Active bacterial strain for promoting formation and development of sessile attached microorganism community and application thereof

Abstract

This invention discloses an active bacterial strain that promotes the formation and development of benthic attached organism communities and its applications. *Bacilus pumilus* MSTI-2023-654, deposited on March 6, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 26745, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, effectively increases the attachment rate of benthic diatoms. Furthermore, the crude extract of this strain can induce diatom cells to produce extracellular polymers containing more polysaccharides, especially acidic polysaccharides, thereby increasing EPS adhesion and the attachment rate of benthic diatoms. The crude extract of this strain can also effectively induce the attachment and metamorphosis of barnacle larvae, which is of great significance for the economic aquaculture of oysters, sea cucumbers, and other marine organisms.

Description

Technical Field

[0001] This invention belongs to the field of biology, specifically relating to an active bacterial strain that promotes the formation and development of benthic attached organism communities and its applications. Background Technology

[0002] In recent years, global marine fishery resources have declined significantly due to factors such as environmental pollution, overfishing, and global climate change. As the potential value of the ocean becomes increasingly apparent, the protection and sustainable use of marine resources are receiving increasing attention from various countries. To alleviate marine habitat degradation, effectively protect endangered species, and achieve sustainable use of marine resources, fisheries and marine ecologists generally believe that the construction of marine ranches is an important way to ensure the sustainable development of marine fisheries. A key aspect of building marine ranches is to establish and develop marine benthic attached organism communities, promoting community succession from initial condition films to microbial mucus and then to macroscopic attached organisms.

[0003] In aquaculture, cultivating benthic diatom communities has a crucial impact on the industry's efficiency and development. The low attachment metamorphosis rate of oyster and mussel larvae, and the problem of juvenile mortality due to "sliding" after attachment metamorphosis, hinder the development of this type of marine aquaculture. Research shows that benthic diatoms not only serve as excellent food for benthic diatom larvae but also induce attachment metamorphosis in these larvae. With the development of benthic diatom communities, marine crustaceans, mollusks, and fish can grow and reproduce. Therefore, the quality of benthic diatoms directly affects the survival rate of seedlings and is one of the key factors for successful seedling production of aquatic economic animals. Thus, cultivating sufficient quantities of high-quality benthic diatoms and enhancing the formation of marine biofilms are the most important aspects of artificial seedling production.

[0004] However, because benthic diatoms have strong attachment characteristics, unlike planktonic microalgae which can utilize water in three dimensions and efficiently utilize nutrients, large-scale production and cultivation are very difficult.

[0005] Therefore, at present, the aquaculture industry in this field urgently needs to find ways to expand the scale of benthic diatom cultivation or increase the attachment rate of benthic diatoms to improve the induction activity of attachment metamorphosis of attached organism larvae. Summary of the Invention

[0006] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0007] In view of the problems existing in the above and / or prior art, the present invention is proposed.

[0008] Therefore, the purpose of this invention is to overcome the shortcomings of the prior art and provide a *Bacilus pumilus* MSTI-2023-654, which was deposited on March 6, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 26745, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0009] Another objective of this invention is to overcome the shortcomings of the prior art and provide an application of Bacilus pumilus MSTI-2023-654 in promoting the formation and development of benthic attached organism communities in aquaculture.

[0010] As a preferred embodiment of the application described in this invention, the benthic attached biological community includes benthic diatoms and barnacle larvae.

[0011] As a preferred embodiment of the application described in this invention, it includes: Bacillus pumilus MSTI-2023-654, which improves the attachment rate of benthic diatoms and induces diatom cells to produce more extracellular polymers of acidic polysaccharides, thereby increasing EPS adhesion and promoting the formation and development of benthic biofilms.

[0012] As a preferred embodiment of the application described in this invention, it includes: Bacillus pumilus MSTI-2023-654 effectively inducing attachment and metamorphosis of barnacle larvae, thereby increasing the attachment rate of larvae.

[0013] Beneficial effects of this invention:

[0014] This invention provides a *Bacilus pumilus* MSTI-2023-654, deposited on March 6, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 26745, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. This strain and its crude extract effectively increase the attachment rate of various marine benthic diatoms and barnacle larvae when treated. They also induce diatom cells to produce extracellular polymers containing more polysaccharides, especially acidic polysaccharides, thereby increasing EPS adhesion and promoting the formation and development of benthic biofilms. Furthermore, the crude extract of this strain can effectively induce the attachment and metamorphosis of barnacle larvae. In practical applications of aquaculture, increasing the attachment rate of diatoms and barnacle larvae is of great significance for promoting the formation and development of benthic biofilm communities. Attached Figure Description

[0015] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein:

[0016] Figure 1 This is a phylogenetic tree diagram of the active strain MSTI-2023-654 in the embodiments of the present invention.

