Acinetobacter with algal inhibition capability and application of acinetobacter

Abstract

The invention discloses acinetobacter with algal inhibition capability and application thereof, and relates to the technical field of microorganism and environmental organic pollutant repair and governance, the acinetobacter with algal inhibition capability is preserved in Guangdong Microbial Culture Collection Center, and the preservation number is GDMCC NO.65654. The acinetobacter with algal inhibition capability is preserved in Guangdong Microbial Culture Collection Center; the 16SrDNA nucleotide sequence of the acinetobacter is shown as SEQ ID NO: 7, and the acinetobacter is applied to treatment of cyanobacterial bloom. The method comprises the following steps: extracting an extracellular culture of cylindrotheca, culturing on a beef extract peptone solid culture medium, extracting a single colony from a culture product, preserving, inoculating into a beef extract peptone liquid culture medium, continuously culturing, and carrying out PCR (Polymerase Chain Reaction) amplification on bacteria separated from the culture product by adopting a specific primer pair. The obtained amplification product is subjected to sequencing and contrastive analysis, bacteria are identified to be the acinetobacter with the algae inhibition capacity, the growth of blue-green algae can be effectively inhibited, and the method has the advantages of being environmentally friendly, low in cost and remarkable in algae control effect.

Description

Technical Field

[0001] The invention relates to the technical field of microorganism and environmental organic pollutant restoration and treatment, and in particular to an acinetobacter with algae inhibition ability and an application thereof. Background Art

[0002] Eutrophication of water bodies is a serious water environment problem that has attracted global attention. Cyanobacteria bloom is the most typical manifestation of eutrophic water bodies. The occurrence of algae bloom not only seriously affects the structure and service functions of aquatic ecosystems, but the cyanobacterial toxins produced by cyanobacteria also pose a major threat to human health. At the same time, it will also seriously restrict regional economic and social development. For example, the Taihu Lake water crisis in 2007 caused direct economic losses of more than 2.8 billion yuan to Wuxi City. Judging from the scale of cyanobacteria blooms and the number of water bodies, my country is the country with the most serious eutrophication and cyanobacteria bloom disasters in the world. Under the dual pressure of global climate change and intensified eutrophication, cyanobacteria bloom will become one of the most important water environment problems in my country and even the world for a considerable period of time in the future.

[0003] Microcystis is the most widely distributed and most frequently occurring water bloom type, and in my country it once became synonymous with water bloom cyanobacteria; and under the background of continuous implementation of basin pollution control for 30 years, the overall pollution level has decreased, resulting in a phenomenon of diversification of cyanobacterial water bloom types. The filamentous cyanobacterial water bloom represented by Cylindrospermopsis has become the second most widely distributed harmful water bloom type after Microcystis in my country. Cylindrospermopsis in my country was first reported in reservoirs and lakes in Guangdong and Yunnan in the early 21st century, and has successively occurred in some water supply reservoirs. Blooms on a reservoir-wide scale have shown a trend of rapid spread across the country, and it is urgently needed to carry out targeted research on the prevention and control technology of filamentous cyanobacterial water blooms. Therefore, the present invention proposes a kind of acinetobacter with algae inhibition ability and its application to solve the problems existing in the prior art. Summary of the invention

[0004] In view of the above problems, the purpose of the present invention is to propose an Acinetobacter with algae inhibition ability and its application, which can fundamentally improve the water environment, better inhibit the growth of cyanobacteria, and achieve the prevention and control of cyanobacterial blooms.

[0005] In order to achieve the purpose of the present invention, the present invention is implemented by the following technical scheme: a kind of Acinetobacter with algae inhibition ability, the preservation number of the Acinetobacter with algae inhibition ability is GDMCC NO.65654, and the 16SrDNA nucleotide sequence of the Acinetobacter with algae inhibition ability is shown in SEQ ID NO:7.

[0006] A further improvement is that the Acinetobacter with algae inhibition ability is in the shape of a coccobacillus, with an average cell length of 4.079 μm and an average width of 2.273 μm.

