CN115975860A - Bacillus parabrevis strain of infectious disease research institute and application thereof - Google Patents

Abstract

The invention relates to the technical field of water body microorganism restoration, in particular to a bacillus parabrevis strain of an infectious disease research institute and application thereof. The strain is Bacillus parabacteroides (Metabacillus idriensis) ADOM08 of infectious disease research institute, which is preserved in China general microbiological culture Collection center on 05.2022 and 08.s, wherein the preservation address is No. 3 of Beijing province No. 1 of North Chen Xilu of the Chaoyang district, and the preservation number is CGMCC No.25498. The bacillus parabrevis ADOM08 of the infectious disease research institute provided by the invention can obviously degrade algae source soluble organic matters and reduce organic pollution of a water bloom water body, and has the effect of efficiently inhibiting the growth of harmful microalgae, so that the strain can be used for controlling the water bloom and has important practical significance and application prospect.

Description

Bacillus parabrevis strain of infectious disease research institute and application thereof

Technical Field

The invention relates to the technical field of water body microorganism remediation, in particular to a bacillus paracasei strain of infectious disease research institute and application thereof.

Background

At present, the eutrophication of water bodies is aggravated due to human activities, and harmful microalgae bloom frequently occurs in the world, for example, the bloom phenomenon is gradually serious along with the development of intensive aquaculture in aquaculture systems, algae is eliminated and released algal toxins pollute the water bodies and aquatic products, and finally the safety and health of human beings are harmed. Therefore, the prevention and control of the water bloom becomes a research hotspot and a leading-edge scientific problem, and the search of a technical method capable of effectively preventing and controlling the growth of harmful microalgae is one of the important contents of water ecological protection and restoration at the present stage.

Although physical and chemical methods have been applied to algal bloom control, external additives cause secondary environmental pollution problems, such as large amount of clay suspended in water to affect biological growth and survival, and chemical algistat copper sulfate causes heavy metal pollution in water. At present, an environment-friendly algal bloom prevention and treatment technology is increasingly emphasized by people, mainly comprising the steps of preventing and treating algal blooms by utilizing large-scale aquatic plants (including large-scale algae plants) or the interaction between microorganisms and microalgae, but the application of the aquatic plants in an aquaculture system, particularly a system mainly using herbivory (such as grass carp), is limited, and the existence and large-scale growth of the aquatic plants are not beneficial to gathering and catching cultured objects. The microorganisms have great potential in the aspect of water bloom prevention and control due to the characteristics of wide distribution, multiple varieties, fast metabolic propagation, small environmental disturbance and the like, and the microorganisms mainly inhibit the growth of microalgae through nutrient salt competition or allelochemicals (secondary metabolites) generation and other ways. For example, in the water bloom generation process, algae cells die to release a large amount of algae-derived dissolved organic matter (ADOM), the ADOM can be converted into small molecular nutrient salt for the algae growth requirement after being degraded by natural light, so that the water bloom is continuously generated and developed, the microorganisms are utilized to accelerate the metabolic utilization of the ADOM, the nutrition competition with the microalgae is enhanced, the algae nutrition supply is reduced, the water bloom process can be greatly shortened, and the purposes of controlling the water bloom and repairing pollution are achieved. Furthermore, some algae-lysing bacteria exist in the water bloom system and natural water body, and can be metabolized to generate some algae-lysing components to directly kill algae cells, which plays an important role in maintaining algae biomass balance. Therefore, in recent years, beneficial microorganisms are studied in the aspect of water bloom prevention and control, for example, the development and application of microbial enzyme preparations with thalli as main components become a great research hotspot, but research and application effects are greatly different due to the potential difference of different strains.

Disclosure of Invention

Aiming at the technical problem of secondary environmental pollution in physical and chemical water bloom control technologies, the invention provides a bacterial strain of Bacillus paracasei of infectious disease research institute and application thereof, wherein the bacterial strain of Bacillus paracasei ADOM08 of the infectious disease research institute can obviously degrade algae source soluble organic matters and reduce organic pollution of water bloom water bodies, and has the function of efficiently inhibiting the growth of harmful microalgae, so that the bacterial strain can be used for controlling water bloom and has important practical significance and application prospect.

