Algicidal bacterium and application thereof

Abstract

The invention discloses algicidal bacteria and application thereof, and relates to the technical field of microorganism application. The algicidal bacterium is classified and named as rhodotorula mucilaginosa, the strain name of the algicidal bacterium is Rhodotorula mucilaginosa TLJ-1, and the preservation number of the algicidal bacterium is GDMCC No: 64271. The algicidal bacterium is applied to treatment of water blooms of cylindropsis, when the algicidal bacterium is applied, an algicidal bacterium solution is added into a water body, the volume ratio of the algicidal bacterium solution to the cylindropsis is (0.75-3): 2, the optimum pH is 7-8, and the temperature is 28 DEG C. According to the present invention, the rhodotorula mucilaginosa TLJ-1 is successfully separated from the plateau lake, i.e., the iso-long lake, and the rhodotorula mucilaginosa TLJ-1 has good algae-lysing activity, particularly has good inhibition and killing effects on cylindrospora, and has good treatment effects on algal bloom caused by water eutrophication.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial application, and in particular to an algae-lytic bacteria and application thereof. Background Art

[0002] Algae bloom refers to the phenomenon that under certain environmental conditions, the number of certain algae increases explosively, causing a significant change in the color of the water body, and eventually forming a floating phenomenon of the dominant algae color on the surface of the water body. Before the bloom that can be observed on the surface occurs in the water body, a large number of algae colonies are already suspended in the water body. Once the corresponding environmental conditions are met, the algae colonies will collectively float up and gather on the surface of the water body to form the bloom that people observe.

[0003] With the rapid development of industrialization, the pollution of plateau lakes has become more and more obvious, mainly reflected in the algae bloom pollution caused by eutrophication of water bodies. The amount of pollutants entering the lake exceeds its self-purification capacity, and economic water use squeezes out environmental and ecological water use, resulting in serious organic pollution in the lake and the emergence of algae bloom pollution.

[0004] Biological methods for algae removal have multiple advantages, such as economy, rationality, environmental protection and high efficiency. They have broad application prospects and are receiving more and more attention and research. As an important component of aquatic ecosystems, algae-dissolving bacteria have an inhibitory effect on a variety of algae, and can regulate the number and type of algae in the water body to keep it at a reasonable density, which is of great significance for maintaining the balance of algae in the water body. In actual investigations, it was found that Yilong Lake, one of the nine plateau lakes in Yunnan, was the most severely eutrophic, and Pseudo-cylindrical algae were significantly enriched. If algae-dissolving bacteria that can dissolve Pseudo-cylindrical algae can be screened out, it will be of great significance for the ecological restoration of plateau lake water quality. Summary of the invention

[0005] The purpose of the present invention is to provide an algae-lytic bacteria and application thereof, so as to solve the problem of algae bloom caused by the enrichment of Pseudocylindrospermum in lakes.

[0006] In order to solve the above-mentioned technical problems, the present invention adopts the following technical scheme: an algae-lytic bacteria, characterized in that: its classification name is Rhodotorula mucilaginosa, the strain name is Rhodotorula mucilaginosa TLJ-1, and the strain was deposited in the Guangdong Provincial Microbiological Culture Collection Center on January 11, 2024, with an address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the deposit number is GDMCC No: 64271.

[0007] A further technical solution is the application of the algicidal bacteria in the treatment of Pseudocylospora blooms.

[0008] A further technical solution is that the application is to add algae-lytic bacteria liquid to the water body, and the volume ratio of the algae-lytic bacteria liquid to the pseudocylindrospermum is 0.75 to 3:2.

[0009] A further technical solution is that the culture method of the algae-lysing bacteria liquid is as follows:

[0010] 1) Solid culture: inoculate Rhodotorula mucilaginosa TLJ-1 on a basic solid culture medium for culture;

[0011] 2) Primary culture: Use an inoculation loop to pick up a single colony of Rhodotorula mucilaginosa TLJ-1 on a solid slant medium and culture it in a basic liquid medium to obtain a primary culture solution;

[0012] 3) Expanding the culture: inoculating the primary culture solution into the basic liquid culture medium for further expanding the culture to obtain the algae-lytic bacteria solution.

