An alkaliphilic Exiguobacterium and its application in purifying water bodies of saline-alkali aquaculture caused by water bloom

Abstract

The present invention relates to the field of functional microbial remediation technology, and specifically to a strain of alkaliphilic Exiguobacterium and its application in purifying water bodies for salt-alkali aquaculture in blooms. Alkaliphilic Exiguobacterium SADOM03 has been deposited in the General Microbiological Center of the China Microbiological Culture Collection Committee on November 4, 2024, with a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with a deposit number of CGMCC No. 32489, and is classified as alkaliphilic Exiguobacterium Exiguobacterium alkaliphilum The alkaliphilic Exiguobacterium SADOM03 provided by the present invention can effectively decompose algae-derived dissolved organic matter in water bodies with alkaline blooms and significantly remove organic pollution and nitrogen and phosphorus nutrients in water bodies, greatly improving water quality and purifying water bodies, and is expected to be applied to in-situ treatment of alkaline bloom pollution in saline-alkali aquaculture water bodies.

Description

Technical Field

[0001] The invention relates to the technical field of functional microbial restoration, and in particular to an alkaliphilic microbacterium and its application in purifying alkaline bloom aquaculture water bodies. Background Art

[0002] Pond aquaculture activities often lead to eutrophication of water bodies due to the deposition of feed residues and high stocking density. In the hot spring and summer seasons, algal blooms are easily induced, which is the aggregation of algae caused by the excessive growth of phytoplankton. Algal bloom waters often contain a large amount of algae-derived dissolved organic matter, which can serve as an organic pollution source and continuously release inorganic nutrients into the water through decomposition, continuously supplying the nitrogen and phosphorus nutrition needs of algae. This is the fundamental reason why the eutrophication of water bodies cannot be improved quickly and the algal blooms continue to exist or occur. Therefore, accelerating the degradation of algae-derived dissolved organic matter in algal bloom waters is conducive to fundamentally achieving the purpose of purifying water bodies and preventing algal blooms.

[0003] Due to the shortage of water resources in saline-alkali areas, water for pond aquaculture has always been limited. Most of them adopt the method of one-time water injection and later water replenishment. After the aquaculture is completed, the water is often not changed or drained. Therefore, this kind of saline-alkali pond aquaculture activities often lead to more serious water pollution and increased incidence of algal blooms. For this reason, the prevention and control of eutrophication pollution and algal blooms in saline-alkali aquaculture water bodies has become a key technology for the purification and regulation of saline-alkali pond aquaculture water bodies, and has received increasing attention in recent years.

[0004] As one of the main biological communities in aquatic ecosystems, microorganisms play a very important role in the degradation of organic matter and the conversion and utilization of nitrogen and phosphorus nutrients, and are often used in the field of biological control of water pollution and algal blooms. For example, CN102206605 A uses a microbacterium that can secrete certain extracellular algae-dissolving substances to dissolve algae. However, the technical solution can only dissolve and inhibit a few types of harmful algae. For example, it does not show a good algae-dissolving effect on eukaryotic algae species (green algae M7 and Chlamydomonas BS3). Therefore, there are certain limitations in its application, and it does not have the function of degrading algae-derived dissolved organic matter. At the same time, current research is mainly focused on the purification of algal bloom freshwater or algal bloom seawater aquaculture water bodies. There is still little research on the purification of algal bloom saline-alkali aquaculture water bodies, and there is a lack of salt-alkali tolerant microorganisms that have good removal effects on organic pollution and nitrogen and phosphorus nutrients in algal bloom saline-alkali aquaculture water bodies. Summary of the invention

[0005] In view of the technical problem of lack of salt-alkali tolerant microorganisms with good removal effect on organic pollution and nitrogen and phosphorus nutrients in water bodies of water bloom saline-alkali aquaculture, the present invention provides an alkaliphilic microbacterium and its application in purifying water bodies of water bloom saline-alkali aquaculture.

