Marine repair bacterium composition, microbial filler and application of marine repair bacterium composition and microbial filler

Abstract

The invention provides a marine remediation bacterium composition, a microbial filler and application of the marine remediation bacterium composition and the microbial filler, and belongs to the technical field of microorganisms. The invention provides a marine repair bacterium composition. The marine repair bacterium composition is prepared from an Alcanivora dieselolei strain B-5 and a Halomonas tianensis strain HTPA169, wherein the viable count ratio of the Alcanivora dieselolei strain B-5 to the Taitanke Halomonas tianensis strain HTPA169 is (2-6): 1. The invention also provides a microbial filler which comprises a filler and the marine remediation bacterium composition loaded on the filler. The microbial filler provided by the invention can efficiently enrich marine microorganisms, promote microbial flora proliferation, improve biomass and biodiversity, and repair the seabed environment.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology, and particularly relates to a marine remediation bacteria composition, a microbial filler and their applications. Background Art

[0002] Marine environment remediation has become an important part of marine resource development. Traditional marine remediation mainly relies on physical and chemical means. However, traditional technologies involve large engineering quantities, high construction costs, and may cause secondary pollution, having a greater impact on the environment. In recent years, bioremediation has become the main alternative means. Bioremediation has the advantages of low cost and high remediation rate. Usually, the cost of bioremediation is only one-fourth or lower than that of traditional remediation, and bioremediation is environmentally friendly and does not cause secondary pollution, which is a generally recognized environmental remediation method. However, at present, there are few materials available for bioremediation, and the remediation effect is not good. Summary of the Invention

[0003] In view of this, the present invention provides a marine remediation bacteria composition, which can efficiently enrich marine microorganisms, promote the proliferation of microbial flora, increase the biomass of microorganisms in the ocean, and repair the seabed environment.

[0004] In order to achieve the above object, the present invention provides the following technical solutions:

[0005] The present invention provides a marine remediation bacteria composition, comprising Alcanivorax dieselolei strain B-5 and Halomonas titanicae strain HT PA16_9;

[0006] The viable count ratio of Alcanivorax dieselolei strain B-5 to Halomonas titanicae strain HT PA16_9 in the marine remediation bacteria composition is (2-6):1.

[0007] Preferably, the viable count ratio of Alcanivorax dieselolei strain B-5 to Halomonas titanicae strain HT PA16_9 is 4:1.

[0008] The present invention provides a microbial filler, comprising a filler and the marine remediation bacteria composition loaded on the filler.

[0009] Preferably, the filler comprises at least one of the following materials: polyethylene, polypropylene, polyurethane, ceramic and activated carbon.

[0010] The present invention provides a preparation method of the microbial filler, which is to mix the filler with the bacterial liquid of the marine remediation bacteria composition.

[0011] Preferably, the mixing time is 1-72 h; the mixing temperature is 14-28 °C;

[0012] The initial concentration of Alcanivorax dieselolei strain B-5 in the bacterial solution is 2×10 6 CFU / mL to 6×10 10 CFU / mL.

[0013] The present invention provides the application of the microbial filler prepared by the repair bacterium composition, the microbial filler or the preparation method in enriching marine microorganisms.

[0014] The present invention provides the application of the microbial filler prepared by the repair bacterium composition, the microbial filler or the preparation method in marine remediation.

[0015] Preferably, the marine remediation includes increasing the biomass of microorganisms.

[0016] The present invention provides a method for marine remediation, which places the microbial filler in seawater.

[0017] The present invention has the following advantages compared with the prior art:

[0018] The present invention provides a marine remediation bacterium composition, which includes Alcanivorax dieselolei strain B-5 and Halomonas titanicae strain HT PA16_9 with an effective bacteria number ratio of (2-6):1. By combining Alcanivorax dieselolei strain B-5 and Halomonas titanicae strain HT PA16_9 in a specific ratio, the obtained marine remediation bacterium composition can efficiently enrich marine microorganisms, promote the proliferation of microbial flora, increase the biomass, and repair the seabed environment.

[0019] The present invention also provides a microbial filler, which includes a filler and the marine remediation bacterium composition loaded on the filler. The microbial filler provided by the present invention can efficiently enrich marine microorganisms, promote the proliferation of microbial flora, increase the biomass and biodiversity, and repair the seabed environment. At the same time, the microbial filler has the characteristics of low repair cost, high repair rate, environmental friendliness, and no secondary pollution.