[0017] Figure 2 This is a graph showing the effect of the active strain MSTI-2023-654 on the attachment rate of nine benthic diatoms in an embodiment of the present invention.

[0018] Figure 3 The figure shows the effect of the active strain MSTI-2023-654 on EPS production of *Nyctaginosa microphylla* in the embodiments of the present invention.

[0019] Figure 4 This is a graph showing the effect of the active strain MSTI-2023-654 on the content of various components in EPS of *Nyctaginosa microcarpa* in this embodiment of the invention.

[0020] Figure 5 This is a graph showing the effect of the active strain MSTI-2023-654 on the time-attachment status of barnacle larvae in this embodiment of the invention. Detailed Implementation

[0021] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0022] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0023] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0024] The strain MSTI-2023-654 used in this study was isolated from seawater samples from the Lianyungang sea area. It was identified as a short-lived Bacillus pumilus by 16S rDNA sequence analysis combined with morphological characteristics.

[0025] The strain MSTI-2023-654 of this invention was deposited on March 6, 2023, at the China General Microbiological Culture Collection Center (CGMCC), classified as *Bacilus pumilus*, with accession number CGMCC No. 26745, and deposited at: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0026] Example 1

[0027] Identification of strain MSTI-2023-654: Genomic DNA of strain MSTI-2023-654 was extracted by SDS-CTAB method and sent to Shanghai Sangon Biotech Co., Ltd. for sequencing analysis.

[0028] The effective length of the strain's 6S rDNA sequence was determined to be 1489 bp. The sequence was submitted for BLAST analysis and compared with the 16S rDNA sequences of other strains in GenBank for homology. A phylogenetic tree was constructed using MEGA 11.0 software (see [link to BLAST]). Figure 1 The strains with the highest evolutionary homology to MSTI-2023-654 are *Bacillus pumilus* SB3002 and *Bacillus pumilus* IHBB9209, with a homology greater than 99.99%. Based on the combined morphological characteristics and gene sequence analysis of the strains, they can be identified as *Bacillus pumilus*.

[0029] Example 2

[0030] (1) Sample collection: Seawater samples separated from the Lianyungang sea area were stored frozen at -80℃.

[0031] Isolation and culture of strain MSTI-2023-654: Seawater samples were serially diluted and spread onto peptone-yeast extract solid medium (i.e., PY medium, formula: 1L of sterile artificial seawater with a salinity of 3% with 3g yeast extract and 5g peptone (solid medium with 20g agar powder added), sterilized by autoclaving at 121℃ for 20min and cooled to room temperature) and cultured at 28℃. From the second day onwards, the colony growth was observed daily, and newly grown actinomycetes were picked and inoculated onto fresh PY solid medium for purification.

[0032] The isolated bacteria were inoculated into PY liquid medium and cultured at 28°C with shaking at 120 rpm for 5 days.

[0033] Centrifugation was used to remove bacterial cells, and the fermentation broth was extracted twice with an equal volume of ethyl acetate. The organic phase was concentrated using a rotary evaporator to obtain crude extracts of each strain, which were then weighed and prepared for analysis.

[0034] Activity screening of strain MSTI-2023-654: The test samples (crude extracts of each of the above strains) were added to *Nitzschia closterium* broth at a concentration of 100 μg / mL. After incubation at 23℃ for 30 days, the adhesion of *Nitzschia closterium* was observed and calculated. An increased adhesion rate indicated that the test strain promoted the adhesion of benthic diatoms. Initial screening showed that the crude extract of one strain at 100 μg / mL significantly promoted the adhesion of *Nitzschia closterium*, and this strain was named MSTI-2023-654.

[0035] Culture and preparation of crude extract of strain MSTI-2023-654: 1 mL of strain cryopreservation solution (containing 50% (v / v) glycerol) was transferred to 10 mL of P-Y medium and cultured in a shaking incubator at 28 °C for 60 h to the logarithmic growth phase. Then, it was transferred to 800 mL of fresh PY medium and cultured at 28 °C and 120 rpm for 84 h to the stationary phase. The entire culture (including bacterial cells) was then collected.

[0036] Add an equal volume of ethyl acetate (containing 5% (v / v) acetone) to the bacterial culture medium and shake vigorously to ensure complete extraction of metabolites;

[0037] After standing and allowing the organic phase and aqueous phase to separate into layers, the organic phase is collected, transferred to a rotary evaporator, concentrated to a small volume under negative pressure at 37°C, and then transferred to a nitrogen blower to dry to obtain the crude extract.