[0013] A further technical solution is that the solid culture conditions in step 1) are: culture at 28°C for 48 hours and store at 4°C; the basic solid culture medium is: sucrose 5g / L, peptone 10g / L, calcium chloride 0.5g / L, agar 20g / L, the above raw materials are mixed and sterilized at 121°C high temperature steam for 20 minutes.

[0014] A further technical solution is that the primary culture conditions in step 2) are: culture at 28°C and 180 rpm on a shaker for 24 hours.

[0015] A further technical solution is that the culture conditions of step 3) are: inoculating the primary culture solution into the basic liquid culture medium at a 10% inoculation rate, and culturing for 72 hours at 28°C and 180 rpm on a shaker; the basic liquid culture medium is: 5 g / L sucrose, 10 g / L trypsin, and 0.5 g / L calcium chloride, and the above raw materials are dissolved in sterile natural lake water and sterilized at 121°C high-temperature steam for 20 minutes.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] 1) The present invention successfully isolated Rhodotorulamucilaginosa TLJ-1 from Yilong Lake, a plateau lake. This strain has good algae-lytic activity, especially good inhibition and killing of Pseudocylindrical spores, and has a good effect on controlling algae blooms caused by eutrophication of water bodies.

[0018] 2) The temperature and pH culture conditions of Rhodotorula mucilaginosa TLJ-1 in the present invention are well matched with the environmental conditions of plateau lakes, and have good compatibility with the environment. It is taken from the lake and then applied to the lake without causing residual pollution, making the algae-lytic bacteria easy to be used in practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the ITS electrophoresis pattern and phylogenetic tree of the alginolytic red yeast Rhodotorula mucilaginosa TLJ-1.

[0020] Figure 2 This is a diagram showing the algae removal effect of different added amounts of Rhodotorula mucilaginosa TLJ-1 bacterial liquid.

[0021] Figure 3 This is a photo of the algae-lysing effect of Rhodotorula mucilaginosa TLJ-1 after 24 hours of culture.

[0022] Figure 4 This is a photo of the algae-lysing effect of Rhodotorula mucilaginosa TLJ-1 on solid culture medium after 24 hours of culture.

[0023] Figure 5 The diagram shows the algae-lyzing effect of Rhodotorula mucilaginosa TLJ-1 at different pH values.

[0024] Figure 6 The diagram shows the algae-lyzing effect of Rhodotorula mucilaginosa TLJ-1 at different temperatures. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0026] Example

[0027] 1. Screening of the alginolytic red yeast Rhodotorula mucilaginosa TLJ-1 strain

[0028] 1-1. Experimental materials:

[0029] The algae were purchased from Wuhan Institute of Hydrobiology, Chinese Academy of Sciences. Zarrouk medium was used to activate and culture the algae. The culture conditions were 28°C, 4000 Lux, and a light-dark ratio of 12:12. The cells were cultured to a cell density of 1×10 7 ~3×10 7 cfu / ml.

[0030] The lake bottom mud was obtained from Yilóng Lake in Shiping County, Honghe Hani and Yi Autonomous Prefecture, Yunnan Province.

[0031] 1-2. Screening of algicidal bacteria:

[0032] Take the bottom mud of Yilong Lake in Shiping County, Honghe Hani and Yi Autonomous Prefecture, Yunnan Province, remove the supernatant, take 1mL of activated sludge, inoculate it into LB medium, and culture it in a constant temperature incubator at 28℃ and 180rpm. Take another 1mL and culture it in a new LB medium. Add the cultured bacterial solution in the logarithmic growth period to the cultured pseudo-cylindrical spores, and culture it in a light incubator at 28℃, 4000Lux of light intensity, and 12:12 light-dark ratio. Shake once in the morning and evening every day to observe the changes in the color of the algae. If the color of the algae becomes lighter, it indicates that algae-lysing bacteria may exist in the system.

[0033] 1-3. Isolation of algicidal bacteria:

[0034] Take 1 mL of the supernatant in the algicidal system, apply 1 ml of the diluted bacterial solution evenly to the LB medium using the gradient dilution method, and culture at 30°C until a single colony grows. Select a single colony and continue to streak it in the LB medium until a pure culture is obtained, inoculate it into a slant medium and store it in a 4°C refrigerator.

