CN111471609B

CN111471609B - Pseudomonas with algae-lysing activity and application thereof

Info

Publication number: CN111471609B
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: 2022-03-15
Anticipated expiration: 2039-12-26
Also published as: CN111471609A

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 a G-bacterium, is short rod-shaped or slightly bent, has a length of about 1.0-8.0 μm and a width of about 0.3-1.0 μm through separation, purification and culture and algae-lysing efficiency screening. The colony formed on the LB solid plate is in a spherical shape with a slightly raised center, is milky white, has loose texture, is easy to be picked up by an inoculating loop, has smooth, opaque and moist surface and regular edges. Through 16S rDNA sequencing and morphological identification, the strain belongs to Pseudomonas (Pseudomonas), is named as Pseudomonas sp (THW 7), and has been deposited in China center for type culture Collection (CCTCC NO) in 12 and 18 months in 2019, with the deposit number of CCTCC NO: m20191065.

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) embedding the pseudomonas thallus into the small spheres, then throwing the small spheres into the cyanobacterial bloom water body, and adding 1.0-1.2g/L 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 the strain THW7 on an LB 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 adjacency method (Neighbor-joining, duplication F1000) and the 16S rDNA sequence of the strain THW7 aligned by BLAST, and the related sequences are analyzed by sequence alignment in Bioedit 7.0.9.1.

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 of separating and purifying algicidal bacteria is shown in FIG. 1. Collecting cyanobacterial bloom water sample from Taihu lake, filtering by 0.8 μm filter membrane (Millipore), diluting supernatant with sterile water by 10 times gradient (the dilution level is determined according to specific conditions), taking 100 μ L of each gradient of diluent, uniformly coating the diluent on LB solid plate, wherein each gradient is three parallel, writing corresponding number, and inversely culturing for 48h at 30 ℃ in biochemical incubator. Selecting a flat plate with uniformly distributed bacterial colonies, selecting single bacterial colonies with different forms, carrying out partition streaking, separating and purifying twice on an LB solid flat plate to obtain a pure culture bacterial strain, and storing the pure culture bacterial strain in a refrigerator at the temperature of-80 ℃ by using a glycerol preservation method.

And (3) selecting the monoclonal antibody, inoculating the monoclonal antibody on an LB solid inclined plane, flushing the monoclonal antibody with sterile water after bacterial colonies grow out, and uniformly blowing and beating the bacterial colonies to prepare a lawn eluent. Adding lawn eluent into Microcystis aeruginosa (PCC 7806) algae solution at a volume ratio of 10%, adding equal volume of sterile water into the algae solution of the blank control group, and co-culturing in a 24-well plate. Placing in a light incubator at 25 deg.C with light intensity of 25 μ E and light-dark period ratio of 12 h: 12 h. After 5d, observing that if the algae liquid is yellowed, the bacteria are algicidal bacteria.

According to the result of primary screening, a single clone of algicidal bacteria is selected and inoculated in LB liquid culture medium, cultured for 48h at 30 ℃ and 140rpm in a temperature-controlled shaking table, the bacterial culture is added into Microcystis aeruginosa (Microcystis aeruginosa PCC 7806) algae liquid in a volume ratio of 10%, and the mixed culture is co-cultured in a 100mL triangular flask by adding sterile water with the same volume as a blank control group. Placing in an illumination incubator, culturing under the same conditions, measuring the content of chlorophyll a after 6d, and calculating the algae-lysing efficiency. The calculation formula of the algae dissolving efficiency is as follows:

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-lysing efficiency is screened out, and the algae-lysing efficiency of the strain on day 6 is 84.61%, which is numbered THW 7. The colony morphology of the strain THW7 on LB solid plates is shown in FIG. 2. Bacterial colonies formed by the strain THW7 on an LB solid plate are spherical with a slightly raised center, are milky white, loose in texture, easy to pick up by inoculating loops, smooth in surface, opaque, moist and neat in edge. The transmission electron micrograph of the strain THW7 is shown in FIG. 3, and is in the shape of a short rod or a little bent, with a length of about 1.0-8.0 μm and a width of about 0.3-1.0 μm.

