CN111471609A

CN111471609A - Pseudomonas with algae-lysing activity and application thereof

Info

Publication number: CN111471609A
Application number: CN201911373674.5A
Authority: CN
Inventors: 甘南琴; 宋立荣; 陈莉婷
Original assignee: Institute of Hydrobiology of CAS
Current assignee: Institute of Hydrobiology of CAS
Priority date: 2019-12-26
Filing date: 2019-12-26
Publication date: 2020-07-31
Anticipated expiration: 2039-12-26
Also published as: CN111471609B

Abstract

The invention discloses pseudomonas (a) with algae-lysing activityPseudomonassp.) THW7 with the preservation number of CCTCC NO: M20191065, has higher algae-lysing efficiency, is prepared into a bacterial embedding pellet and then is put into a cyanobacterial bloom water sample, and is supplemented with glucose with a certain concentration as energy supplement, thereby having good effect of controlling the cyanobacterial bloom.

Description

Pseudomonas with algae-lysing activity and application thereof

Technical Field

The invention belongs to the field of microbial algae control, and particularly relates to pseudomonas with algae-lysing activity and application thereof in blue algae bloom control.

Background

In the last two decades, the cyanobacterial bloom in the fresh water body of China has the characteristics of high biomass, large range, high frequency and high harm on the whole. The cyanobacteria forming the water bloom mainly comprise Microcystis (Microcystis), long-noded algae (Dolichospermum), Aphanizomon (Aphanizomenon), flounder (Planktothrix), nodulococcus (Nodularia), Oscillatoria (Oscillatonia) and the like, the Microcystis water bloom is the most important water bloom type in China, and the water bloom of the long-noded algae and the water bloom of the Aphanizomenon are the second most important water bloom types in China.

The wantonly outbreak of cyanobacterial bloom directly or indirectly affects human health, causes imbalance of the structure and function of the water ecosystem, and is a major water environment problem facing and urgently needing to be solved currently and even in the future in China for a long time. The research and development of key technologies for controlling and inhibiting algae have important theoretical and practical significance for promoting the development of lakes to the healthy and stable direction. Compared with the conventional physical method and chemical method algae control technology, the microorganism algae control technology based on the modern ecological environment protection concept has the advantages of economy, specificity, safety and maintenance of ecological balance of water body, and is an ecological restoration technology for controlling algae bloom by utilizing organisms such as bacteria, actinomycetes, fungi, protozoa and viruses to solve the water environment problem. The method reasonably utilizes a microbiological method to control the bloom-forming cyanobacteria, and has very important significance for improving water quality and relieving the harm of the bloom.

Throughout the research at home and abroad, the most reported method in the microbial algae control method is to control the cyanobacterial bloom by using algae-lysing bacteria. However, in view of the fact that the actual algae-lysing effect is easily affected by various biological factors and non-biological factors in the water ecosystem and the ecological safety of some microorganisms is yet to be further scientifically demonstrated, the research on algae-lysing bacteria is mainly focused on the aspects of separation identification, description of algae-lysing phenomena and algae-lysing modes and mechanisms, the research on the field application method is still in the primary stage, and there are few cases about successful application of the algae-lysing bacteria. Therefore, the method for screening the algae-lysing bacteria with high-efficiency algae-lysing efficiency and primarily trying the practical application of the algae-lysing bacteria has very important significance for treating the water bloom.

Disclosure of Invention

The invention aims to provide pseudomonas with algae-lysing activity, which has higher algae-lysing efficiency, has better effect when being prepared into bacteria embedding pellets for controlling cyanobacterial bloom, has longer service life, can be repeatedly used, and is safe and environment-friendly.

In order to achieve the purpose, the invention adopts the following technical scheme:

the inventor collects a water sample from a Taihu lake where cyanobacterial bloom occurs, and obtains an algae-lysing bacterium THW7 which is G-bacterium, is short rod-shaped or slightly bent, is about 1.0-8.0 μ M long and about 0.3-1.0 μ M wide, wherein a colony formed on a L B solid plate is in a round sphere shape with a slightly raised center, is milk-white and loose in texture, is easy to pick up by an inoculating loop, has a smooth, opaque and moist surface, and has a neat edge.

