CN108624521B - Novel bacillus thuringiensis mutant strain and application thereof - Google Patents

Novel bacillus thuringiensis mutant strain and application thereof Download PDF

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CN108624521B
CN108624521B CN201710161848.6A CN201710161848A CN108624521B CN 108624521 B CN108624521 B CN 108624521B CN 201710161848 A CN201710161848 A CN 201710161848A CN 108624521 B CN108624521 B CN 108624521B
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陈昌傑
白明德
吴劭易
郭欣慈
卢文章
万皓鹏
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Abstract

The invention provides a novel Bacillus thuringiensis mutant strain with the collection number DSM 32419.

Description

Novel bacillus thuringiensis mutant strain and application thereof
Technical Field
The invention relates to a novel Bacillus thuringiensis mutant strain and application thereof.
Background
Since microalgae can effectively utilize light energy, carbon dioxide and inorganic salts to synthesize proteins, fats, carbohydrates and various bioactive substances with high added values, the microalgae culture technology can be applied to the production of biofuels, health foods, food additives, feeds and other chemicals.
With the development of bioenergy technology in recent years, part of oil-containing microalgae has the advantages of high oil production capacity (about 20-60% of the dry weight of cells), high photosynthetic efficiency, short growth period, capability of growing in different regional environments and the like, and can absorb carbon dioxide in factory exhaust gas to achieve the benefit of carbon reduction, so that countries in the world do not invest a large amount of capital and research and development energy to research and develop related technologies.
However, obtaining microalgae biomass (bioglass) in large scale, low cost and high efficiency is the biggest bottleneck in the industrialization of microalgae bioenergy at present. According to analysis, the cost of obtaining microalgae biomass accounts for about 60% of the overall biodiesel production cost, so that the improvement of microalgae yield and the shortening of production time are key ways for improving the utilization of microalgae industry.
Therefore, a new technology capable of promoting the growth of microalgae cells is needed.
Disclosure of Invention
The invention provides a novel Bacillus thuringiensis mutant strain with the collection number DSM 32419.
The invention also provides a preparation method of the accelerant for promoting the growth of microalgae cells, which comprises the following steps: (a) inoculating a Bacillus thuringiensis (Bacillus thuringiensis) mutant strain into a culture solution to obtain a strain suspension, wherein the Bacillus thuringiensis mutant strain has a deposit number of DSM 32419; (b) culturing the bacillus thuringiensis mutant in the bacterial suspension at least until a stationary phase of growth to obtain a culture solution for culturing the bacillus thuringiensis mutant, wherein the temperature for culturing the bacillus thuringiensis mutant is about 20-40 ℃; and (c) subjecting the culture solution of the cultured bacillus thuringiensis mutant strain to a reduced pressure heating process to obtain a promoter for promoting the growth of microalgae cells, wherein the promoter for promoting the growth of microalgae cells contains active substances for promoting the growth of microalgae cells, and the heating temperature of the reduced pressure heating process is about 40-90 ℃, and the pressure of the reduced pressure heating process is about 10-400 mmHg.
The invention also provides a promoter for promoting the growth of microalgae cells, which is prepared by the preparation method of the promoter for promoting the growth of microalgae cells.
The invention also provides a method for promoting the growth of microalgae cells, which comprises the following steps: culturing the microalgae cells in the presence of the promoter for promoting the growth of the microalgae cells to promote the growth of the microalgae cells.
In order to make the aforementioned and other objects, features and advantages of the present invention comprehensible, preferred embodiments accompanied with figures are described in detail below.
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FIG. 1 shows a transposon-carrying plasmid used in the transposon (transposon) mutagenesis of Bacillus thuringiensis (deposited at the German Collection of microorganisms and strains, collection No. DSM29807, 12.11.2014) in one embodiment of the present invention.
FIG. 2 shows the effect of different concentrations of the inventive microalgae cell growth promoter on the growth of Haematococcus pluvialis.
FIG. 3A shows the effect of the microalgae cell growth promoter of the present invention on the growth of Synechococcus cultured on a basal medium containing urea and ammonium nitrogen as the main nitrogen sources.
FIG. 3B shows the effect of the microalgae cell growth promoter of the present invention on the growth of Synechococcus cultured on a basal medium containing nitrate as the main nitrogen source.
FIG. 4 shows the effect of the microalgae cell growth promoting agent of the present invention on the growth of chlorella.
FIG. 5 shows the effect of the microalgal cell growth promoters of the invention on the growth of Pantoea zhouensis.
FIG. 6 shows the effect of the growth promoter for microalgae cells on the growth of Isochrysis.
FIG. 7 shows the effect of the microalgae cell growth promoter of the present invention on the growth of Dunaliella.