[0007] A further improvement is that the method for culturing the Acinetobacter with algae inhibition ability comprises the following steps:

[0008] Step 1: Cultivate Pseudocylarvae in the laboratory until all algal cells die, and then extract 0.1 mL of the culture for later use;

[0009] Step 2: Spread the extracted culture on beef extract peptone solid medium plate and culture overnight;

[0010] Step 3: using the dilution plate spreading method to separate the colonies on the beef extract peptone solid culture medium plate, and randomly extracting seven single colonies, preserving them and inoculating them into beef extract peptone liquid culture medium, and culturing them on a shaker overnight;

[0011] Step 4: After the culture is completed, the culture is centrifuged and the bacteria are collected. PCR amplification is then performed using a specific primer pair for the bacterial 16S rDNA. The obtained amplified product is sequenced and compared and analyzed to identify the bacteria as Acinetobacter.

[0012] A further improvement is that in step 2, the culture on the beef extract peptone solid culture medium plate is cultured overnight at 28°C.

[0013] A further improvement is that in step three, the single bacterial colony on the beef extract peptone liquid culture medium is cultured overnight at 28° C. on a shaker at a speed of 150 r / min.

[0014] A further improvement is that: in the step 4, the specific primer pair includes primer 27F and primer 1492R, wherein the nucleotide sequence of primer 27F is shown in SEQ ID NO: 8, and the nucleotide sequence of primer 1492R is shown in SEQ ID NO: 9;

[0015] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3';

[0016] 1492R: 5'-TACGGCTACCTTGTTACGACTT-3'.

[0017] The invention discloses an application of Acinetobacter with algae inhibition ability, wherein the Acinetobacter with algae inhibition ability is applied to the treatment of blue algae blooms.

[0018] The beneficial effects of the present invention are as follows: the present invention extracts the culture of Pseudocylospora and cultures it on a beef extract peptone solid culture medium, then extracts a single colony from the culture product, preserves it, and inoculates it into a beef extract peptone liquid culture medium for further culture, then uses a specific primer pair to perform PCR amplification on the bacteria separated from the culture product, and the obtained amplification products are sequenced and compared and analyzed to identify the bacteria as Acinetobacter with algae inhibition ability, which can effectively inhibit the growth of cyanobacteria, has the advantages of being environmentally friendly, low cost, and having a significant algae control effect, and has a good application prospect in the field of prevention and treatment of cyanobacterial blooms. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] 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, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 is a schematic diagram of a microscopic photograph of the C7 strain in Example 1 of the present invention stained with crystal violet;

[0021] Figure 2 is a schematic diagram of the growth curve of the C7 strain in Example 1 of the present invention;

[0022] Figure 3 1 is a schematic diagram comparing the inhibition efficiency of adding different C7 bacterial solutions on the growth of Pseudocylospora in Example 1 of the present invention;

[0023] Figure 4 It is a schematic diagram of microscopic photographs of Pseudocylospora in the control group and the group with 10% bacterial solution addition in the first embodiment of the present invention.

[0024] Biological Deposit Description

[0025] Acinetobacter sp.C7 with algae inhibition ability; deposited in Guangdong Microbial Culture Collection Center on December 19, 2024; address: 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou; deposit number: GDMCCNO.65654 DETAILED DESCRIPTION

[0026] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions 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 are within the scope of protection of the present invention.

[0027] At present, the treatment methods of cyanobacteria blooms are mainly divided into physical methods, chemical methods and biological methods. Among them, physical methods mainly include salvage, aeration and shading, which are costly and may cause certain disturbances to aquatic ecosystems. Chemical methods mainly include the addition of flocculants and the use of algaecides, which may have toxic effects on aquatic organisms. Biological methods mainly include the release of filter-feeding fish, the planting of aquatic plants and microbial algae control, which are environmentally friendly, have little impact on aquatic ecosystems, can fundamentally improve the water environment, and inhibit the growth of cyanobacteria for a long time, so they are widely used.