[0006] The technical solution of the present invention is as follows:

[0007] In the first aspect, the present invention provides a strain of alkaliphilic Exiguobacterium SADOM03, which has been deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on November 4, 2024, with a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with a deposit number of CGMCC No. 32489, and is classified as alkaliphilic Exiguobacterium Exiguobacterium alkaliphilum .

[0008] In the second aspect, the present invention provides an application of the above-mentioned alkaliphilic Exiguobacterium SADOM03 to purify water bodies for algae bloom saline-alkali aquaculture, preferably, including specific applications such as degrading algae-derived dissolved organic matter in water bodies for algae bloom saline-alkali aquaculture and / or removing organic pollution and nitrogen and phosphorus nutrients in water bodies (i.e., reducing COD, TN, TP in water bodies).

[0009] Furthermore, a bacterial suspension of alkaliphilic Exiguobacterium SADOM03 was added to the bloom saline-alkali aquaculture water.

[0010] Furthermore, the preparation method of the bacterial suspension of Exiguobacterium alkaliphilum SADOM03 is as follows: after inoculating Exiguobacterium alkaliphilum SADOM03 into a sterile LB medium for activation and culture for 2 days, the bacterial bodies are centrifuged and resuspended in the same volume of sterile pure water.

[0011] Furthermore, the viable count of the alkaliphilic Exiguobacterium SADOM03 bacterial suspension was 3×10 8 -6×10 8 Pieces / mL.

[0012] Furthermore, the viable count was 3 × 10 8 -4×10 8 The alkaliphilic Exiguobacterium SADOM03 bacterial suspension with a concentration of 100 / mL was used to remove organic pollution and nitrogen and phosphorus nutrients from alkaline bloom aquaculture water.

[0013] Furthermore, the viable count was 5 × 10 8 -6×10 8 The alkaliphilic Exiguobacterium SADOM03 bacterial suspension with a concentration of cells / mL can degrade algae-derived dissolved organic matter in algae bloom aquaculture water.

[0014] Furthermore, the alkaliphilic Exiguobacterium SADOM03 bacterial suspension is added to the bloom saline-alkali aquaculture water body at a volume ratio of 1%-5%, preferably at a volume ratio of 2%.

[0015] Furthermore, the purification time is more than 3 days. When used to remove organic pollution and nitrogen and phosphorus nutrients in water bodies caused by water bloom and saline-alkali aquaculture, the purification time is preferably more than 5 days.

[0016] The beneficial effects of the present invention are:

[0017] The alkaliphilic Exiguobacterium SADOM03 provided by the present invention can effectively decompose algae-derived dissolved organic matter in algae bloom saline-alkali aquaculture water and significantly remove organic pollution and nitrogen and phosphorus nutrients in the water, greatly improving water quality and purifying the water. It is expected to be applied to the in-situ treatment of algae bloom pollution in saline-alkali aquaculture water. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0019] Figure 1 This is a colony picture of the alkaliphilic Exiguobacterium SADOM03 in Example 1.

[0020] Figure 2 This is the phylogenetic tree obtained after 16S rDNA gene sequence analysis of the alkaliphilic Exiguobacterium SADOM03 in Example 1.

[0021] Figure 3 It is a three-dimensional fluorescence spectrum diagram of the degradation of algae-derived dissolved organic matter in the bloom saline-alkali aquaculture water by the alkaliphilic Exiguobacterium SADOM03 in Example 2, wherein the upper figure is the three-dimensional fluorescence spectrum of the algae-derived dissolved organic matter solution, and the lower figure is the three-dimensional fluorescence spectrum of the SADOM03 experimental group.