[0020] The present invention provides an application of the microbial filler prepared by the repair bacterium composition, the microbial filler or the preparation method in enriching marine microorganisms and / or marine restoration. In the embodiments of the present invention, taking biomass as an index, the effects of the microbial filler, the random microbial filler and the blank filler on the seabed microorganisms and the marine environment were compared. The results showed that the protein content in the microbial filler was significantly higher than that in the random microbial filler and the blank filler, and a variety of microorganisms were enriched in the microbial filler. Except for Halomonas titanicae strain HT PA16_9 and Alcanivorax dieselolei strain B-5, other microorganisms accounted for 59% of the total biomass. It can be seen that the method of the present invention can efficiently enrich marine microorganisms, promote the proliferation of microbial flora in the ocean, increase the biomass, and achieve the purpose of repairing the marine environment. Description of the Drawings

[0021] Figure 1 It is a graph of the biomass measurement results;

[0022] Figure 2 It is a graph of the proportion of different microorganisms in the total amount of microorganisms. Detailed Embodiments

[0023] The present invention provides a marine repair bacterium composition, including Alcanivorax dieselolei strain B-5 and Halomonas titanicae strain HT PA16_9;

[0024] The viable cell number ratio of Alcanivorax dieselolei strain B-5 to Halomonas titanicae strain HT PA16_9 in the marine repair bacterium composition is (2-6):1.

[0025] In the present invention, the Alcanivorax dieselolei strain B-5 is disclosed in the prior art: Anzhang Li; Zongze Shao. Degradation of halogenated compounds by Alcanivorax dieselolei B-5 and its haloalkane dehalogenase DadA [J]. Acta Microbiologica Sinica, 2014, 54(09): 1063-1072. DOI: 10.13343 / j.cnki.wsxb.2014.09.011. The preferred culture method of the Alcanivorax dieselolei strain B-5 is to pick colonies and inoculate them in 2216E liquid medium for culture. The preferred temperature for the culture is 22-24°C, more preferably 23°C. The preferred rotation speed during the culture is 120-180 rpm, more preferably 140-160 rpm, and most preferably 150 rpm. The culture method of the present invention is beneficial to improving the activity of the Alcanivorax dieselolei strain B-5, thereby effectively enriching marine microorganisms and repairing the marine environment.

[0026] In the present invention, the Halomonas titanicae strain HT PA16_9 is disclosed in the Chinese patent with the publication number CN110669700B and the title "A highly efficient petroleum hydrocarbon-degrading bacterium PA16_9 and its screening method and application". The cultivation method of the Halomonas titanicae strain HT PA16_9 is preferably the same as that of the above-mentioned Alcanivorax dieselolei strain B-5, which will not be elaborated herein. The cultivation method of the present invention is beneficial to improving the activity of the Halomonas titanicae strain HT PA16_9, thereby effectively enriching marine microorganisms and repairing the marine environment.

[0027] In the present invention, the viable cell number ratio of the Alcanivorax dieselolei strain B-5 to the Halomonas titanicae strain HT PA16_9 is preferably 4:1. This viable cell number ratio is beneficial to the stable and long-term colonization of the marine remediation bacterium composition, and improves the effect of enriching marine microorganisms and repairing the marine environment. Examples of the present invention show that by combining the Alcanivorax dieselolei strain B-5 and the Halomonas titanicae strain HT PA16_9 in a specific ratio, the obtained composition can efficiently enrich marine microorganisms, promote the proliferation of the microbial flora, increase the biomass, and repair the seabed environment.

[0028] The present invention provides a microbial filler, which includes a filler and the marine remediation bacterium composition loaded on the filler.

[0029] In the present invention, the filler preferably includes at least one of the following: polyethylene, polypropylene, polyurethane, ceramics, and activated carbon, and more preferably includes MBBR filler. The filler can not only provide a suitable growth environment for microorganisms, improve the efficiency of enriching marine microorganisms and repair the marine environment, but also facilitate the statistical analysis of the repair effect of the marine remediation bacterium composition. In the examples of the present invention, K6 biological filler is used as the filler, and the K6 biological filler is purchased from Henan Jiekang Environmental Protection Technology Co., Ltd., and the commodity name is fluidized bed MBBR filler K6.