[0028] Microbial algae control refers to the use of microorganisms to control excessive algae growth. This method is considered to be ecologically friendly because it reduces dependence on chemicals and thus reduces potential harm to the environment.

[0029] Embodiment 1

[0030] The present embodiment provides an acinetobacter with algae inhibition ability. The acinetobacter with algae inhibition ability of the present embodiment was deposited in Guangdong Microbial Culture Collection Center (GDMCC) on December 19, 2024, with a deposit number of GDMCC NO.65654, and a deposit address of 5F, Building 59, No. 100, Xianlie Middle Road, Guangzhou. The 16SrDNA nucleotide sequence of the acinetobacter with algae inhibition ability is shown in SEQ ID NO:7, and the culturing method thereof comprises the following steps:

[0031] Step 1: Cultivate Pseudocylarvae in the laboratory until all algal cells die, and then extract 0.1 mL of the culture for later use;

[0032] Step 2: Spread the extracted culture on a beef extract peptone solid culture medium plate and culture it overnight at 28°C. After the overnight culture, a large amount of bacteria grows on the beef extract peptone solid culture medium plate;

[0033] Step 3: The colonies on the beef extract peptone solid medium plate were separated by dilution plate spreading method, and seven single colonies (numbered C1-7) were randomly extracted and stored in beef extract peptone liquid medium, and cultured overnight on a shaker at 150 r / min at 28°C;

[0034] In this embodiment, the nucleotide sequence of C1 is shown in SEQ ID NO: 1, the nucleotide sequence of C2 is shown in SEQ ID NO: 2, the nucleotide sequence of C3 is shown in SEQ ID NO: 3, the nucleotide sequence of C4 is shown in SEQ ID NO: 4, the nucleotide sequence of C5 is shown in SEQ ID NO: 5, the nucleotide sequence of C6 is shown in SEQ ID NO: 6, and the nucleotide sequence of C7 is shown in SEQ ID NO: 7;

[0035] Step 4: After the culture is completed, the culture is centrifuged and the bacteria are collected. PCR amplification is then performed using a specific primer pair for bacterial 16S rDNA. The amplified product is sent to Guangzhou Aiki Biotechnology Co., Ltd. for sequencing. The seven sequences are compared by nucleotide sequence Blast on the website of the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov). The homology of the seven strains with the gene sequences of other Acinetobacter in the NCBI database is more than 99.7%. Therefore, the seven isolated strains are all identified as Acinetobacter sp. The C1 and C7 strains are selected for the effect experiment on the growth of Pseudomonas spp. It is found that C7 has a more obvious algae inhibition ability. The C7 strain is stained with crystal violet and its morphology is observed under an oil microscope (16×100 times), as shown in FIG. Figure 1 As shown, the bacterial body is club-shaped, existing singly or in pairs, with an average cell length of 4.079 μm and an average width of 2.273 μm;

[0036] The specific primer pair in this embodiment includes primer 27F and primer 1492R, wherein the nucleotide sequence of primer 27F is shown in SEQ ID NO:8, and the nucleotide sequence of primer 1492R is shown in SEQ ID NO:9;

[0037] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3';

[0038] 1492R: 5'-TACGGCTACCTTGTTACGACTT-3'.

[0039] See also Figure 2 In this example, a 24-well cell culture plate was used to prepare the growth curve of the C7 strain. The culture medium was beef extract peptone liquid culture medium. 2 ml of bacterial solution was added to each well of the 24-well plate. The initial OD 600nm The OD value was 0.1, 4 parallel wells were added with beef extract peptone solution as blank control, the OD value was measured every hour, the culture temperature was set to 28℃, and the shaker speed was set to 150rpm / min. The results showed that the C7 strain grew rapidly after inoculation and entered a stable growth period after 11 hours, with the maximum OD value of 1.44.