[0022] Figure 4 This is a bar graph of the removal effect of alkaliphilic Exiguobacterium SADOM03 on organic pollution and nitrogen and phosphorus nutrients in aquaculture water in Example 3. * indicates that there is a significant difference between the data. P <0.05. DETAILED DESCRIPTION

[0023] In order to enable those skilled in the art to better understand the technical solutions in the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0024] The algae-derived dissolved organic matter solution used in the following examples was prepared as follows:

[0025] Water samples were taken from saline-alkali aquaculture ponds where algal blooms occurred. The main algae and composition abundance of algal blooms in the water body were as follows: Anabaena 55%, Oscillatoria 21%, and Microcystis 8%. The algal slurry was obtained by centrifugation, and the algal slurry was washed with ultrapure water for later use. Another water sample was filtered through a 0.22μm filter membrane, and the filtrate was taken to obtain pond clean water for later use. Take 20g of algal slurry with a wet weight and add it to 1000mL of pond clean water and mix well. Then, the concentrated green sample was first ultra-low-temperature frozen and then autoclaved (121℃, 20min) to break the algal cells. The sample was filtered through a 0.45μm filter membrane to obtain a brown-yellow algae-derived dissolved organic matter solution, which was sealed and stored at low temperature away from light. When used, adjust the pH to 9 and autoclave at 121℃ for 20min.

[0026] The test solution for degradation of saline-alkali aquaculture water used for bloom was prepared as follows:

[0027] Water samples were collected from saline-alkali aquaculture ponds where algal blooms occurred, filtered through a 10µm filter membrane to remove most protozoa and other particulate matter, while retaining most microalgae. The filtrate was then ultra-frozen, autoclaved at 121°C for 20 min, and filtered through a 0.45μm filter membrane. The pH value of the filtrate was adjusted to 9, divided into 100mL conical flasks, autoclaved at 121°C for 20 min, and set aside.

[0028] The LB medium used was prepared as follows:

[0029] Weigh 10 g of tryptone, 6 g of beef extract, 2.5 g of yeast extract, and 5 g of NaCl, add them to 1 L of deionized water, adjust the pH to 8.5, and sterilize at 121°C for 20 min to prepare LB liquid culture medium; add 20 g of agar powder to the above formula to prepare LB plate / slant culture medium.

[0030] Example 1 Obtaining alkaliphilic Exiguobacterium SADOM03

[0031] In August 2024, it was isolated and purified from the water bloom water body of the largemouth black bass breeding pond in Leling, Dezhou, Shandong. Specifically, a water sample was obtained from the water bloom water body (water body salinity of 6, total alkalinity of 290 mg / L, pH value of 9) of the largemouth black bass breeding pond in Leling, Dezhou, Shandong. 1 mL was taken from it and added to 100 mL of algae-derived dissolved organic matter solution for enrichment and domestication culture. After 10 days, the dominant strains were separated and purified on the LB plate medium by the coating plate method and streak separation method, and finally 6 dominant bacteria were obtained. Each bacterial strain was placed in 100 mL LB liquid culture medium, activated and cultured at 30°C and 200 rpm for 2 days, and then 2 mL was taken from it and added to 100 mL of water bloom saline-alkali aquaculture water degradation test solution, and cultured at 30°C, 200 rpm, and light-proof conditions. The removal effect of each bacteria on organic pollution and nitrogen and phosphorus nutrients in water bloom saline-alkali aquaculture water was investigated, and finally the strain SADOM03 with strong pollution control ability was screened and compared.

[0032] The strain SADOM03 was identified as facultative anaerobic, Gram-positive, multi-rod-shaped, flagellated, motile, non-spore-forming, with a flat, light orange colony and a moist surface on the LB plate medium ( Figure 1 ).

[0033] The 16 S rDNA gene sequence of strain SADOM03 was analyzed, and the gene sequence (sequence 1) of the strain was obtained as follows:

[0034]

[0035] The gene sequence of SADOM03 was submitted to GenBank for comparison and the phylogenetic tree of the strain was constructed ( Figure 2 ), the strain SADOM03 was named as alkaliphilic Exiguobacterium SADOM03.