The application of Pseudomonas (Pseudomonas sp.) THW7 in controlling cyanobacterial bloom comprises the following steps:

(1) collecting pseudomonas thallus, washing and re-suspending with sterile water;

(2) adding the pseudomonas heavy suspension into a 3-4% sodium alginate solution, uniformly stirring, dropwise adding the mixed solution into a 3.5-4.5% calcium chloride solution, and performing crosslinking reaction to obtain an embedded pellet with a smooth surface and a diameter of 4-6 mm;

(3) after embedding the small balls in the pseudomonas thalli, throwing the pseudomonas thalli into the cyanobacterial bloom water body, and adding 1.0-1.2 g/L of glucose into the water body.

Compared with the prior art, the invention has the following advantages and beneficial effects:

aiming at the problems of large culture consumption in the actual algae control by using algae-lysing bacteria and additional nutrient load on a water body ecosystem in the actual application, the invention uses cheap and easily obtained sodium alginate and calcium chloride for crosslinking to embed thalli, and adds glucose with a certain concentration as energy supplement to be put into algae solution for algae lysing experiment, thereby having very obvious algae lysing effect on unicellular microcystis aeruginosa, and being capable of leading the cyanobacteria bloom to sink obviously, being a method for effectively treating the cyanobacteria bloom, and providing a new idea for treating the cyanobacteria bloom. The embedded pellet is freely collected and released, has long service life, can be repeatedly used, simplifies the algae control operation flow, reduces the cost of strain culture consumption and the like, reduces the extra nutrient load brought to the water body by the algae dissolving process, and is relatively environment-friendly.

Drawings

FIG. 1 is a flow chart of the separation and purification of algicidal bacteria.

FIG. 2 is a photograph of a colony of strain THW7 on a L B solid plate.

FIG. 3 is a transmission electron micrograph of the strain THW 7.

FIG. 4 shows a phylogenetic tree constructed by using MEGA6.0 and using the Neighbor-joining method (duplication F1000) and the 16S rDNA sequence of the strain THW7 through B L AST alignment and the related sequence in Bioedit 7.0.9.1 for sequence alignment analysis.

FIG. 5 is a growth curve of strain THW 7.

FIG. 6 is a photograph of embedded pellets of THW7 cells.

FIG. 7 is a photograph of the algal solution in the 10 th flask after adding the embedded pellet of THW7 bacteria into the unicellular Microcystis aeruginosa.

FIG. 8 is a histogram of the lysing efficiency of encapsulated beads of THW7 bacteria against unicellular Microcystis aeruginosa.

FIG. 9 is a histogram of the change of chlorophyll a concentration in algal fluid after adding embedded globules of THW7 thallus into the algal sample of the field population.

FIG. 10 is a photograph of the algal solution in a triangular flask at 7 days after adding embedded pellets of THW7 cells to the algal sample of the field population.

FIG. 11 is a photograph of 20 days old algal solution in a triangular flask after adding embedded pellets of THW7 cells to an algal sample of a field population.

Detailed Description

The following description of the embodiments of the present invention is provided in connection with the accompanying drawings, which are included to illustrate and not to limit the scope of the invention. The technical scheme of the invention is a conventional scheme in the field if not specifically stated. The reagents or materials, if not specifically mentioned, are commercially available.

Example 1: isolation, purification and culture of Pseudomonas sp (THW 7)

1. Separation, purification and preservation of pseudomonas THW7

The process flow of the separation and purification of the algae-lysing bacteria is shown in figure 1. a cyanobacterial bloom water sample is collected from Taihu lake, filtered by a 0.8 mu m filter membrane (Millipore), the supernatant is diluted by 10 times of gradient with sterile water (the dilution level is determined according to the specific situation), the diluent of 100 mu L gradient is uniformly coated on a L B solid plate, three gradients are parallel, corresponding numbers are written, the plate with uniform colony distribution is selected in a biochemical incubator at 30 ℃, single bacterial colonies with different forms are selected and separated and purified twice in a partition and streak way on a L B solid plate, and the pure cultured bacterial strains are obtained and stored in a refrigerator at-80 ℃ by a glycerol preservation method.

Selecting a monoclonal, inoculating the monoclonal on an L B solid inclined plane, flushing the monoclonal with sterile water when bacterial colonies grow out, blowing and beating the bacterial colonies uniformly to prepare bacterial lawn eluent, adding the bacterial lawn eluent into Microcystis aeruginosa (Microcystis aeruginosa PCC7806) algae liquid according to the volume ratio of 10%, adding the sterile water with the same volume into the algae liquid of a blank control group, co-culturing the Microcystis aeruginosa in a 24-hole plate, placing the Microcystis aeruginosa liquid in an illumination incubator, observing the Microcystis aeruginosa liquid after the culture temperature is 25 ℃, the light intensity is 25 mu E and the light-dark period ratio is 12 h: 12 h.5d, and if the algae liquid is yellowed, determining the bacteria to be algicidal bacteria.