In a first aspect, the invention provides a strain of Bacillus parabuis (Metabacillus idrensis) ADOM08, which has been deposited in China general microbiological culture Collection center at 08-05.2022, with the deposition address of No. 3, siro-1, beijing, the rising area, and the deposition number of CGMCC No.25498.

In a second aspect, the invention provides an application of bacillus parabrevis ADOM08 of infectious disease research institute in prevention and treatment of microalgae aquaria.

Further, the infectious disease research institute, bacillus parabrevis ADOM08 is used for degrading the blue algae source to dissolve organic matters and/or inhibit the growth of microcystis aeruginosa.

Furthermore, the bacterial suspension of the bacillus parabrevis ADOM08 of the infectious disease research institute is used for degrading blue algae source dissolved organic matters in the water body.

Further, adding the thallus suspension into a water body containing blue algae source dissolved organic matters according to the volume ratio of 3-10% for degradation, wherein the viable count of the thallus suspension is 1 multiplied by 10 ⁸ -2×10 ⁸ one/mL, and the degradation time is more than 12 h.

Further, adding the thallus suspension into a water body containing blue algae source dissolved organic matters according to the volume ratio of 5 percent for degradation, wherein the viable count of the thallus suspension is 1.44 multiplied by 10 ⁸ -2×10 ⁸ one/mL, and the degradation time is more than 72 h.

Further, the bacterial suspension is obtained according to the following preparation method:

inoculating Bacillus parabacteroides ADOM08 of institute of infectious diseases into sterilized LB culture medium, shake culturing at 28 deg.C and 250rpm for 2 days, centrifuging, and resuspending in physiological saline to obtain the final product.

Furthermore, the fermentation liquor of the bacillus parabrevis ADOM08 of the institute of infectious diseases is used for inhibiting the growth of the microcystis aeruginosa.

Further, the fermentation liquor is added into the water body containing the microcystis aeruginosa according to the volume ratio of 0.3-5 percent for inhibiting the growth of the algae, and the viable count of the fermentation liquor is 8 multiplied by 10 ⁸ -9×10 ⁸ Per mL, and the algae inhibiting culture time is more than 2 days.

Further, the algae inhibiting culture conditions are static culture at 30 ℃, under the illumination intensity of 4000lx and the light-dark ratio of 1698 h.

Further, the fermentation liquor is added into the water body containing the microcystis aeruginosa according to the volume ratio of 0.5 to 2 percent for inhibiting the growth of the algae, and the viable count of the fermentation liquor is 8.94 multiplied by 10 ⁸ -9×10 ⁸ One cell per mL, and the algae inhibiting culture time is more than 5 days.

Further, the fermentation liquor is obtained according to the following preparation method:

inoculating Bacillus parabuisticis ADOM08 of infectious disease research institute into sterilized LB culture medium, and shake culturing at 30 deg.C and 250rpm for 5 days.

The invention has the beneficial effects that:

the bacillus paracasei ADOM08 provided by the infectious disease research institute has stronger degradation and pollution remediation capabilities on blue algae source dissolved organic matters, has a remarkable inhibition effect on the growth of Microcystis aeruginosa (Microcystis aeruginosa), can greatly improve the ecological condition of a water body, and is expected to develop an environment-friendly algae inhibiting microbial inoculum for biological control of water bloom.

Drawings

In order to more clearly illustrate the embodiments or technical solutions in the prior art of the present invention, the drawings used in the description of the embodiments or prior art will be briefly described below, and it is obvious for those skilled in the art that other drawings can be obtained based on these drawings without creative efforts.

FIG. 1 is a graph showing the degradation effect of 5 active strains selected in example 2 on the dissolved organic matter of the cyanobacteria source.

FIG. 2 is a graph showing the growth inhibitory effect of 5 active strains selected in example 2 on Microcystis aeruginosa.

FIG. 3 is a phylogenetic tree of strain ADOM08 of example 2.

Fig. 4 is a graph showing the change in the optical absorption coefficient in example 3.

FIG. 5 is a curve showing the change of chemical oxygen demand in example 3.

FIG. 6 is a graph showing the effect of the volume of the ADOM08 strain fermentation broth on the growth of Microcystis aeruginosa in example 4.

