CN111269855A

CN111269855A - Algicidal bacteria, screening method and application

Info

Publication number: CN111269855A
Application number: CN202010101591.7A
Authority: CN
Inventors: 王美娟; 张文艺; 张小梅; 薛静静; 许明宸
Original assignee: Changzhou University
Current assignee: Changzhou University
Priority date: 2020-02-19
Filing date: 2020-02-19
Publication date: 2020-06-12

Abstract

The invention discloses algicidal bacteria, a screening method and application thereof, and bacillus (B) of the algicidal bacteriaPaenibacillus sp.) The strain XXG is preserved in China general microbiological culture Collection center (CGMCC) of the institute of microbiology, China academy of sciences, and the preservation time is as follows: 4/1/2019, accession number: CGMCC No. 17503. Is screened from soil and provides the application of the algicidal bacteria in removing microcystis aeruginosa in a water system. The algicidal bacterium XXG screened by the invention has the advantages of good ecological adaptability, high safety, low extraction cost, simple and feasible method and the like. And is suitable for the condition of a natural water system, and can effectively control the outbreak of blue algae.

Description

Algicidal bacteria, screening method and application

Technical Field

The invention belongs to the field of algae control microbiology, and particularly relates to algicidal bacteria, a screening method and application.

Background

In summer and autumn, water blooms in Tai lake, nest lake, Dian lake and other important areas of China frequently occur every year, and Harmful Algae Blooms (HAB) seriously affect fishery production, water safety of residents and regional ecological safety. Microcystis aeruginosa is one of the dominant algae species in the Taihu basin HAB, and has become a public nuisance in water environment because the produced algal toxin has high toxicity and is difficult to degrade and can be biologically enriched. Later in practice, during the outbreak of HAB in the lake tai water system, local farmers irrigate the paddy field with HAB-containing algae water, and blue algae do not appear in the paddy field water, and it is hypothesized that the plant roots or microorganisms in the paddy field have an algae-lysing effect. Therefore, the method has feasibility in screening the algicidal bacteria with good ecological adaptability from the natural paddy field soil.

However, the research of algae control of algae-lysing bacteria firstly aims to separate high-efficiency algae-lysing bacteria from soil. The method for separating and screening the algicidal bacteria at the present stage mainly comprises the steps of infecting algae cells by using a bacterial culture solution, and inspecting the algae lysis rate by measuring the concentration of chlorophyll a (Chl-a), but the ecological adaptability and the application value of the algicidal bacteria screened by the method need to be considered, and the influence of a culture medium on the growth of the algae cells cannot be eliminated.

Disclosure of Invention

Aiming at the problems, the invention aims to screen a strain with good algae-dissolving performance from soil, particularly rice root system soil, and apply the strain to control dominant algae, namely microcystis aeruginosa, in blue algae so as to reduce the burst probability of the blue algae.

The technical purpose is achieved, the technical effect is achieved, and the invention is realized through the following technical scheme:

the invention provides an algicidal bacterium XXG which is identified as Paenibacillus sp, is currently preserved in No. 3 of the national institute of culture and management of microorganisms (CGMCC) of the institute of microbiology of China institute of academy of sciences, and has the preservation time as follows: 4/1/2019, accession number: CGMCC No. 17503.

The invention also provides a method for screening the algicidal bacteria, which comprises the following steps:

1) mixing and culturing soil and algae cell sap, and shaking and mixing for 5-10 days at the temperature of 28-30 ℃ until the color of the culture solution changes;

2) taking a certain amount of the culture solution after the step 1), using fresh algae cell sap to perform volume fixing according to a proportion, and repeating the operation for at least 3 times by adopting the same culture conditions as the step 1) to obtain enriched bacterial liquid;

3) diluting the enriched bacterial liquid, performing plate coating on the diluted bacterial liquid, further culturing for 2-3d, selecting different single bacterial colonies, and performing plate streaking culture for multiple times to obtain a purified bacterial strain;

4) and (4) detecting the bacteriolysis effect of each single colony purified and separated by using the algae cell sap, and screening out the phycolytics.

As a further improvement of the invention, the soil is selected from rice root system soil.

As a further improvement of the invention, the algae is selected from the blue algae bloom algae species, and the microcystis aeruginosa is mainly adopted.