According to the primary screening result, selecting a monoclonal of algicidal bacteria, inoculating the monoclonal into L B liquid culture medium, culturing for 48h at 140rpm and 30 ℃ in a temperature-controlled shaking table, adding bacterial culture into microcystis aeruginosa (Microcystoerynginsa PCC7806) algae liquid according to the volume ratio of 10%, adding sterile water with the same volume as a blank control group, co-culturing in a 100m L triangular flask, placing the microcystis aeruginosa liquid culture in a light incubator, culturing under the same conditions, measuring the content of chlorophyll a after 6d, and calculating the algicidal efficiency, wherein the algicidal efficiency has the following calculation formula:

algae-lysing efficiency (%) [ (C)₀-C)/C₀]×100％

In the formula C₀The initial chlorophyll-a concentration in the treatment group, and C the chlorophyll-a concentration measured in the treatment group.

Through the method, a strain with high algae dissolving efficiency is screened, the algae dissolving efficiency of the strain on the 6 th day is 84.61%, the number is THW7, the colony form of the strain THW7 on a L B solid plate is shown in figure 2, the colony formed by the strain THW7 on a L B solid plate is in a spherical shape with a slightly raised center, is milk white, loose in texture, easy to pick up by an inoculating loop, smooth, opaque and moist in surface and neat in edge, and a transmission electron microscope photo of the strain THW7 is shown in figure 3 and is in a short rod shape or slightly bent shape, the length is about 1.0-8.0 mu m, and the width is about 0.3-1.0 mu m.

A primer is designed, 16S rRNA genes of the primer are amplified for sequencing, B L AST comparison is carried out, a phylogenetic tree is constructed by adopting a Neighbor-joining method (repetition F1000) as shown in figure 4, the genetic relationship between a strain THW7 and Pseudomonas tescens tescenosis type strain PR65T (AM419154.2) is nearest, the similarity of the 16S rDNA genes is 99.79 percent, and the phylogenetic tree shows that Pseudomonas is an independent branch, and the molecular biology identification strain THW7 belongs to Pseudomonas (Pseudomonas).

The strain is preserved in glycerol at the temperature of minus 80 ℃ and is named as pseudomonas THW7, the Latin school name is Pseudomonas sp.THW7 (hereinafter referred to as strain THW7), the strain is preserved in China center for type culture Collection in 12-18 months in 2019, and the preservation number is CCTCCNO: m20191065.

2. Culture of Pseudomonas THW7

Selecting single clone, performing amplification culture in fresh sterile L B culture medium (Table 1), inoculating the amplified bacterial liquid into fresh L B liquid culture medium, and controlling initial OD_600nm0.088, at 30 ℃, 140rpm, sampling periodically, the OD was determined with a spectrophotometer_600nm. The growth curve is plotted as shown in FIG. 5, and the growth of the strain THW7 is divided into three stages, namely, a lag phase (0-1h), a logarithmic phase (1-24h), and a stationary phase (24-34 h). After 34h, strain THW7 entered the decline phase.

TABLE 1L B Medium composition

Example 2: application of pseudomonas THW7 in control of cyanobacterial bloom

Logarithmic phase (20h, OD)_600nm1.8) at room temperature (5000rpm, 6min), collecting the cells, washing with sterile water for 2-3 times to wash out L B medium on the cell surface, adding sterile water (as small as possible) to the obtained cells, and resuspending to obtain a cell weight suspension.

And adding the thallus heavy suspension into a sterilized sodium alginate solution with the mass concentration of 3%, stirring and uniformly mixing by using a clean glass rod, dropwise adding the mixed solution into a calcium chloride solution with the mass concentration of 4% by using an injector, and crosslinking for 30 min. The embedded pellet is washed with sterile water for 3-5 times to remove the calcium chloride solution on the surface, and the thallus embedded pellet with the diameter of about 4-6mm is prepared for later use, as shown in figure 6.