[0036] On November 4, 2024, SADOM03 was sent to the General Microbiological Center of China Microbiological Culture Collection Administration for preservation. The preservation address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The preservation number is CGMCC No.32489, and the classification name is alkaliphilic Exiguobacterium Exiguobacterium alkaliphilum .

[0037] Example 2 Degradation of Algae-derived Dissolved Organic Matter in Algae Bloom Aquaculture Water by Exiguobacterium alkaliphilum SADOM03

[0038] After activation on LB slant medium, the alkaliphilic Exiguobacterium SADOM03 was washed with sterile saline and then inoculated into 100 mL sterilized LB liquid medium and continued to shake and activate the culture for 48 h to allow the bacteria to enter the exponential growth phase. 2 mL of the bacterial solution was centrifuged at 10,000 rpm for 30 min to obtain bacterial cells, which were then resuspended in sterile pure water to obtain a bacterial cell density of 5.81×10 8 / mL of bacterial suspension.

[0039] Take 2mL of the above bacterial suspension and add it to 100mL of algae-derived dissolved organic matter solution as the SADOM03 experimental group; take 100mL of algae-derived dissolved organic matter solution and add 2mL of sterile pure water as the blank control (control group). Each treatment was repeated 3 times, and all treatments were placed in a shaking incubator at 30℃ and 200rpm in the dark for 3 days.

[0040] After the culture was completed, the culture fluid was collected from each treatment group, filtered through a 0.22 μm filter membrane, and then mixed with the filtrate of the same treatment. The three-dimensional fluorescence spectrum was measured using an F-4500 fluorescence spectrometer. The parameters were set as follows: excitation wavelength 220-400 nm, interval 3 nm; emission wavelength 250-550 nm, interval 2 nm; slit width 5 nm; scanning speed 12000 nm / min, photomultiplier tube (PMT) voltage 700 V.

[0041] Figure 3 The above figure shows the three-dimensional fluorescence spectrum of the algae-derived dissolved organic matter solution. It can be seen that the main components of the algae-derived dissolved organic matter are macromolecular organic matter such as humic acid-like (excitation wavelength > 250nm and emission wavelength > 380nm) and fulvic acid-like (excitation wavelength < 250nm and emission wavelength > 380nm). Figure 3The figure below is the three-dimensional fluorescence spectrum of the SADOM03 experimental group. It can be found that the algae-derived dissolved organic matter in the solution is decomposed into more subtle components, which fully reveals that the alkaliphilic Exiguobacterium SADOM03 has a significant degradation effect on the algae-derived dissolved organic matter in the bloom saline-alkali aquaculture water.

[0042] Example 3: Purification effect of alkaliphilic Exiguobacterium SADOM03 on water bodies containing saline-alkali blooms:

[0043] After activating the alkaliphilic Exiguobacterium SADOM03 on the LB slant medium, it was washed with sterile saline and then inoculated into 100 mL of sterilized LB liquid medium and continued to be shaken and activated for 48 h to allow the bacteria to enter the exponential growth phase. 2 mL of the bacterial solution was centrifuged at 10,000 rpm for 30 min to obtain bacterial cells, which were then resuspended in sterile pure water to obtain a bacterial cell density of 3.20 × 10 8 / mL of bacterial suspension.

[0044] Take 2mL of the above bacterial suspension and add it to 100mL of the water body degradation test solution for salt-alkali aquaculture in water bloom as the SADOM03 experimental group; add 2mL of sterile pure water to the water body degradation test solution for salt-alkali aquaculture in water bloom as the blank control (control group). Each treatment was repeated 5 times, and all treatments were placed in a shaking incubator at 30℃ and 200rpm in the dark for 5 days.