As a further improvement of the invention, the step 1) and the step 2) are used for observing whether the culture solution is yellowed or not; and 4) comparing the yellowing degrees of the detection solutions corresponding to different strains in the step 4).

The invention also provides application of the algicidal bacteria in removing microcystis aeruginosa in a water system.

As a further improvement of the invention, the algicidal bacteria in the logarithmic growth phase are selected to remove the microcystis aeruginosa in the water system.

As a further improvement of the invention, the volume ratio of the added algicidal bacteria to the microcystis aeruginosa is 5.0-6.0%, and the time interval of the adding is 5-8 days.

The invention has the beneficial effects that: (1) the algicidal bacterium XXG with the algae lysing effect is separated from the soil of the paddy field and belongs to Paenibacillus sp.

(2) Through the influence on the algae dissolving effect of the strain under different ages, different algae concentrations, different algae volume fractions, different temperatures and different pH values, the survival conditions of the strain in the active period are all suitable for the conditions of a natural water system and are matched with the blue algae outbreak period, meanwhile, the bacteria dissolving capacity of the strain is enhanced along with the increase of the algae concentration, and the outbreak of the blue algae can be effectively controlled.

Drawings

FIG. 1 is a gram-stained microscopic view of strain XXG;

FIG. 2 is a phylogenetic tree of strain XXG;

FIG. 3 shows the effect of different ages on the algicidal effect of strain XXG;

FIG. 4 shows the effect of Microcystis aeruginosa liquid with different concentrations on the algae-lysing effect of XXG strain;

FIG. 5 shows the effect of different algal volume ratios on the algae lysing effect of the XXG strain;

FIG. 6 shows the effect of different temperatures on the algae lysing effect of strain XXG;

FIG. 7 shows the effect of different pH values on the algicidal effect of strain XXG.

A bacillus (Paenibacillus sp.) XXG of algicidal bacteria is deposited in China general microbiological culture Collection center (CGMCC) of China institute of microbiology, and the preservation time is as follows: 4/1/2019, accession number: CGMCC No. 17503.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

The following detailed description of the principles of the invention is provided in connection with the accompanying drawings.

In the embodiment, the rice root soil irrigated in the Taihu lake is taken as a strain source, the efficient algicidal strain XXG is screened and separated from the strain source, and the strain is used for an algae lysing experiment.

Firstly, screening algicidal bacteria XXG comprises the following specific steps:

1) weighing 5g of the mixture and 100mL of the microcystis aeruginosa liquid, mixing and culturing the mixture, placing the mixture in a shaking table at the temperature of 28-30 ℃ and the rotating speed of 120-150 r/min, and oscillating for 5-10 days to find that the algae liquid is yellowed.

2) Taking 10mL of etiolated algae solution, diluting the volume to 100mL with fresh Microcystis aeruginosa solution, and repeating the operation for 3 times under the same culture condition to obtain enriched bacteria solution.

3) The enriched bacterial liquid is treated according to the proportion of 10-10⁶The method comprises the following steps of (1) carrying out gradient dilution, carrying out plate coating on 100 mu L of diluted bacterial liquid with different concentrations, carrying out inverted culture for 2-3d, picking different single bacterial colonies by using an inoculating loop, carrying out plate streaking for multiple times to obtain purified strains, and separating 5 different purified strains with the serial numbers of 1# to 5 #.

4) A small number of cells (about 6.9X 10) were picked up by sterile inoculating loops⁶cells)5 purified bacteria (1# to 5#) are mixed with 100mL of microcystis aeruginosa liquid with the concentration of chlorophyll a (Chl-a) of 1648 mu g/L, the mixture is placed in a shaking table with the temperature of 28 ℃ to 30 ℃ and the rotating speed of 120 r/min to 150r/min for shaking culture for 20 to 30 days, the culture solution is yellowed to different degrees, wherein the 4# bacterial strain has the highest yellowing degree and the best removal effect on the chlorophyll a, and the culture solution is determined to be the efficient algae-dissolving bacteria XXG.