[0035] 1-4. Rescreening of algicidal bacteria:

[0036] The bacterial liquids cultured to the logarithmic growth phase were added to the cultured Pseudocylindrospermum, and cultured at 28°C, 4000 Lux of light, and a light-dark ratio of 12:12. The growth of the algae was observed, and those whose color turned yellow or light were selected as algicidal bacteria.

[0037] 2. Identification of algicidal bacteria

[0038] The genomic DNA was released using a PCR rapid identification kit and used as a template. 16s and ITS universal primers were used for PCR amplification. The PCR products were detected by agarose gel electrophoresis and recovered for sequencing. The sequencing results were analyzed and compared with BLAST of the NCBI database to analyze sequence homology. The phylogenetic tree was constructed using the neighbor-joining method.

[0039] The sequencing results were compared by Blastn on NCBI, and it was found that the ITS rDNA similarity between strain Rhodotorula mucilaginosa TLJ-1 and Rhodotorula mucilaginosa was the highest, reaching 99.83%. Figure 1 As shown, the phylogenetic tree further showed that the strain TLJ-1 was located on the Rhodotorula mucilaginosa branch, and the screened TLJ-1 was identified as Rhodotorula mucilaginosa.

[0040] 3. Cultivation of algicidal bacteria strains

[0041] 1) Solid culture: The strain of Rhodotorula mucilaginosa TLJ-1 was inoculated on a basic solid culture medium, cultured at 28° C. for 24 hours, and stored at 4° C. The basic solid culture medium comprises 5 g / L sucrose, 10 g / L tryptone, 0.5 g / L calcium chloride, and 20 g / L agar. The above raw materials were mixed and sterilized at 121° C. for 20 min.

[0042] 2) Primary culture: a single colony of Rhodotorulamucilaginosa TLJ-1 on a solid slant medium was picked up with an inoculation loop and placed on a basal liquid medium, and cultured at 28°C with a shaker at 180 rpm for 24 h to obtain a primary culture solution; the basal liquid medium contained 5 g / L sucrose, 10 g / L tryptone, and 0.5 g / L calcium chloride, and the above raw materials were dissolved in sterile natural lake water and sterilized at 121°C for 20 min.

[0043] 3) Expansion: The primary culture solution is inoculated into the basic liquid culture medium for further expansion. The primary culture solution is inoculated into the basic liquid culture medium at an inoculation rate of 10%, and cultured at 28° C. and 180 rpm for 72 hours to obtain an active algae-lytic bacteria solution.

[0044] 4. Algae-inhibiting effect of algae-lysing bacteria:

[0045] Processing steps: Take 2 ml (1×10 7 ~3×10 7cfu / ml), 0.75ml, 1.5ml and 3.0ml bacterial solution of Rhodotorula mucilaginosa TLJ-1 (OD600 nm is 1-1.5) were added respectively, and the total volume was fixed with sterile water. After the bacterial solution reacted for 6 hours, the supernatant was removed by centrifugation and the corresponding volume of methanol was added to incubate in a constant temperature water bath at 45℃ for 24 hours. The chlorophyll content was measured at the absorbance of OD470 nm, and the algae lysis rate was calculated after comparison with the control group ( Figure 2 , Figure 3 ).

[0046] The specific formula of algae lysis rate is as follows: algae lysis rate % = (1-absorbance at OD470 nm of the experimental group / absorbance at OD470 nm of the control group) × 100%.

[0047] Lysis of algae by algae-lysing bacteria in solid culture medium: Rhodotorula mucilaginosa TLJ-1 was applied to Zarrouk solid culture medium containing glucose and Pseudocylospora. The volume ratios of Pseudocylospora and Rhodotorula mucilaginosa TLJ-1 were 1:0.5, 1:1, and 1:3, respectively. A control group was set up without adding Rhodotorula mucilaginosa TLJ-1 ( Figure 4 ).

[0048] The experimental results are as follows Figure 2 , Figure 3 , Figure 4 As shown. Among them, Figure 3 The left side in the middle indicates that only Pseudomonas was added without the addition of algicidal bacteria, while the right side test tube indicated that the algicidal bacteria was added at a volume ratio of 3:2. Figure 4 In the figure, the lower left is the control group (no algicidal bacteria were added, only Pseudocylospora was added), while the volume ratios of Pseudocylospora and the algicidal bacteria Rhodotorulamucilaginosa TLJ-1 in the lower right, upper right, and upper left are 1:0.5, 1:1, and 1:3, respectively.

