on the standard curve, measure for 2 days, record the experimental data and analyze.

[0058] 2. Experimental Results

[0059] (1) Nitrite content and OD 550 Standard curve creation, such as Figure 3 The figure shows the standard curve of sodium nitrite content. The linear correlation coefficient obtained is 0.9998, indicating that the sodium nitrite concentration and OD550 of the standard curve are strongly correlated.

[0060] (2) Figure 4 and Figure 5 As shown, the *Salmonella exostreatus* CGMCC No. 40095 inoculant exhibited significant nitrite degradation effects under both dark and light conditions. Under dark conditions, after 48 hours, the nitrite content decreased from 2.5 mg / L to a minimum of 0.195 mg / L; under light conditions, after 48 hours, the sodium nitrite content decreased from 2.5 mg / L to 0.217 mg / L.

Claims

1. A strain of *Ectothiorhodospira shaposhnikovii* Cherni, with accession number CGMCC No. 40095.

2. A microecological preparation containing *Exothromyces salviae* as described in claim 1.

3. The method for preparing the probiotic preparation according to claim 2, characterized in that, The process includes the following steps: Inoculate the seed culture of *Exothromyces salsa* CGMCC No. 40095 into the fermentation medium at a volume ratio of 1-10%, and culture under light for 2-5 days.

4. The application of the *Exothromyces salsa* as described in claim 1 in the degradation of nitrite in water.

5. The application of the microecological preparation according to claim 2 in the degradation of nitrite in water.

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