[0040] See also Figure 3 , select a single clone of the C7 strain and expand it in liquid beef extract peptone medium to control its OD 600nm The value was between 1.2 and 1.4, making it in the late logarithmic growth phase, and the volume ratio of 2.5%, 5% and 10% was added to the algae solution of Pseudocylospora, and Pseudocylospora was cultured in WC medium. The culture conditions were temperature 28±1℃, light intensity 35μmol / (m 2 ·s), the light-dark cycle was 12h:12h, and fresh beef extract peptone liquid culture medium was used as blank control. After three days, the algae cell concentration was counted under a microscope, and the algae inhibition efficiency was calculated according to the following formula:

[0041] Algae inhibition efficiency (%) = (1-algae cell concentration in treatment group / algae cell concentration in control group) × 100

[0042] Depend on Figure 3 It can be seen that 2.5% bacterial solution has no inhibitory effect on the growth of Pseudo-Cylinderella, 5% bacterial solution has a certain effect on the growth of Pseudo-Cylinderella, with an inhibition rate of 23.7%, and after 10% C7 bacterial solution and Pseudo-Cylinderella co-cultured for three days, the inhibition efficiency reached 82.3%;

[0043] Figure 4 Microscopic photos of the control group and the group with 10% bacterial solution added. Figure 4 It can be seen that the concentration of Pseudocylospora in the treatment group was significantly lower than that in the control group.

[0044] Table 1 WC medium composition

[0045]

[0046]

[0047] Embodiment 2

[0048] This embodiment provides an application of Acinetobacter with algae inhibition ability, and the Acinetobacter with algae inhibition ability is applied to the control of cyanobacterial blooms.

[0049] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. An Acinetobacter with algae-inhibiting ability, characterized in that: The deposit number of the Acinetobacter with algae inhibition ability is GDMCC NO.65654, and the 16SrDNA nucleotide sequence of the Acinetobacter with algae inhibition ability is shown in SEQ ID NO:

7.

2. The Acinetobacter with algae-inhibiting ability according to claim 1, characterized in that: The Acinetobacter with algae inhibition ability is in the shape of a coccobacillus, with an average cell length of 4.079 μm and an average cell width of 2.273 μm.

3. The Acinetobacter with algae-inhibiting ability according to claim 1, characterized in that: The method for culturing the Acinetobacter with algae inhibition ability comprises the following steps: Step 1: Cultivate Pseudocylarvae in the laboratory until all algal cells die, and then extract 0.1 mL of the culture for later use; Step 2: Spread the extracted culture on beef extract peptone solid medium plate and culture overnight; Step 3: using the dilution plate spreading method to separate the colonies on the beef extract peptone solid culture medium plate, and randomly extracting seven single colonies, preserving them and inoculating them into beef extract peptone liquid culture medium, and culturing them on a shaker overnight; Step 4: After the culture is completed, the culture is centrifuged and the bacteria are collected. PCR amplification is then performed using a specific primer pair for the bacterial 16S rDNA. The obtained amplified product is sequenced and compared and analyzed to identify the bacteria as Acinetobacter.

4. The Acinetobacter with algae-inhibiting ability according to claim 3, characterized in that: In the step 2, the culture on the beef extract peptone solid culture medium plate is cultured overnight at 28°C.

5. The Acinetobacter with algae-inhibiting ability according to claim 3, characterized in that: In the step 3, the single bacterial colony on the beef extract peptone liquid culture medium is cultured overnight at 28° C. on a shaker at a speed of 150 r / min.

6. The Acinetobacter with algae-inhibiting ability according to claim 3, characterized in that: In the step 4, the specific primer pair includes primer 27F and primer 1492R, wherein the nucleotide sequence of primer 27F is shown in SEQ ID NO: 8, and the nucleotide sequence of primer 1492R is shown in SEQ ID NO: 9; 27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; 1492R: 5'-TACGGCTACCTTGTTACGACTT-3'.

7. An application of Acinetobacter with algae inhibition ability, characterized in that: The acinetobacter with algae inhibition ability is applied to the treatment of blue algae blooms.

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