II, biological characteristic identification:

(1) and (3) morphology observation: the screened algicidal bacteria XXG were gram-stained and observed in a microscope (see fig. 1). Through morphological observation, after the strain is cultured for 2d, the strain XXG has an irregular colony shape, a diameter of about 5mm, a gray color, a smooth surface, a slight bulge, a regular edge and a quick growth. The observation of an electron microscope shows that the strain is rod-shaped; the gram staining result shows that the bacillus subtilis is a positive bacterium.

(2) Physiological and biochemical experiments: experiments were performed according to the identification items and methods in Bergey's Manual of bacteria identification (eighth edition) and Manual of identification of common bacteria systems (east elegans bead et al, 2001). The physiological and biochemical characteristics are shown in table 1 below.

TABLE 1 physiological and biochemical characteristics of alga lysing bacteria XXG

Note "+" indicates positive and "-" indicates negative.

(3) And (3) DNA identification: the selected strains are sent to bioengineering (Shanghai) GmbH for sequence determination, a phylogenetic tree (see figure 2) is constructed by utilizing Mega5.0 software, and XXG is identified to be Paenibacillus (Paenibacillus sp.) and the DNA sequence of the Paenibacillus sp is shown in a sequence table in detail.

Application of algicidal bacterium XXG

(1) Activation of the strain: inoculating algicidal bacteria XXG into a beef extract peptone liquid culture medium from a slant solid culture medium, carrying out constant-temperature shaking culture at 30 ℃ and 150r/min for 2d to prepare a seed solution, transferring the seed solution into a fresh beef extract peptone culture medium according to the addition of 1% of the seed solution by volume ratio, and respectively extracting strains in a lag phase (0-8h), a log phase (8-52h) and a stabilization phase (52-62h) for later use;

(2) the bacterial strain algae dissolving process comprises the following steps: adding the bacterial liquid into 100mL of microcystis aeruginosa liquid, respectively carrying out algae dissolving reaction under the conditions of different ages, different algae concentrations, different volume fractions of bacteria and algae, different temperatures and different pH values, measuring the chlorophyll a concentration of the bacteria and the algae, and inspecting the removal effect of the strain XXG on the microcystis aeruginosa under different conditions. Specific examples are as follows:

example 1

In order to examine the influence of different bacterial ages on the algae dissolving effect of the XXG bacterial strain, 5.6mL of XXG bacterial suspension in a retardation stage, a logarithmic stage and a stationary stage is respectively inoculated into a conical flask filled with 100mL of microcystis aeruginosa liquid with Chl-a concentration of 1294.22 mu g/L for shaking culture for 6 days, and the algae dissolving rate after 6 days is 40.28%, 50.13% and 47.19% respectively. It can be seen that the bacterial strain at the logarithmic phase is superior in algicidal effect to the bacterial strains at the other phases (see FIG. 3).

Example 2

In order to examine the influence of different concentrations of microcystis aeruginosa liquid on the algae dissolving effect of XXG strains, 5.6mL of XXG bacterial suspension in a logarithmic growth phase is inoculated into conical flasks filled with 100mL of microcystis aeruginosa liquid with Chl-a concentrations of 659.68 [ mu ] g/L, 1294.22 [ mu ] g/L and 4259.88 [ mu ] g/L respectively for shaking culture for 6d, the algae dissolving rate is 77.1%, 50.13% and 18.60% respectively after 6d, and the Chl-a concentrations in the microcystis aeruginosa liquid are reduced by 508.61 [ mu ] g/L, 648.79 [ mu ] g/L and 792.33 [ mu ] g/L respectively through conversion (see figure 4). It can be seen that the bacteriolytic capacity of the XXG strain to the microcystis aeruginosa is enhanced with the increase of the number of the microcystis aeruginosa.

Example 3

To examine the influence of different algae volume ratios on the algae dissolving effect of the XXG strain, the XXG strain suspension in the logarithmic growth phase is inoculated into a conical flask filled with 100mL of microcystis aeruginosa liquid with Chl-a concentration of 1294.22 mu g/L for shaking culture for 6d, the algae volume ratios are respectively 5.0%, 5.2%, 5.4%, 5.6%, 5.8% and 6.0%, and the algae dissolving rates after 6d are respectively 36.03%, 41.66%, 42.24%, 50.13%, 44.56% and 47.09% (see figure 5).