[0049] As shown in the figure, under the mixed culture conditions of Rhodotorula mucilaginosa and Pseudocylospora in liquid, the removal rate of Pseudocylospora by Rhodotorula mucilaginosa TLJ-1 reached 56% at the highest after 6 hours of culture. Under solid culture conditions, the algae-lysing effect increased with the increase of the proportion of algae-lysing bacteria.

[0050] By changing the pH and temperature of the algae-lysing effect of Rhodotorula glutinosae, its algae-lysing effect in actual water was tested. 7 ~3×10 7cfu / ml), add 1ml of Rhodotorulamucilaginosa TLJ-1 (OD600 nm is 1-1.5), after 5 days of reaction, centrifuge to remove the supernatant and add the corresponding volume of methanol to incubate in a constant temperature water bath at 45℃ for 24 hours, measure the chlorophyll content at OD470 nm absorbance, and calculate the algae lysis rate after comparison with the control group ( Figure 5 , Figure 6 ). As can be seen from the figure, under the mixed culture conditions of red yeast and Pseudocylospora in water, the algae lysis effect is best under pH 7 and pH 8. After 5 days of cultivation, the removal rate of Rhodotorula mucilaginosa TLJ-1 on Pseudocylospora can reach up to 81%, and the optimal algae lysis temperature is 28℃.

[0051] The above are only some specific embodiments of the present invention. The known specific contents or common sense in the scheme are not described in detail here (including but not limited to abbreviations, abbreviations, and units commonly used in the art). It should be pointed out that the above embodiments do not limit the present invention in any way. For those skilled in the art, all technical solutions obtained by equivalent replacement or equivalent transformation fall within the protection scope of the present invention. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. An algicidal bacterium, characterized in that: Its classification name is Rhodotorula mucilaginosa, and the strain name is Rhodotorula mucilaginosa TLJ-1. The strain was deposited in Guangdong Provincial Microbiological Culture Collection Center on January 11, 2024, and the preservation number is GDMCC No: 64271.

2. Use of the algicidal bacteria as claimed in claim 1 in treating a bloom of Pseudocylospora spp.

3. The use according to claim 2, characterized in that: The application is to add algae-lysing bacteria liquid to the water body, and the volume ratio of the algae-lysing bacteria liquid to the pseudocylindrospermum is 0.75-3:

2.

4. The use according to claim 3, characterized in that: The culture method of the algae-lysing bacteria liquid is as follows: 1) Solid culture: inoculate Rhodotorulamucilaginosa TLJ-1 on a basic solid culture medium for culture; 2) Primary culture: Use an inoculation loop to pick up a single colony of Rhodotorula mucilaginosa TLJ-1 on a solid slant medium and culture it in a basic liquid medium to obtain a primary culture solution; 3) Expanding the culture: inoculating the primary culture solution into the basic liquid culture medium for further expanding the culture to obtain the algae-lytic bacteria solution.

5. The use according to claim 4, characterized in that: The solid culture conditions of step 1) are: culture at 28° C. for 48 hours and store at 4° C. The basic solid culture medium is: 5 g / L sucrose, 10 g / L tryptone, 0.5 g / L calcium chloride, and 20 g / L agar. The above raw materials are mixed and sterilized at 121° C. high-temperature steam for 20 minutes.

6. The use according to claim 4, characterized in that: The primary culture conditions of step 2) are: culture at 28° C. and 180 rpm on a shaker for 24 hours.

7. The use according to claim 4, characterized in that: The culture conditions of step 3) are as follows: inoculate the primary culture solution into the basic liquid culture medium at a 10% inoculation rate, and culture at 28° C. and 180 rpm on a shaker for 72 hours; the basic liquid culture medium is: 5 g / L sucrose, 10 g / L trypsin, and 0.5 g / L calcium chloride. The above raw materials are dissolved in sterile natural lake water and sterilized at 121° C. high-temperature steam for 20 minutes.

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