[0030] The present invention provides a preparation method of the microbial filler, which is to mix the filler with the bacterial liquid of the marine remediation bacterium composition.

[0031] In the present invention, the initial concentration of the Alcanivorax dieselolei strain B-5 in the bacterial liquid is preferably 2×10 6 CFU / mL to 6×10 10 CFU / mL, more preferably 2×10 7 CFU / mL to 6×10 9 CFU / mL, further preferably 2×10 8 CFU / mL to 6×10 8 CFU / mL, and most preferably 4×10 8CFU / mL. The initial concentration of Halomonas titanicae strain HT PA16_9 in the bacterial liquid is preferably 0.5×10 6 CFU / mL - 1.5×10 10 CFU / mL, more preferably 0.5×10 7 CFU / mL - 1.5×10 9 CFU / mL, further preferably 0.8×10 8 CFU / mL - 1.2×10 8 CFU / mL, most preferably 1×10 8 CFU / mL. The mixing time is preferably 1 - 72 h, more preferably 2 - 48 h, further preferably 3 - 12 h, most preferably 4 h; the mixing temperature is preferably 14 - 28 °C, more preferably 18 - 26 °C, preferably 23 °C. During the mixing, rotation is involved, and the rotation speed is preferably 120 - 180 rpm, more preferably 140 - 160 rpm, most preferably 150 rpm. The volume ratio of the mixed liquid materials is preferably (1 - 2):(3 - 6), more preferably 2:3. After mixing, the strains in the marine restoration bacterial composition adhere to the filler and colonize on the filler to obtain the microbial filler. The filler provides a large number of attachment sites and a stable growth environment for the strains in the marine restoration bacterial composition and other microorganisms in the ocean, increasing the number and activity of microorganisms per unit volume. After the strains in the marine restoration bacterial composition adhere to the filler, a stable microenvironment can be formed with the filler, which is beneficial to giving full play to the activity of enriching marine microorganisms and restoring the ocean. The microbial filler prepared by the preparation method can effectively improve the effect of enriching marine microorganisms, which is beneficial to increasing the biomass of microorganisms and restoring the seabed environment.

[0032] The present invention provides an application of the restoration bacterial composition, the microbial filler or the microbial filler prepared by the preparation method in enriching marine microorganisms.

[0033] The present invention provides an application of the restoration bacterial composition, the microbial filler or the microbial filler prepared by the preparation method in marine restoration.

[0034] In the present invention, the marine restoration preferably includes increasing the biomass of microorganisms. The marine restoration preferably further includes increasing the diversity of microorganisms. The ocean preferably includes the ocean where the diversity of microorganisms is reduced and the biomass of microorganisms is reduced due to various engineering behaviors.

[0035] The present invention provides a method for marine restoration, which comprises placing the microbial filler in seawater.

[0036] In the present invention, there are no restrictions on the method of placing the microbial filler in seawater, including the depth of placement in seawater and the placement density of the microbial filler in seawater. Conventional methods in the art or decisions based on actual needs can be adopted. As an alternative embodiment, the method of placing the microbial filler in seawater preferably mounts the microbial filler on a PVC net, with the surface of the microbial filler facing upwards and immersed in the seawater body. After immersion, the distance of the microbial filler from the water surface is preferably 2.5 - 3.5 m, more preferably 2.8 - 3.2 m, and most preferably 3 m. The length, width, and height of the PVC net are preferably 30 cm × 30 cm × 0.6 cm. The seawater preferably includes seawater with nutrient resource persecution and / or seawater polluted by resource exploitation. The time for placing the microbial filler in seawater is preferably greater than 7 days, more preferably 7 - 50 days, further preferably 10 - 40 days, and most preferably 28 days. Using the method of the present invention can effectively play the role of the microbial filler, improve the efficiency of enriching marine microorganisms, increase marine microbial diversity, and repair the marine environment.

[0037] The present invention provides a method for enriching marine microorganisms, which is preferably the same as the above-mentioned method for marine restoration and will not be elaborated here.