Detailed Description
In one aspect of the present invention, the present invention provides a novel Bacillus thuringiensis (Bacillus thuringiensis) mutant strain. The Bacillus thuringiensis mutant strain has the capability of promoting the growth of microalgae cells, and can generate active substances or metabolites for promoting the growth of the microalgae cells.
The microalgae cells described herein can be eukaryotic or prokaryotic cells of unicellular or multicellular microalgae, have cell walls, are viable for photosynthesis, and can grow in freshwater or saltwater environments, and common microalgae include green algae, blue algae, brown algae, red algae, and the like. Examples of the microalgae cells may include, but are not limited to, Haematococcus pluvialis (Haematococcus pluvialis), Synechococcus sp, Chlorella sp, Tetraselis sp, Isochrysis galbana (Isochrysis galbana), Dunaliella sp, and the like.
In one embodiment, the microalgae cell can be Haematococcus pluvialis. In another embodiment, the microalgae cell can be a Synechococcus. In yet another embodiment, the microalgae cell can be chlorella. Also, in one embodiment, the microalgae cells may be Platymonas subcordiformis, and the Platymonas subcordiformis may be Tetraselmis chui (Tetraselmis chui), but is not limited thereto. In addition, in one embodiment, the microalgae cell can be Isochrysis. In addition, in one embodiment, the microalgae cell may be dunaliella.
In one embodiment, the bacillus thuringiensis mutant strain may be a mutant strain mutated from bacillus thuringiensis DSM29807 (deposited at the german collection of microorganisms and cultures 2014 12-11), but is not limited thereto. The 16S rRNA gene of the Bacillus thuringiensis mutant strain can at least comprise the following genes: 1 has a sequence similarity of at least 95%, but is not limited thereto.
In another embodiment, the mutant strain of bacillus thuringiensis can be obtained via natural mutation or artificial mutagenesis. In a particular embodiment, the mutant strain of bacillus thuringiensis is obtained by artificial mutagenesis with a transposon (transposon).
In a particular embodiment, the mutant strain of Bacillus thuringiensis may be the mutant strain ITRI-BtM101, deposited in the German collection of microorganisms and cultures at 30.1.2017 under the accession number DSM 32419. The Bacillus thuringiensis mutant ITRI-BtM101 was obtained by transposon (transposon) mutagenesis of Bacillus thuringiensis DSM 29807. Also, in this particular example, the 16S rRNA gene of the bacillus thuringiensis mutant strain ITRI-BtM101 can be seq id no: 1.
In another aspect of the present invention, the present invention provides a method for preparing a promoter for promoting the growth of microalgae cells.
The microalgae cells described herein can be eukaryotic or prokaryotic cells of unicellular or multicellular microalgae, have cell walls, are viable for photosynthesis, and can grow in freshwater or saltwater environments, and common microalgae include green algae, blue algae, brown algae, red algae, and the like. Examples of the microalgae cells may include, but are not limited to, haematococcus pluvialis, coccidioida, chlorella vulgaris, tetraselmis, isochrysis galbana, dunaliella and the like.
In one embodiment, the microalgae cell can be Haematococcus pluvialis. In another embodiment, the microalgae cell can be a Synechococcus. In yet another embodiment, the microalgae cell can be chlorella. Furthermore, in one embodiment, the microalgae cell may be Platymonas subcordiformis, and the Platymonas subcordiformis may be Platymonas sobria, but is not limited thereto. In addition, in one embodiment, the microalgae cell can be Isochrysis. In addition, in one embodiment, the microalgae cell may be dunaliella.
The preparation method of the promoter for promoting the growth of microalgae cells of the present invention may include the following steps, but is not limited thereto.
Firstly, inoculating a bacillus thuringiensis mutant strain into a culture solution to obtain a thallus suspension, wherein the bacillus thuringiensis mutant strain has the capability of promoting the growth of microalgae cells or can generate active substances or metabolites for promoting the growth of the microalgae cells.
In one embodiment, the bacillus thuringiensis mutant strain may be a mutant strain mutated from bacillus thuringiensis DSM29807, but is not limited thereto. The 16S rRNA gene of the Bacillus thuringiensis mutant strain can at least comprise the following genes: 1 has a sequence similarity of at least 95%, but is not limited thereto.
In another embodiment, the mutant strain of bacillus thuringiensis can be obtained via natural mutation or artificial mutagenesis. In a particular embodiment, the mutant strain of bacillus thuringiensis is obtained by artificial mutagenesis with a transposon (transposon).
In a particular embodiment, the mutant strain of Bacillus thuringiensis may be the mutant strain ITRI-BtM101, deposited in the German collection of microorganisms and cultures at 30.1.2017 under the accession number DSM 32419. The Bacillus thuringiensis mutant ITRI-BtM101 was obtained by transposon (transposon) mutagenesis of Bacillus thuringiensis DSM 29807. In this particular example, the 16S rRNA gene of the bacillus thuringiensis mutant strain ITRI-BtM101 can be seq id no: 1.