The method comprises the steps of subpackaging Microcystis aeruginosa (Microcystis aeruginosa PCC7806) algae liquid in a logarithmic growth phase into 250m L triangular flasks, each 150m L, 9 flasks in total, 3 flasks in one group, namely a blank control group, a glucose group and a thallus embedding pellet group, adding a glucose solution with the concentration of 30 g/L into the algae liquid of the glucose group and the thallus embedding pellet group to enable the final concentration to be 1 g/L, putting the thallus embedding pellet into the algae liquid of a corresponding experiment group, culturing in a light incubator under the same conditions, measuring the content of chlorophyll a of the glucose solution after 10d, calculating the algae dissolving efficiency, and taking a picture of the algae liquid in a 10 th flask as shown in figure 7.

After one round of algae-lysing experiment is finished, the thallus embedding pellets are harvested, the surfaces of the pellets are washed with sterile water for 4-5 times, and the next round of algae-lysing experiment is reserved, and the experimental steps are the same as above. Repeating the process until the thallus embedding pellet has no algae dissolving activity or poor algae dissolving efficiency. And analyzing the change of algae dissolving efficiency and the service life of the thallus embedding pellet each time. Six times of algae-lysing experiments are carried out in total, and the histogram of the algae-lysing efficiency of each time is shown in fig. 8. The first four algae-lysing effects show a slightly rising trend without significant difference, and the algae-lysing efficiency is between 45% and 65%, wherein the fourth algae-lysing efficiency is 62.85%. The fifth and sixth algae-lysing efficiencies were all reduced to different degrees compared with the fourth time, but the reduction at the sixth time was significant, and the algae-lysing efficiency was reduced to 37.32%. Therefore, after the bacteria are embedded into the pellets, the bacteria can be repeatedly harvested for algae lysis, the best effect is achieved 1-5 times, the algae lysis efficiency is obviously reduced at the 6 th time, and the service life of the embedded pellets is 68 days by taking the time from the beginning of the first algae lysis experiment to the end of the fifth experiment.

The method comprises the steps of collecting a field cyanobacteria bloom water sample, subpackaging the field cyanobacteria bloom water sample into clean and sterile 1L triangular flasks, wherein each flask is 600m L, 6 flasks in total, 3 flasks are a group, namely a control group and a treatment group, the experiment is started, THW7 thallus embedding pellets with the thallus embedding amount of 40m L are added into the treatment group, the experiment phenomenon is observed after 7d, the chlorophyll a concentration is sampled and measured, the algae dissolving effect is analyzed, meanwhile, a certain volume of glucose solution is added, the final concentration is 1 g/L, the change condition of algae in the experiment process is continuously observed, the chlorophyll a concentration is measured after 20d, the fig. 9 is a bar chart of the change of the chlorophyll a concentration of algae solution after adding the THW7 embedding pellets into a field colony algae sample, the algae solution is a photograph of the chlorophyll a concentration change of the algae solution after adding the THW7 thallus pellets into the field colony algae sample, the algae solution in the triangular flask is a photograph of the algae sample, the algae solution after adding the THW7 embedding pellets into the field colony sample, the field colony algae sample, the algae solution, the algae sample, the algae solution is added with the chlorophyll a concentration change, the chlorophyll a concentration of the algae solution is reduced, the algae suspension is reduced, the algae suspension culture process is a water sample, the algae suspension culture process is a, the algae suspension culture process is reduced, the algae suspension culture process is a water sample, the algae suspension culture process is reduced, the algae suspension culture process is reduced, the algae culture process is reduced, the process is the process.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims

1. Pseudomonas (A) with algicidal activityPseudomonassp.), characterized in that the preservation number of the pseudomonas is CCTCC NO: M20191065.

2. An embedded pellet comprising the pseudomonad of claim 1.

3. The method for preparing the pseudomonas embedded pellet as claimed in claim 2, which comprises the following steps:

(2) adding the pseudomonas heavy suspension into a 3-4% sodium alginate solution, uniformly stirring, dropwise adding the mixed solution into a 3.5-4.5% calcium chloride solution, and performing crosslinking reaction to obtain an embedded pellet with a smooth surface and a diameter of 4-6 mm.

4. The use of the pseudomonads of claim 1 for the control of cyanobacterial bloom.

5. The use as claimed in claim 4, wherein the Pseudomonas is prepared into bacteria embedding pellet, and then is thrown into cyanobacterial bloom water body, and 1.0-1.2 g/L glucose is added into the water body.