CN113462574B - Marine nannochloropsis LAMB205 and ciliates algae preventing and treating agent and preventing and treating method - Google Patents
Marine nannochloropsis LAMB205 and ciliates algae preventing and treating agent and preventing and treating method Download PDFInfo
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- CN113462574B CN113462574B CN202110642565.XA CN202110642565A CN113462574B CN 113462574 B CN113462574 B CN 113462574B CN 202110642565 A CN202110642565 A CN 202110642565A CN 113462574 B CN113462574 B CN 113462574B
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/32—Biological treatment of water, waste water, or sewage characterised by the animals or plants used, e.g. algae
- C02F3/322—Biological treatment of water, waste water, or sewage characterised by the animals or plants used, e.g. algae use of algae
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/12—Unicellular algae; Culture media therefor
Abstract
The invention relates to the technical field of microorganisms, in particular to a marine nannochloropsis LAMB205, a ciliate algae prevention and treatment agent and a prevention and treatment method. The marine Nannochloropsis oceanica LAMB205 is classified and named as Nannochloropsis oceanica, the preservation number is CGMCC NO.20714, the preservation date is 2020, 10 and 23 days, the preservation unit is the common microorganism center of China general microbiological culture Collection center, and the preservation address is No. 3 of No. 1 Hospital of West Chen of the sunward area in Beijing; the ciliate algae preventing and treating agent comprises marine nannochloropsis LAMB204 and LAMB205; the ciliate control method comprises the step of adding the ciliate control algicide into a water body containing ciliates. The invention utilizes the characteristic that marine nannochloropsis LAMB204 and LAMB205 have resistance to disease ciliates to inhibit exponential growth of ciliates.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to a marine nannochloropsis LAMB205, a ciliate algae prevention and treatment agent and a prevention and treatment method.
Background
The nannochloropsis has simple structure and small size. The nannochloropsis oculata generally contains chloroplasts, can perform photosynthesis and produce a large amount of organic matters and oxygen, and is beneficial to orderly operation of a water body ecological system. The nannochloropsis has high protein content and contains various inorganic elements and vitamins, so that the nannochloropsis has important application value in food, nutrition, health care and other fields.
At present, the engineering culture of nannochloropsis is still faced with great challenge, and how to control the ingestion of the nannochloropsis by the primary plants such as ciliates and reduce the economic loss of culturists due to the light eating of the microalgae is a problem which must be solved for further open utilization of nannochloropsis. At present, research mostly centers on physicochemical culture conditions for controlling protozoa harm in nannochloropsis oculata outdoor culture, mainly focuses on changing the aspects of pH, temperature, nitrogen source and the like of a culture water body, for example, ammonium bicarbonate is added into a fresh water body, so that the reproduction rate of the protozoa is slowed down, and the purpose of temporarily inhibiting ingestion of nannochloropsis oculata is achieved. However, due to the influence of the buffer capacity of seawater itself, the method for controlling the physicochemical culture conditions is difficult to exert its effect in the marine environment, and is difficult to realize the control of protozoan pollution in the engineering culture of marine nannochloropsis.
Disclosure of Invention
Aiming at the technical problem that the control effect of protozoan pollution in the engineering culture of marine nannochloropsis is not ideal, the invention provides a strain of marine nannochloropsis LAMB205, a ciliate algae control agent and a control method, wherein the marine nannochloropsis LAMB205 is obtained by mutagenizing and screening wild nannochloropsis, has resistance to disease ciliates, can obviously reduce the ingestion of the disease ciliates, and is very suitable for large-scale culture; the invention also prepares an algicide capable of inhibiting ciliates from the marine nannochloropsis LAMB205 and the marine nannochloropsis LAMB204, and after the algicide is added into a water body containing ciliates, an obvious exponential growth period does not appear in the propagation process of the ciliates.
In a first aspect, the invention provides a strain of anti-disease ciliate marine Nannochloropsis oceanica LAMB205, which is classified and named as marine Nannochloropsis oceanica, with the collection number of CGMCC NO.20714, the preservation date of 10-23 days of 2020, the preservation unit is the China general microbiological culture Collection center, the preservation address is No. 3 of Beijing ministerial district North Chen West Lu No. 1, the postal code: 100101.
furthermore, the inhibition rate of the marine nannochloropsis LAMB205 on the growth of the paraquat flora is 66.67%.