Moreover, the composition of the culture solution is not particularly limited. In one embodiment, the components of the culture solution may include peptone (peptone) and yeast extract (yeast extract), etc., but are not limited thereto. The concentration of the peptone in the culture solution may be about 0.5-10g/L, and the concentration of the yeast extract in the culture solution may be about 0.1-5g/L, but is not limited thereto. In a particular embodiment, the composition of the broth may include 5g/L peptone and 3g/L yeast extract.
Subsequently, the Bacillus thuringiensis mutant strain in the cell suspension is cultured at least until a stationary phase (stationary phase) of growth to obtain a culture solution in which the Bacillus thuringiensis mutant strain is cultured. In one embodiment, the OD of the bacterial suspension600When the value reaches above 2.0, the bacillus thuringiensis mutant strain in the strain suspension reaches the growth stable period. In one embodiment, a colony of a mutant strain of Bacillus thuringiensis is cultured in a bacterial suspension for about 12 to 72 hours to allow the mutant strain of Bacillus thuringiensis to grow to stationary phase. In a particular embodiment, the bacillus thuringiensis mutant strain is grown to stationary phase by culturing the bacillus thuringiensis mutant strain for about 18 hours.
The temperature for culturing the mutant strain of Bacillus thuringiensis is about 20-40 ℃, but not limited thereto. In one embodiment, the mutant strain of Bacillus thuringiensis is cultured at 30 ℃.
And then, carrying out a reduced pressure heating procedure on the culture solution for culturing the bacillus thuringiensis mutant strain to obtain the promoter for promoting the growth of the microalgae cells, wherein the promoter for promoting the growth of the microalgae cells contains active substances for promoting the growth of the microalgae cells.
The heating temperature of the reduced pressure heating process may be about 40 to 90 ℃, but is not limited thereto. In one embodiment, the heating temperature may be about 40 ℃, about 50 ℃, about 60 ℃, about 75 ℃, or about 90 ℃. In one particular embodiment, the heating temperature of the reduced pressure heating procedure may be about 50 ℃. The reduced pressure heating process may have a pressure of about 10-400mmHg, but is not limited thereto. In one embodiment, the pressure may be about 10-50mmHg, about 50-100mmHg, about 100-. In one particular embodiment, the reduced pressure heating process may have a pressure of about 80 mmHg.
In one embodiment, the heating temperature of the reduced pressure heating process may be about 50 ℃, and the pressure of the reduced pressure heating process may be about 80 mmHg.
In addition, in one embodiment, the method for preparing the promoter for promoting the growth of microalgae according to the present invention may further include a step of removing cells from the culture solution in which the mutant bacillus thuringiensis is cultured, between the step of obtaining the culture solution in which the mutant bacillus thuringiensis is cultured and the step of subjecting the culture solution in which the mutant bacillus thuringiensis is cultured to a reduced-pressure heating process.
The method for removing the bacterial cells from the culture medium in which the bacillus thuringiensis mutant strain is cultured is not particularly limited, as long as the bacterial cells can be removed from the culture medium in which the bacillus thuringiensis mutant strain is cultured without damaging the components of the culture medium in which the bacillus thuringiensis mutant strain is cultured, and the method can be performed by, for example, centrifugation, filtration, or the like, but is not limited thereto.
In another embodiment, the present invention also provides a promoter for promoting growth of microalgae cells. The promoter for promoting the growth of microalgae cells of the present invention can be obtained by any of the above-mentioned methods for preparing the promoter for promoting the growth of microalgae cells of the present invention.
In another embodiment, the invention also provides a method of promoting growth of a microalgae cell. The method for promoting the growth of microalgae cells according to the present invention described herein may include, but is not limited to, the following steps.
First, the microalgae cells are cultured in the presence of any of the above-mentioned promoters for promoting the growth of microalgae cells of the present invention to promote the growth of microalgae cells.
The microalgae cells described herein can be eukaryotic or prokaryotic cells of unicellular or multicellular microalgae, have cell walls, are viable for photosynthesis, and can grow in freshwater or saltwater environments, and common microalgae include green algae, blue algae, brown algae, red algae, and the like. Examples of the microalgae cells may include, but are not limited to, Haematococcus pluvialis, Haematococcus sp, Chlorella sp, Platymonas sp, Isochrysis sp, Dunaliella sp, and the like.
In one embodiment, the microalgae cell can be Haematococcus pluvialis. In another embodiment, the microalgae cell can be a Synechococcus. In yet another embodiment, the microalgae cell can be chlorella. Furthermore, in one embodiment, the microalgae cell may be Platymonas subcordiformis, and the Platymonas subcordiformis may be Platymonas sobria, but is not limited thereto. In addition, in one embodiment, the microalgae cell can be Isochrysis. In addition, in one embodiment, the microalgae cell may be dunaliella.