[0045] After the cultivation, the culture fluid was taken from each treatment group, filtered through a 0.22 μm filter membrane, and the chemical oxygen demand (COD), total nitrogen (TN) and total phosphorus (TP) of the filtrate were determined. COD was determined by the acid potassium permanganate method, and the degree of organic pollution in the water body was evaluated based on the consumption of potassium permanganate solution; TN and TP were determined by spectrophotometry after potassium persulfate digestion; the data results were expressed as mean ± standard deviation and statistically analyzed ( P <0.05 is a significant difference level), and the removal rates of COD, TN, and TP were calculated using the following formula:

[0046] .

[0047] The results are as follows Figure 4 As shown in the figure, alkaliphilic Exiguobacterium SADOM03 has a significant effect on removing organic pollution and nitrogen and phosphorus nutrients in water bodies of alkaline bloom aquaculture. The removal rates of COD, TN and TP are 44.63%, 58.73% and 26.96%, respectively, which can significantly reduce the eutrophication degree of water bodies.

[0048] In summary, the alkaliphilic Exiguobacterium SADOM03 of the present invention has the effect of fundamentally improving and controlling the water quality pollution of alkaline bloom aquaculture water bodies.

[0049] Although the present invention has been described in detail with reference to the accompanying drawings and in combination with the preferred embodiments, the present invention is not limited thereto. Without departing from the spirit and essence of the present invention, a person of ordinary skill in the art may make various equivalent modifications or substitutions to the embodiments of the present invention, and these modifications or substitutions shall be within the scope of the present invention. Any person of ordinary skill in the art may easily think of changes or substitutions within the technical scope disclosed by the present invention, and these shall be within the scope of protection of the present invention.

Claims

1. A strain of Exiguobacterium alkaliphilum ( Exiguobacterium alkaliphilum ) SADOM03, characterized in that, Alkaliphilic Exiguobacterium SADOM03 was deposited in the General Microbiology Center of China Culture Collection Administration on November 4, 2024. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.32489.

2. An application of the alkaliphilic Exiguobacterium SADOM03 as claimed in claim 1 to purify saline-alkali aquaculture water bodies, characterized in that: Purification is to remove organic pollution and nitrogen and phosphorus nutrients in water bodies caused by algae blooms and saline-alkali aquaculture and / or degrade algae-derived dissolved organic matter in water bodies caused by algae blooms and saline-alkali aquaculture.

3. The purification application according to claim 2, characterized in that: Add the alkaliphilic Exiguobacterium SADOM03 bacterial suspension into the bloom saline-alkali aquaculture water.

4. The purification application according to claim 3, characterized in that: The preparation method of the bacterial suspension of Exiguobacterium alkaliphilum SADOM03 is as follows: after inoculating Exiguobacterium alkaliphilum SADOM03 into a sterile LB medium for activation and culture for 2 days, the bacterial bodies are centrifuged and resuspended in the same volume of sterile pure water.

5. The purification application according to claim 3, characterized in that: The viable count of the alkaliphilic Exiguobacterium SADOM03 bacterial suspension was 3×10 8 -6×10 8 Pieces / mL.

6. The purification application according to claim 5, characterized in that: The number of viable cells used was 3 × 10 8 -4×10 8 The alkaliphilic Exiguobacterium SADOM03 bacterial suspension with a concentration of 100 / mL was used to remove organic pollution and nitrogen and phosphorus nutrients from alkaline bloom aquaculture water.

7. The purification application according to claim 5, characterized in that: The viable count was 5 × 10 8 -6×10 8 The alkaliphilic Exiguobacterium SADOM03 bacterial suspension with a concentration of cells / mL can degrade algae-derived dissolved organic matter in algae bloom aquaculture water.

8. The purification application according to claim 2, characterized in that: The bacterial suspension of Exiguobacterium alkaliphilum SADOM03 is added into the bloom saline-alkali aquaculture water body at a volume ratio of 1%-5%.

9. The purification application according to claim 2, characterized in that: The purification time is more than 3 days.

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