Detailed Description

In order to make those skilled in the art better understand the technical solution of the present invention, the technical solution in the embodiment of the present invention will be clearly and completely described below with reference to the drawings in the embodiment of the present invention, and it is obvious that the described embodiment is only a part of the embodiment of the present invention, and not all embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, shall fall within the protection scope of the present invention.

Example 1 preparation of ADOM Medium

A water sample is taken from a grass carp culture pond with the occurrence of cyanobacterial bloom, and the main algae composition of a sampled water body is shown in the following table 1. Centrifuging a water sample to obtain algae slurry, cleaning the algae slurry with ultrapure water, adding 10g (wet weight) of the algae slurry into 50mL of ultrapure water, uniformly mixing, ultrasonically crushing by using an ultrasonic cell crusher, quickly freezing in liquid nitrogen, thawing again, repeating for 2 times, breaking algae cells, filtering by using a 0.45-micrometer filter membrane, sterilizing by high pressure and damp heat (121 ℃, 20 min) to obtain 10 times of algae source dissolved organic matter stock solution, sealing, preventing bacteria, keeping out of the sun and storing at low temperature. When in use, a proper amount of stock solution is taken and diluted by 0.9 percent of normal saline to working concentration, the pH value is adjusted to 7.8, and the stock solution is subpackaged in conical bottles for sterilization (121 ℃, 20 min) for later use.

TABLE 1 Main algal composition of sampled Water

EXAMPLE 2 isolation of active Strain

Collecting water body of the water bloom generating grass carp culture pond as a degrading bacteria separation source, carrying out enrichment culture on the water body by adopting the ADOM culture medium prepared in the embodiment 1 for 7 days, and then separating and purifying strains by using a coating plate and a scribing separation method to finally obtain 19 strains of bacteria, which are named as ADOM01-ADOM19 in sequence.

The bacteria are respectively placed in 100mL of sterilized nutrient broth culture medium (commodity), activated and cultured for 1-2 days under the conditions of 28 ℃ and 250rpm, then 5mL of bacteria culture solution is respectively taken out from the bacteria culture medium and respectively added into 100mL of ADOM culture medium for culture under the conditions of 28 ℃, 250rpm and dark, the sterilized nutrient broth culture medium with the same volume is used as a blank control to replace the bacteria culture solution, samples are taken after 48 hours, and the ultraviolet-visible spectrum absorption curve of the ADOM is measured after the filtration through a 0.22 mu m filter membrane.

The detection conditions are as follows: wavelength 230-500nm, 1cm quartz cuvette, interval 1nm, and pure water as reference. With a light absorption coefficient alpha at 280nm ₍₂₈₀₎ Characterizing the ADOM content and calculating the degradation rate, the formula is as follows:

the effect of each strain on the degradation of ADOM was compared. As shown in fig. 1, it is found that 5 strains of bacteria (ADOM 05, ADOM08, ADOM09, ADOM11, and ADOM 18) have high capability of promoting the decomposition of organic matter dissolved by cyanobacteria source, and their 48h degradation rates on ADOM are respectively 50.72%, 61.16%, 56.96%, 65.46% and 54.42%, and the degradation effect difference between strains is significant, with ADOM11> ADOM08> ADOM09= ADOM18> ADOM05. Inoculating 5 strains of bacteria into LB slant culture medium, and storing at 4 ℃ for later use.

The 5 strains of bacteria were prepared into fermentation broth, 10mL of each strain was inoculated separately into 100mL of sterilized LB medium, and shake-cultured at 30 ℃ and 250rpm for 5 days. And then inoculating 1mL of fermentation liquor into 50mL of microcystis aeruginosa liquid in the logarithmic phase, placing the microcystis aeruginosa liquid in an illumination incubator for standing culture under the culture conditions of 30 ℃, illumination intensity of 4000lx and light-dark ratio of 1697, replacing the fermentation liquor with a sterilized LB culture medium with the same volume as a blank control, and shaking regularly for 3-5 times every day. Sampling and microscopic examination are carried out after 5 days of culture to determine the cell density of algae, and the algae inhibition rate of each strain is calculated by the following formula:

comparing the growth inhibition effect of the microcystis aeruginosa of each strain. The results are shown in fig. 2, when the fermentation liquor of 5 strains of bacteria (ADOM 05, ADOM08, ADOM09, ADOM11, ADOM 18) is added at a volume ratio of 2%, the inhibition rates of Microcystis aeruginosa for 5 days are 81.49%, 89.31%, 79.54%, 70.31% and 53.51 respectively, and the algae inhibition effect difference among strains is obvious, namely ADOM08> ADOM05= ADOM09> ADOM11> ADOM18.