In a second aspect, the present invention provides a ciliate control algicide comprising marine nannochloropsis LAMB204 and marine nannochloropsis LAMB205;
the marine Nannochloropsis oceanica Lamb204 is classified and named as marine Nannochloropsis oceanica, the preservation number is CGMCC NO.20713, the preservation date is 2020, 10 and 23 days, the preservation unit is the common microorganism center of China microorganism culture preservation management committee, the preservation address is No. 3 of West Lu No. 1 of Beijing Kogyo area north Chen, the postal code: 100101.
further, the ciliate control algicide is a mixed algae liquid of marine nannochloropsis LAMB204 and marine nannochloropsis LAMB205.
Further, the preparation method of the ciliate algae control agent comprises the following steps:
respectively culturing marine nannochloropsis LAMB204 and marine nannochloropsis LAMB205 at 20-30 deg.C under 100 μmol phos/s/m 2 irradiation, with an illumination period of 12:12, when the density of the algae reaches 4 multiplied by 10 7 -5×10 7 After cells/mL, the two algae solutions were mixed in equal volumes.
Further, the total density of Nannochloropsis oceanica in the ciliate controlling algicide is 4 × 10 7 -5×10 7 cells/mL。
In a third aspect, the present invention provides a ciliate control method of adding the ciliate control algicide according to claim 3 to a water body containing ciliates, to inhibit the proliferation of ciliates.
Further, the ciliate is a mythimna separata.
The invention has the beneficial effects that,
the invention provides a strain of disease-resistant ciliate-resistant marine nannochloropsis LAMB205, which can inhibit the growth of a peripheral population, and the inhibition effect is greatly improved compared with that of a wild strain, and the marine nannochloropsis LAMB205 is suitable for large-scale culture;
the invention also provides a ciliate prevention and treatment algicide prepared by using the marine nannochloropsis LAMB205 and the marine nannochloropsis LAMB204 and a prevention and treatment method.
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 comparing the inhibitory effect of wild strains and LAMB205 on the growth of a wild-type parasite population;
FIG. 2 is a graph comparing the inhibitory effect of LAMB204, LAMB205 and a prophylactic/therapeutic algaecide on the growth of a parasite population.
Detailed Description
In order to make those skilled in the art better understand the technical solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
(1) Wild marine nannochloropsis oculata (purchased from Australia CSIRO Collection of Living Microalgae, htttp:// www.cmar. Au/mixrolgae) is separated and purified by adopting a stepwise dilution method and a solid plate method, and then cultured in f/2 seawater culture medium at the temperature of 25 ℃ and the light intensity of 100 mu mol phosns/s/m 2 irradiation, with an illumination period of 12:12, salinity of 30 per mill, and pH value of 7.8. Mutagenesis experiments were performed when the algae grew to the mid-exponential phase.
f/2 seawater culture medium contains the following substances in weight per 1L of seawater: 75 mg NaNO 3 5 mg of NaH 2 PO 4 ·H 2 O, 20 mg of Na 2 SiO 3 ·9H 2 O, 4.36 mg Na 2 EDTA, 3.16 mg FeCl 3 ·6H 2 O, 0.01 mg CuSO 4 ·5H 2 O, 0.023 mg ZnSO 4 ·7H 2 O, 0.012 mg CoCl 2 ·6H 2 O, 0.18 mg MnCl 2 ·4H 2 O, 0.07 mg Na 2 MoO 4 ·2H 2 O, 0.1 mg vitamin B1, 0.5 μ g vitamin B12, 0.5 μ g biotin.
(2) The method for mutagenizing the wild marine nannochloropsis oculata by using nitrosoguanidine with the concentration of 3.5g/L comprises the following steps:
collecting, cleaning and resuspending the algae solution in step (1), counting with a blood counting chamber, and preparing to obtain the solution with concentration of 1 × 10 6 cells/mL, and nitrosoguanidine is added to make nitrosoguanidine in the algae solutionThe concentration of the obtained product reaches 3.5g/L to obtain a reaction system;
placing the whole reaction system in a shaking table, carrying out shaking incubation for 1h at 300rpm under the dark condition at room temperature, then stopping the reaction by using phosphate buffer solution, centrifuging at 5000rpm for 10min, collecting algae liquid, carefully taking the supernatant, washing again by using phosphate buffer solution, and repeating for 2 times to remove residual nitrosoguanidine;
and (3) suspending the obtained algae cells in an f/2 culture medium, keeping the culture medium in the dark at room temperature overnight, coating the mutagenized algae cells on an f/2 solid plate the next day, observing and recording the growth condition of the cells every day, randomly selecting a group of monoclonal algae colonies from the solid plate to 50mL conical flasks for amplification culture when the monoclonal algae colonies grow on the solid plate, and naming the monoclonal algae colonies as marine nannochloropsis LAMB205.