Example 4

In order to examine the influence of different temperatures on the algae dissolving effect of the XXG strain, 5.6mL of XXG bacterial suspension in the logarithmic growth phase is inoculated into a conical flask filled with 100mL of microcystis aeruginosa liquid with Chl-a concentration of 1294.22 mu g/L, the pH is 7.5, the rotating speed of a shaking table is 150r/min, and the shake culture is carried out under the conditions of 15 ℃, 30 ℃ and 45 ℃ respectively to examine the algae dissolving effect of the strain 6 d. The degradation result shows that: the concentration of chlorophyll a in the microcystis aeruginosa liquid with the initial concentration of 1294.22 mug/L is respectively reduced to 441.20 mug/L, 9.45 mug/L and 483.52 mug/L by the strain within 6d, and the algae dissolving rate is 65.91%, 99.27% and 62.64% (see figure 6). Meanwhile, the temperature of 30 ℃ is the temperature interval corresponding to the blue algae outbreak, so that the XXG strain is suitable for effectively controlling the blue algae outbreak in the natural water system.

Example 5

To examine the influence of different pH values on the algae-lysing effect of the XXG strain, OD was taken₆₀₀5.6mL of XXG bacterial suspension with the concentration of 0.75 is inoculated into a conical flask filled with 100mL of microcystis aeruginosa liquid with the concentration of Chl-a of 1294.22 mu g/L, the temperature is 30 ℃, the rotating speed of a shaking table is 150r/min, shaking culture is carried out under the conditions that the pH is 6.5, 7.5 and 8.5 respectively, and the algae dissolving effect of the strain 6d is examined. The degradation result shows that: the concentration of chlorophyll a in the microcystis aeruginosa liquid with the initial concentration of 1294.22 mu g/L is respectively reduced to 343.10 mu g/L, 9.45 mu g/L and 328.21 mu g/L by the strain within 6d, and the algae dissolving rate is 73.49%, 99.27% and 74.64% (see figure 7). From this, it can be seen that the alga lysing bacterium XXGThe pH of the activated water is about 7.5, and the activated water is suitable for a natural water system.

The foregoing shows and describes the general principles and broad features of the present invention and advantages thereof. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

Sequence listing

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cgccgtaaac gatgagtgct aggtgttagg ggtttcgata cccttggtgc cgaagttaac 780

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Claims

1. Bacillus (B) of algicidal bacteriaPaenibacillus sp.) XXG, the strain is preserved in China general microbiological culture Collection center (CGMCC) of China institute of microbiology, China academy of sciences, and the preservation time is as follows: 4/1/2019, accession number: CGMCC No. 17503.

2. A method of screening for the phycolytics of claim 1, comprising the steps of:

mixing and culturing soil and algae cell sap, and shaking and mixing for 5-10 days at the temperature of 28-30 ℃ until the color of the culture solution changes;

taking a certain amount of the culture solution after the step 1), using fresh algae cell sap to perform volume fixing according to a proportion, and repeating the operation for at least 3 times by adopting the same culture conditions as the step 1) to obtain enriched bacterial liquid;

diluting the enriched bacterial liquid, performing plate coating on the diluted bacterial liquid, further culturing for 2-3d, selecting different single bacterial colonies, and performing plate streaking culture for multiple times to obtain a purified bacterial strain;

and (4) detecting the bacteriolysis effect of each single colony purified and separated by using the algae cell sap, and screening out the phycolytics.

3. The screening method according to claim 2, wherein: the soil is selected from rice root system soil.

4. The screening method according to claim 2, wherein: the algae is selected from cyanobacterial bloom algae species, including microcystis aeruginosa.

5. The screening method according to claim 4, wherein: the step 1) and the step 2) are used for observing whether the culture solution is yellowed or not; and 4) comparing the yellowing degrees of the detection solutions corresponding to different strains in the step 4).

6. The use of the phycolytics of claim 1 for removing microcystis aeruginosa from water systems.

7. The use of claim 6, wherein the water system is treated with microalgae-lysing bacteria in logarithmic growth phase to remove microcystis aeruginosa.

8. The use of claim 6, wherein the volume ratio of the algicidal bacteria to the microcystis aeruginosa is 5.0-6.0%, and the time interval is 5-8 days.