[0038] Examples of the present invention compared the effects of microbial fillers, random microbial fillers, and blank fillers on seabed microorganisms and the marine environment. The results showed that the protein content in the microbial filler was significantly higher than that in the random microbial filler and the blank filler, and a variety of microorganisms were enriched in the microbial filler. Except for Halomonas titanicae strain HT PA16_9 and Alcanivorax dieselolei strain B-5, other microorganisms accounted for 59% of the total biomass. It can be seen that the method of the present invention can efficiently enrich marine microorganisms, promote the proliferation of microbial flora, and increase the biomass. At the same time, the prior art Sitthi, A., Pimple, U., Piponiot, C. et al. Assessing the effectiveness of mangrove rehabilitation using above-ground biomass and structural diversity. Sci Rep 15, 7839 (2025). https: / / doi.org / 10.1038 / s41598-025-92514-7 shows that biomass is an important indicator for the restoration of the marine environment. Therefore, the method of the present invention can repair the seabed environment.

[0039] To further illustrate the present invention, the following describes the solution of the present invention in detail with reference to the accompanying drawings and examples, but they should not be construed as limiting the protection scope of the present invention.

[0040] Example 1

[0041] 1. Preparation method of marine remediation bacteria composition

[0042] Bacterial suspensions of Halomonas titanicae strain HT PA16_9 and Alcanivorax dieselolei strain B-5 at a concentration of 10 4 CFU / ml were inoculated into 2216E liquid medium at an inoculation ratio of 1:100 and cultured at 23 °C and 150 rpm to obtain PA16_9 bacterial suspension and B-5 bacterial suspension.

[0043] The PA16_9 bacterial suspension and the B-5 bacterial suspension were mixed, and the concentration of Alcanivorax dieselolei strain B-5 in the mixed bacterial suspension was 4×10 8 CFU / ml, and the concentration of Halomonas titanicae strain HT PA16_9 was 1×10 8 CFU / ml. The mixed bacterial suspension was designated as the marine remediation bacteria composition.

[0044] 2. Preparation method of microbial packing material

[0045] K6 high-density polyethylene packing material was mixed with the above-mentioned marine remediation bacteria composition at a volume ratio of 2:3 so that the K6 high-density polyethylene packing material was completely immersed, and the mixture was cultured in a shaker at 23 °C and 150 rpm for 4 h to obtain a packing material loaded with the marine remediation bacteria composition, designated as the microbial packing material.

[0046] Comparative Example 1

[0047] Preparation method of random microbial packing material

[0048] Escherichia coli isolated from the ocean was used as the random bacterial agent. Bacterial suspension at a concentration of 10 4 CFU / ml was inoculated into 2216E liquid medium at an inoculation ratio of 1:100 and cultured at 23 °C and 150 rpm to obtain a random bacterial suspension;

[0049] K6 high-density polyethylene packing material was mixed with the random bacterial suspension at a concentration of 5×10 8 CFU / ml at a volume ratio of 2:3 so that the K6 high-density polyethylene packing material was completely immersed, and the mixture was cultured in a shaker at 23 °C and 150 rpm for 4 h to obtain a packing material loaded with the random bacterial agent, designated as the random microbial packing material.

[0050] Example 2

[0051] Application of microbial packing material in seabed remediation

[0052] The microbial fillers prepared in Example 1, the random microbial fillers prepared in Comparative Example 1, and the blank fillers (K6 high-density polyethylene fillers without bactericide) were evenly mounted on a PVC net (30 cm × 30 cm, 0.6 cm thick) at a loading amount of 100 particles per plate. The PVC net was suspended at the dock, and the plate was immersed in the seawater about 3 m below the water surface. The microbial fillers and random microbial fillers were recovered every 7 days, and the biomass in the microbial fillers was measured until 28 days. Biomass refers to the protein content per cubic centimeter of microbial filler, and the biological composition refers to the relative content of attached organisms. The specific measurement methods are as follows: After rinsing the recovered microbial fillers, random microbial fillers, and blank fillers with sterilized seawater, the biomass and biological composition of the microbial fillers, random microbial fillers, and blank fillers were measured respectively.

[0053] The method for measuring biomass is as follows: After ultrasonic disruption (ice bath, power 30 W, time 3 s, interval 10 s, repeated 30 times) in a PBS bacterial lysis solution containing 8% lysozyme and 10% triton X-100 to recover proteins, filter with filter paper, and centrifuge the filtrate at 12,000 rpm and 4 °C for 10 min. Take 1 ml of the supernatant, add 1 ml of Bradford staining solution, incubate at room temperature for 30 min, and then measure the absorbance at 590 nm with a spectrophotometer. Calculate the protein concentration according to the standard curve obtained from the standard protein staining.