The f/2 solid plate was prepared by mixing f/2 seawater culture medium with agar, wherein 10 g of agar was used per 1L of f/2 seawater culture medium.
(3) Culturing marine Nannochloropsis LAMB205 and wild marine Nannochloropsis in a light culture chamber at 25 deg.C under the intensity of 100 μmol photons/s/m 2 irradiation, illumination period 12:12 until the density of algae is 4.5X 10 7 cells/mL. Ciliate inhibition assays were performed in 24-well plates, 3 replicates were added to each well with 2mL of algal solution and 20 independent parasites, which were then incubated at 25 ℃ in the light until the parasite density reached a relatively steady state.
As shown in FIG. 1, in ciliate medium feeding on wild marine Nannochloropsis, the density of the wild type was increased from the initial 10ind/mL to 78ind/mL at the end of the index after 60 hours and finally maintained at an average density of 56 ind/mL. Compared with wild marine nannochloropsis, marine nannochloropsis LAMB205 has obvious inhibition effect on the growth of the paragonimus population, and during the propagation process of the paragonimus, the maximum density of ciliates reaches 26ind/mL, and during the plateau stage, the density of the paragonimus is maintained at about 10ind/mL.
Example 2
The marine Nannochloropsis oceanica LAMB204 was cultured in the same manner as in example 1, and the LAMB204 was obtained by mutagenesis of the wild Nannochloropsis oceanica isolated and purified in example 1 with ethyl methanesulfonate as a mutagen at 1mol/L, followed by washing with sodium thiosulfate solution and collecting the centrifuged supernatant as in example 1.
Culturing LAMB204 and LAMB205 to 4.5 × 10 7 When cells/mL, mixing the two algae solutions in equal volume to obtain the algaecide.
Ciliate inhibition assays were performed in 24-well plates, 3 replicates of 2mL of control algicide and 20 of myxobolus per well, followed by light culture at 25 ℃ until the myxobolus density reached a relatively steady state and control with LAMB204, LAMB205 alone as shown in figure 2. It can be seen that the combination of the two algal solutions has more stable effect of inhibiting the growth of the parasite population, the number of the parasites hardly increases exponentially in the process of propagation, and the number of the parasites is basically stabilized at 10ind/mL.
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 (7)
1. Sea nannochloropsis (A) (B)Nannochloropsis oceanica) The LAMB205 is characterized in that the preservation number of the marine nannochloropsis LAMB205 is CGMCC NO.20714, the preservation date is 2020, 10 and 23 days, the preservation unit is the common microorganism center of China Committee for culture Collection of microorganisms, the preservation address is No. 3 of the Beijing West Lu No. 1 of the morning-Yan district, the postal code: 100101.
2. the marine Nannochloropsis LAMB205 of claim 1, wherein the inhibition of the growth of the paracoccus animalis LAMB205 by the swarm is 66.67%.
3. A ciliate algae preventing and treating agent is characterized by comprising marine nannochloropsis (A) and (B)Nannochloropsis oceanica) LAMB204 and the marine Nannochloropsis oceanica LAMB205 of claim 1;
the marine nannochloropsis LAMB204 is named as marine nannochloropsisNannochloropsis oceanicaThe preservation number is CGMCC NO.20713, the preservation date is 2020, 10 and 23 days, the preservation unit is China general microbiological culture Collection center, the preservation address is No. 3 of Xilu No. 1 of Beijing, chaoyang, and the preservation address is as follows: 100101.
4. the ciliate control algicide of claim 3, wherein said control algicide is a mixed algae liquid of marine nannochloropsis LAMB204 as set forth in claim 3 and marine nannochloropsis LAMB205 as set forth in claim 1.
5. The ciliate control algicide of claim 4, wherein the control algicide is prepared by a method comprising:
separately culturing the marine Nannochloropsis LAMB204 of claim 3 and the marine Nannochloropsis LAMB205 of claim 1 at 20-30 deg.C under 100 μmol phos/s/m of illumination intensity 2 irradiation, with an illumination period of 12:12, when the density of the algae reaches 4 multiplied by 10 7 -5×10 7 After cells/mL, the two algae solutions were mixed in equal volumes.
6. The ciliate control algicide of claim 5, wherein the total density of nannochloropsis oceanica in the algicide is 4 x 10 7 -5×10 7 cells/mL。
7. Use of the ciliate control algicide of claim 3, in the preparation of a ciliate control medicament, wherein the ciliate is a slap.
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