[0038] The crude extract sample was dissolved in dimethyl sulfoxide (DMSO) to prepare a test solution of a certain concentration for use in biological testing.

[0039] (2) Experiment on inducing benthic diatom attachment:

[0040] Diatoms tested: Phaeodactylumtricornutum, Chaetocerosgracilis, Nitzschiaclosterium, Nitzschiafrustulum, Stauroneis sp., Amphorasp. M3, Staurophorasp. WH6, Amphorasp. WH8, and Amphorasp. Amp.

[0041] The diatoms in the solution were counted using a hemocytometer, and the density was adjusted to 3–6 × 10⁻⁶ using artificial seawater. 5 cells / mL;

[0042] Add 2 mL of the test algal solution to a 24-well plate, and add 2 μL of DMSO solution containing the crude extract to be tested, so that the crude extract test concentration is 100 μg / mL. Add the same volume of blank seawater and DMSO without crude extract to the control.

[0043] Meanwhile, take the bacterial culture capable of producing a crude extract of 100 μg / mL, centrifuge at 4500 rpm for 5 min, remove the supernatant, wash twice with the same volume of artificial seawater, and pipette evenly for later use.

[0044] Washed bacterial cells and the original bacterial solution were added separately to 2 mL of the test algal solution (the bacterial cells were plated before the test to confirm their viability). Temperature: 23℃; Light intensity: 100 μmol photons·m⁻¹ -2 ·s -1 Light-dark ratio 12h:12h, cultured for 3 days.

[0045] Place the 24-well plate on a shaker at 70 rpm for 10 minutes. Use a pipette to remove the unattached algae from the top layer. Rinse the test wells three times with artificial seawater to remove any unattached diatoms. Observe and count the algae cells still attached to the bottom of each well using a hemocytometer to calculate the diatom attachment rate. Each test should be repeated at least four times.

[0046] The results are expressed as diatom adhesion rate:

[0047]

[0048] The isolated active strains can effectively promote the attachment of different benthic diatoms, see Figure 2 Among them, Figures a to i are respectively: Triangular Brown Finger Algae, Fine Chaetoceros Algae, Small Crested Rhomboid Algae, Fragmented Rhomboid Algae, Radial Algae, Dipylidium M3, Heterodoxae WH6, Dipylidium WH8, and Dipylidium Amp.

[0049] It can be seen that the adhesion rates of *Phaeodactylum triangularis*, *Chaetoceros slenderus*, *Nyctaginus simonii*, and *Diamondii M3* were significantly improved under treatment with MSTI-2023-654 bacterial solution and its crude extract. The adhesion rates of *Nyctaginus fragments* and *Diamondii Amp* were also significantly improved under treatment with the active bacterial solution, while the effect was slightly less pronounced under treatment with the crude extract, but still significantly improved compared to the control.

[0050] The adhesion rates of *Hymenochrysis radiata*, *Heterodactylus spp.* WH6, and *Dendrocalamus spp.* WH8 were originally around 80-90%, so this strain did not have a significant impact on their adhesion. However, the results of experiments on other diatoms showed that the crude extract of this strain had a significant effect on improving the diatom adhesion rate, and after washing away the extracellular material, the cells of this strain themselves also had a certain promoting effect on diatom adhesion.

[0051] Example 3

[0052] Experiment on the effect of crude extract on different EPS components of diatoms:

[0053] The experiment used f / 2 medium containing Na2SiO3 (composition: 1L of 3% salinity artificial sterile seawater with 75mg NaNO3, 5mg NaH2PO4·H2O, 30mg Na2SiO3·9H2O, 1.3mg FeCl3·6H2O, 8.7mg Na2EDTA·2H2O, 9.8μg CuSO4·5H2O, 6.3μg Na2MoO4·H2O, 22μg ZnSO4·7H2O, 10μg CoCl2·6H2O, 0.18mg MnCl2·4H2O, and 1μg Vitamin B1). 12 1 μg Biotin, 2 mg Thiamine HCl) were autoclaved at 121°C for 20 min and then cooled to room temperature before use.

[0054] Inoculate at a ratio of 8% (v / v), add DMSO solution containing the crude extract to be tested to make the crude extract test concentration 100 μg / mL, add the same volume of DMSO without crude extract to the control, and set up at least three parallel groups for each treatment;

[0055] Temperature 23℃, light intensity 100 μmol photons·m -2 ·s -1 Light-dark ratio 12h:12h, cultured for 30 days.