[0054] The method for measuring biological composition is as follows: Use the Fast DNA TM Spin Kit for Soil kit (MP BIO) to extract the genome. The steps are briefly described as follows. All the following related solutions and filter columns are provided by the kit: Transfer all biological samples to an EP tube, add 978 μl of Sodium Phosphate Buffer and 122 μl of MT Buffer, place it on a shaker and mix for 40 s, then centrifuge at 14,000 g for 5 min to take the supernatant. After adding 250 μl of PPS and mixing, add 1 ml of Binding Matrix solution, manually invert and mix well, and then let it stand for 3 min. After removing 500 μl of the supernatant, resuspend and transfer it into the SPIN TM Filter, centrifuge at 14,000 g for 1 min, discard the filtrate, then add 500 μl of SEWS-M solution, centrifuge at 14,000 g for 1 min, and discard the filtrate. Centrifuge the SPIN TM Filter at 14,000 g for 2 min, replace the collection tube, place it at room temperature for 5 min, and finally add 50 μl of sterile water to the SPIN TM Filter, centrifuge at 14,000 g for 1 min, and elute the DNA into the collection tube. Entrust the sample to a sequencing company (Sangon Biotech (Shanghai) Co., Ltd.) for 16s diversity sequencing to determine the microbial composition.

[0055] The biomass measurement results are shown in Figure 1 , and the proportions of different microorganisms in the microbial packing prepared in Example 1 accounting for the weight of microorganisms are shown in Figure 2 . From Figure 1 , it can be seen that the biomass of microorganisms in the microbial packing is significantly higher than that of the blank packing and the random microbial packing. From Figure 2 , it can be seen that a variety of microorganisms are enriched in the microbial packing. Except for Halomonas titanicae strain HT PA16_9 and Alcanivorax dieselolei strain B-5, other microorganisms account for 59% of the total biomass, indicating that the microbial packing can efficiently enrich marine microorganisms, promote the proliferation of microbial communities, and increase the biomass. Existing technologies have shown that biomass is an important indicator for the restoration of the marine environment (Sitthi, A., Pimple, U., Piponiot, C. et al. Assessing the effectiveness of mangrove rehabilitation using above-ground biomass and structural diversity. Sci Rep 15, 7839 (2025). https: / / doi.org / 10.1038 / s41598-025-92514-7). Therefore, the microbial packing of the present invention can effectively repair the seabed environment.

[0056] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. Other embodiments can also be obtained based on these embodiments without creative work, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A marine restoration bacterium composition, characterized in that, It includes Alcanivorax dieselolei strain B-5 and Halomonas titanicae strain HT PA16_9; In the marine remediation bacterium composition, the viable count ratio of Alcanivorax dieselolei strain B-5 to Halomonas titanicae strain HT PA16_9 is (2-6):

1.

2. The marine repair bacteria composition according to claim 1, characterized in that, The viable count ratio of Alcanivorax dieselolei strain B-5 to Halomonas titanicae strain HT PA16_9 is 4:

1.

3. A microbial filler, characterized in that, It includes a filler and the marine remediation bacterium composition according to claim 1 or 2 loaded on the filler.

4. The microbial filler according to claim 3, characterized in that, The filler includes at least one of the following materials: polyethylene, polypropylene, polyurethane, ceramic, and activated carbon.

5. A method for preparing the microbial filler according to claim 3 or 4, characterized in that, Mix the filler with the bacterial liquid of the marine remediation bacterium composition according to claim 1 or 2.

6. The preparation method according to claim 5, characterized in that, The mixing time is 1-72 h; the mixing temperature is 14-28 °C; The initial concentration of the diesel-degrading bacterium strain B-5 in the bacterial solution is 2×10 6 CFU / mL to 6×10 10 CFU / mL.

7. Application of the remediation bacterium composition according to claim 1 or 2, the microbial filler according to claim 3 or 4, or the microbial filler prepared by the preparation method according to claim 5 or 6 in enriching marine microorganisms.

8. Application of the remediation bacterium composition according to claim 1 or 2, the microbial filler according to claim 3 or 4, or the microbial filler prepared by the preparation method according to claim 5 or 6 in marine remediation.

9. The application according to claim 8, characterized in that The marine remediation includes increasing the biomass of microorganisms.

10. A method for marine restoration, characterized in that, Place the microbial filler according to claim 3 or 4 in seawater.

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