Finally, the ADOM08 is considered to have stronger capability of degrading the blue algae source dissolved organic matters and inhibiting the growth of microcystis aeruginosa and has optimal activity by comprehensive comparison.

Gram staining was performed on the strain ADOM08, and colony morphology and microbial microscopic morphology were observed. Strain ADOM08 is aerobic or facultative anaerobic, gram-positive, rod-shaped, motile, sporulating, round, orange, opaque, flat, surface-moist when grown in nutrient broth.

The strain ADOM08 after purification and fermentation culture is sent to the company of biological engineering (Shanghai) to carry out 16S rDNA molecular fragment amplification sequencing.

The primer is 27F:5'-AGAGTTTGATCMTGGCTCAG-3' and 1492R:5'-TACGGYTACCTTGTTA CGACTT-3'.

The 16S rDNA sequence of the strain ADOM08 is finally obtained as follows:

GGTGAGTAACACGTGGGCAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGAGCTAATACCGGATACTATGTCAAACCGCATGGTTTGACATTCAAAGACGGTTTCGGCTGTCACTTACAGATGGGCCCGCGGCGCATTAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAACTCTGTTGTCAGGGAAGAACAAGTGCCGGAGTAACTGCCGGCACCTTGACGGTACCTGACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGAAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTTTGCCACTTCTAGAGATAGAAGGTTCCCCTTCGGGGGACAAAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAAAGGGCTGCGAGACCGCGAGGTTTAGCCAATCCCATAAAACCATTCTCAGTTCGGATTGCAGGCTGCAACTCGCCTGCATGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAG。

the sequencing sequence of ADOM08 was submitted to GenBank for alignment, and a phylogenetic tree of this strain was constructed, as shown in FIG. 3, to identify the strain ADOM08 as a Bacillus parabuis (Metabacillus idriensis) institute for infectious disease.

Example 3 degradation and contamination remediation Effect of ADOM by Strain ADOM08

Activating strain ADOM08 on LB slant culture medium, inoculating into sterilized LB culture medium, shake culturing at 28 deg.C and 250rpm for 2 days, centrifuging 5mL bacterial solution (10000 r/min,30 min) to obtain thallus cells, and resuspending in 5mL physiological saline to obtain viable count of 1.44 × 10 ⁸ Cell suspension per mL.

The whole cell suspension is added into 100mL sterilized ADOM culture medium, and cultured in the dark at 28 ℃ and 250rpm, and sterilized normal saline with the same volume is used to replace the cell suspension as a blank control, and samples are respectively taken at 0h, 12h, 24h, 48h, 72h and 96h and filtered by a 0.22 mu m filter membrane, and then the ultraviolet-visible spectrum absorption curve of ADOM is determined, and the detection conditions are the same as those in example 2. Measuring Chemical Oxygen Demand (COD) of filtrate by acid potassium permanganate method _Mn ) And evaluating the change of the organic pollution degree of the water body.

The results are shown in FIGS. 4 and 5. The blank control has no change in the ultraviolet-visible spectrum absorption curve during the culture period; in the treatment group added with ADOM08 thallus suspension, blue algae source dissolved organic matters show a rapid degradation trend within 72 hours, the 72 hAMOM degradation rate reaches 69.23 percent, and the degradation rate is increased to 96 hours and is possibly related to bacterial dissolved organic matters released by thallus cell death. Chemical Oxygen Demand (COD) _Mn Has similar change trend, 72h organic pollution (COD) _Mn ) The removal rate reaches 72.40 percent.