[0056] Place the conical flask on a shaker and shake at 70 rpm for 10 minutes. Pour off the upper layer of phytoplankton (top), add an equal volume of artificial seawater, and use a brush to remove all the algae cells attached to the bottom of the conical flask (bottom). Centrifuge at 4000 rpm for 10 minutes and collect the supernatant, which are the SL-EPS solutions of phytoplankton (top) and attached algae cells (bottom), respectively.

[0057] Add an equal volume of phosphate buffer solution (10 mL, 0.1 mmol / mL, pH = 7.5) to the centrifuged algal cell pellet, mix thoroughly, and let stand at room temperature for 20 min.

[0058] Incubate in a water bath at 70℃ for 1 hour. After the system cools to room temperature, centrifuge at 4000 rpm for 10 minutes and collect the supernatant to obtain B-EPS solutions of phytoplankton (bottom) and attached algae (bottom).

[0059] The polysaccharide content in EPS was determined by the phenol-sulfuric acid method, the protein content in EPS solution was quantitatively analyzed by Coomassie brilliant blue staining, the glycosamine content in EPS was determined by the Elson-Morgan reaction, the uronic acid content in EPS was measured by the sulfuric acid-carbazole method, and the sulfate group content in EPS was measured by the barium sulfate turbidimetric method.

[0060] Treatment with crude extract of strain MSTI-2023-654 significantly increased the production of B-EPS in attached diatom cells (bottom). Figure 3 The content of different EPS components in *Nyctaginosa* changed after treatment with crude extract of strain MSTI-2023-654. Figure 4 In the SL-EPS of phytoplankton cells (top), the contents of polysaccharides and proteins were significantly increased, while other components did not change significantly. In the B-EPS of phytoplankton cells (top) and the SL-EPS of benthic algae cells, the protein content was significantly increased. In the B-EPS of benthic algae cells (bottom), which plays a crucial role in the attachment and biofilm formation of benthic diatoms, the polysaccharide content was significantly increased, the uronic acid content was increased, while the protein content, glycosamine content and sulfate content decreased.

[0061] Example 4

[0062] Test on the attachment activity of barnacle larvae: The barnacle used in this study was Amphibalanus amphitrite. Adult barnacles were collected from the intertidal zone of Lianyungang (34°88'N, 119°19'E). After obtaining nauplius larvae through artificial stimulation, the nauplius larvae were raised in fresh artificial seawater in the laboratory of the Institute of Marine Science and Technology, Yangzhou University, and fed with Chaetoceros filamentosa. After being cultured at room temperature for about one week, Venus larvae were obtained.

[0063] The Venus larvae used in this experiment were stored in a 4°C refrigerator for 48 hours before being used for biological testing.

[0064] During the test, 1 mL of artificial seawater was added to each well of a 24-well plate, followed by 15–20 golden snub-nosed larvae. Using the same volume of blank DMSO as a control, DMSO solution containing the crude extract to be tested was added to achieve a crude extract concentration of 100 μg / mL. Six replicates were performed for each group.

[0065] The 24-well plate used for testing was covered with aluminum foil and placed in a dark, room-temperature environment. The attachment of *Amanita muscaria* larvae was counted every 12 hours, and the attachment rate was calculated. The formula for calculating the attachment rate is as follows:

[0066]

[0067] Treatment with crude extract of strain MSTI-2023-654 for 12 to 96 hours resulted in higher attachment rates of barnacle larvae compared to the control group. Figure 5 When the attachment status was observed for 48 hours, the attachment rate of Venus larvae induced by the crude extract of the strain was significantly increased, showing a significant difference from the control group (p<0.01).

[0068] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the present invention.

Claims

1. Bacillus pumilus ( Bacillus pumilus MSTI-2023-654 was deposited on March 6, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 26745. The deposit address is: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

2. The *Bacillus pumilus* as described in claim 1 (… Bacillus pumilus MSTI-2023-654 Application in Aquaculture to Promote the Formation and Development of Benthic Attached Organism Communities, characterized by: The benthic attachment community consists of benthic diatoms and barnacle larvae. The application is to improve the attachment rate of benthic diatoms and to induce the attachment and metamorphosis of barnacle larvae. The benthic diatoms mentioned are *Phaeodactylum triangularis*, *Chaetoceros slenderis*, *Rhomboidella fragilis*, or *Rhomboidella tinctoria*.

3. The application as described in claim 2, characterized in that: The application aims to improve the attachment rate of benthic diatoms and induce diatom cells to produce more extracellular polymers of acidic polysaccharides, thereby increasing EPS adhesion and promoting the formation and development of benthic biofilms. The benthic diatom is *Rhomboidea microcarpa*.

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