The 16S rRNA gene is amplified by designing a primer for sequencing, then BLAST comparison is carried out, a phylogenetic tree is constructed by adopting an adjacent approach (Neighbor-join, repeatability F1000) as shown in figure 4, the relative relationship between a strain THW7 and Pseudomonas stutzeri textilis type strain PR65T (AM419154.2) is nearest, and the similarity of the 16S rRNA gene is 99.79%. As can be seen from the phylogenetic tree, the genus 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 chemical 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

Single clones were picked for expansion culture in fresh sterile LB medium (Table 1). Inoculating the expanded bacterial liquid into a fresh LB liquid culture medium, and controlling the 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 1 LB Medium composition

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

Logarithmic phase (20h, OD)_600nm1.8) was centrifuged at room temperature (5000rpm, 6min), and the cells were collected and washed with sterile water 2 to 3 times to wash off the LB medium on the cell surface. The obtained thalli is added with sterile water (the volume is as small as possible) for re-suspension, and a thalli weight suspension is prepared for standby.

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 Microcystis aeruginosa (Microcystis aeruginosa PCC 7806) algae liquid in logarithmic growth phase is respectively filled in 250mL triangular bottles, each bottle is 150mL, 9 bottles and 3 bottles are taken as a group, and the group is respectively a blank control group, a glucose group and a thallus embedding pellet group. Adding 30g/L glucose solution into the algae solution of glucose group and thallus embedding pellet group to make its final concentration be 1g/L, adding thallus embedding pellet into the algae solution of corresponding experimental group, placing in illumination incubator to culture under the same culture conditions. And measuring the content of chlorophyll a after 10 days, and calculating the algae-lysing efficiency. FIG. 7 shows photographs of algal solutions in the 10 th flask, where glucose had no effect on algae, the glucose group and the blank control group showed good growth of algae, the concentration of algal solution increased and the color remained green, while the growth of algae in the bacteria-embedded cocci group was severely inhibited, the concentration of algal solution decreased, the color became whitish, and a large number of algal cells died. The glucose used in the experiment has no influence on the growth of algae and meets the requirement of energy required by the growth of thalli, and meanwhile, compared with components of nutrient-rich thalli culture solution such as peptone, yeast powder or beef extract, the glucose is relatively oligotrophic, and is a relatively simple and environment-friendly additive.

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.

Collecting a field cyanobacterial bloom water sample, and subpackaging the field cyanobacterial bloom water sample into clean and sterile 1L triangular bottles, wherein each bottle contains 600mL, 6 bottles in total, and 3 bottles are taken as a group, and the group is respectively a control group and a treatment group. At the beginning of the experiment, THW7 thallus embedding pellets with the thallus embedding amount of 40mL are added into a treatment group, the experimental phenomenon is observed after 7d, and sampling is carried out to determine the chlorophyll a concentration. Meanwhile, a certain volume of glucose solution is added, the final concentration is 1g/L, the change condition of the algae in the experimental process is continuously observed, samples are taken at 20 days to measure the chlorophyll a concentration, and the algae dissolving effect is analyzed. FIG. 9 is a histogram of changes in chlorophyll a concentration of algal fluid after adding embedded globules of THW7 cells to a field population algal sample, and FIGS. 10 and 11 are photographs of algal fluid in a triangular flask at different time points (7d, 20d) after adding embedded globules of THW7 cells to a field population algal sample. Experiments show that under the condition of not adding any energy, the thallus embedding pellets have no algae dissolving effect on field population algae, but the phenomenon that part of algae sinks to the bottom can be seen in the culture process, and part of algae is attached to the wall of the triangular flask. After glucose supplementation, the treated algal population almost completely sunk to the bottom, with very few floating on the water surface. The chlorophyll a content of the water body begins to reduce, and the chlorophyll a concentration of the treated chlorophyll at 20d is reduced by 31.50 percent compared with that at 7 d. The result shows that the thallus embedding pellet has a good effect in field water sample algal bloom control, has strong practicability, and particularly has an obvious effect in the aspect of sinking a large amount of colony algae. Meanwhile, the thallus embedding pellet can be freely retracted and extended, has a long service life, can be repeatedly used, simplifies the algae control operation process, 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.

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 with algae-lysing activity（Pseudomonas sp.) THW7, wherein the preservation number of the pseudomonas is CCTCC NO: M20191065.

2. An embedded pellet comprising Pseudomonas THW7 according to claim 1.

3. The use of pseudomonas THW7 of claim 1 for the control of cyanobacterial bloom.

4. The use of claim 3, wherein the pseudomonas is prepared into bacteria embedding pellets, and then is put into the cyanobacterial bloom water body, and glucose is added into the water body to make the final concentration of the cyanobacterial bloom water body be 1.0-1.2 g/L.