Example 4 algal inhibition effect of ADOM08 fermentation broth of Strain on Microcystis aeruginosa

Activating strain ADOM08 on LB slant culture medium, inoculating 10mL into 100mL sterilized LB culture medium, shake culturing at 30 deg.C and 250rpm for 5 days to obtain viable count of 8.94 × 10 ⁸ one/mL of fermentation broth. Taking 100 mu L of fermentation liquor,Respectively adding 250 mu L, 500 mu L and 1000 mu L into 50mL of microcystis aeruginosa liquid in a logarithmic growth phase, enabling the addition volume ratio to be four gradients of 0.2%, 0.5%, 1% and 2%, treating by taking fermentation liquor not added as a blank control, then placing the microcystis aeruginosa liquid in an illumination incubator for standing culture, wherein the culture conditions are 30 ℃, the illumination intensity is 4000lx and the light-dark ratio is 16h, shaking for 3-5 times every day at regular time, sampling and microscopic examination are performed every day to detect the cell density of algae, the algae inhibition rate is calculated, and the continuous monitoring is performed for 5 days.

The result is shown in fig. 6, except 0.2%, the ADOM08 fermentation liquor has significant inhibition effect on the growth of microcystis aeruginosa under the test addition volume ratio of 0.5%, 1% and 2%, the greater the addition ratio is, the more obvious the algae inhibition effect is, the algae inhibition rate of 5 days of each gradient is 34.21%, 78.02% and 82.53%, respectively, and the best algae inhibition effect is under the addition volume ratio of 2%.

Although the present invention has been described in detail by referring to the drawings in connection with the preferred embodiments, the present invention is not limited thereto. Various equivalent modifications or substitutions can be made on the embodiments of the present invention by those skilled in the art without departing from the spirit and scope of the present invention, and these modifications or substitutions are within the scope of the present invention/any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention.

Claims (10)

1. A strain of Bacillus paracasei (Metabacillus idriensis) ADOM08 of infectious disease research institute is characterized in that the strain is preserved in China general microbiological culture Collection center on 08-05-month 2022, the preservation address is No. 3 of Xilu No. 1 of Beijing Korean district, beijing, and the preservation number is CGMCC No.25498.

2. The use of the infectious disease institute for the prevention and treatment of microalgae aquaria, as claimed in claim 1, wherein the infectious disease institute for the prevention and treatment of microalgae aquaria comprises using the bacillus parabrevis ADOM08 to degrade cyanobacterial sources to solubilize organic matter and/or inhibit the growth of microcystis aeruginosa.

3. The use of claim 2, wherein the bacterial suspension of Bacillus parabacteroides ADOM08 is used for degrading blue algae-derived dissolved organic matters in water.

4. The use as claimed in claim 3, wherein the bacterial suspension is added into the water containing blue algae source dissolved organic matter for degradation according to the volume ratio of 3% -10%, and the viable count of the bacterial suspension is 1 x 10 ⁸ -2×10 ⁸ one/mL, and the degradation time is more than 12 h.

5. The use according to claim 3, wherein the bacterial suspension is added into the water containing the blue algae source dissolved organic matter for degradation according to the volume ratio of 5 percent, and the viable count of the bacterial suspension is 1.44 x 10 ⁸ -2×10 ⁸ one/mL, and the degradation time is more than 72 h.

6. The use according to claim 3, wherein the bacterial suspension is obtained by the following preparation method:

inoculating Bacillus parabuisticis ADOM08 of infectious disease research institute into sterilized LB culture medium, shake culturing at 28 deg.C and 250rpm for 2 days, centrifuging, and resuspending in physiological saline to obtain the final product.

7. The use according to claim 2, wherein the growth of microcystis aeruginosa is inhibited by a fermentation broth of bacillus parabrevis ADOM08 of the institute for infectious disease.

8. The use according to claim 7, wherein the fermentation broth is added to the water containing Microcystis aeruginosa in an amount of 0.3-5% by volume for algal growth inhibition, the viable count of the fermentation broth being 8 x 10 ⁸ -9×10 ⁸ One cell per mL, and the algae inhibiting culture time is more than 2 days.

9. Use according to claim 7, wherein the fermentation broth is added to the microcapsules containing Aerugo in a volume ratio of 0.5% to 2%Inhibiting algae growth in algae water body, and the viable count of the fermentation liquid is 8.94 × 10 ⁸ -9×10 ⁸ One cell per mL, and the algae inhibiting culture time is more than 5 days.

10. The use according to claim 7, wherein the fermentation broth is obtained as follows:

inoculating Bacillus parabacteroides ADOM08 of institute of infectious diseases into sterilized LB culture medium, and shake culturing at 30 deg.C and 250rpm for 5 days.

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