The method of culturing the microalgae cells in the presence of any of the above-described promoters for promoting the growth of microalgae cells of the present invention is not particularly limited, and it is sufficient if the microalgae cells are grown in the presence of the promoters for promoting the growth of microalgae cells or if the microalgae cells are in contact with the promoters for promoting the growth of microalgae cells, and for example, the promoters for promoting the growth of microalgae cells may be directly added to a solution or a culture medium containing the microalgae cells, and the solution or the culture medium may be cultured.
In one embodiment, the method of culturing the microalgae cells in the presence of any of the above-mentioned promoters for promoting the growth of microalgae cells of the present invention may include, but is not limited to, adding the promoter for promoting the growth of microalgae cells of the present invention to a solution or a culture medium containing the microalgae cells to form a mixed solution, and culturing.
The amount of the promoter for promoting the growth of microalgae cells of the present invention is not particularly limited, and for example, the promoter for promoting the growth of microalgae cells of the present invention may account for 1-60% (v/v) of the above-mentioned mixed solution, for example, 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 55%, 60%, but is not limited thereto.
In one embodiment, the method for promoting the growth of microalgae cells of the present invention can increase the number of microalgae cells by at least about 5%. In one embodiment, the method for promoting the growth of microalgae cells of the present invention can increase the number of microalgae cells by more than about 50%.
Examples
Example 1
Obtaining of mutant strains
Transposon (transposon) Artificial mutagenesis
The mutant strain of the present invention is obtained by artificially mutating Bacillus thuringiensis (deposited in the German Collection of microorganisms and cell cultures, accession number DSM29807, 12/11/2014) with a transposon (transposon).
Transposons, also known as "jumping genes", are small fragments of DNA that can be transferred between molecules of DNA, from plasmid DNA to chromosomal DNA. The action of transposons transferred between genes often results in recombination of genes, which results in a change in cell function.
The transposon mutagenesis procedure performed in this experiment was as follows:
1. making competent cells
A single colony of Bacillus thuringiensis (Bacillus thuringiensis) DSM29807 was picked and inoculated into LB medium and shake-cultured at 30 ℃ and 150rpm overnight.
Then, the cultured broth was transferred to a fresh LB medium at a ratio of 1/200 and cultured with shaking at 150rpm at 30 ℃ to OD550The value was 0.2, and the bacterial solution was centrifuged to collect the cells.
The obtained cells were treated with electroporation buffer (400mM sucrose, 1mM MgCl.)27mM phosphate buffer, pH 6.0) were resuspended to wash the cells. Repeating the cleaning step for 2 times, wherein the cleaning process is carried out at 4 ℃. Finally, the thalli are re-suspended uniformly by an electroporation buffer solution to be used as bacillus thuringiensis competent cells.
2. Transposon plasmid transfection
Transposon plasmid transfection of Bacillus thuringiensis DSM29807 was carried out according to the literature-described method for DNA transfection of Bacillus thuringiensis by electroporation (Walter Schurter et al, 1989).
The plasmid carrying the transposon used in this experiment is shown in FIG. 1. The plasmid has resistance genes Amp of antibiotics ampicillin (ampicillin) and erythromycin (erythromycin)R、ErmRIt can be used to select strains with successful transfection. The transposon has a resistance gene kan for kanamycin (kanamycin)RCan be used for screening the mutant strain with transposition (transposition) completed.
The competent cells of Bacillus thuringiensis DSM29807 prepared as described above were mixed with the transposon plasmid and then placed on ice for 10 minutes. Subsequently, the mixture of the competent cells and the transposon plasmid was transferred to a pre-cooled electroporation cuvette (electroporation cuvette) and the cells were subjected to electric pulses at a voltage of 1.3 to 2.0kV, a capacitance of 25. mu.F, and a resistance of 100. omega.).
The electrically pulsed thallus is set on ice for 10 min and then added into LB culture medium for vibration culture to repair and reproduce the thallus. After 2 hours of culture, the cells were collected and spread evenly on a medium plate (medium plate) containing 5. mu.g/ml of erythromycin, and cultured overnight to select strains that were successfully transfected.
3. Transposon mutant strain screening
Since the transposon on the transposon plasmid carries the resistance gene for kanamycin, an antibiotic (kan)R) Therefore, when transposition occurs, the transposon carries the kanamycin resistance gene (kan)R) Moved and inserted into the genomic DNA of the strain, and subsequently, kanamycin was used to screen for strains that succeeded in transposon mutagenesis (transposon mutagenisis). Therefore, for the selection of transposon mutants, the previously successfully transfected strains were spread on a medium plate containing 20. mu.g/ml kanamycin and cultured at 45 ℃ to induce transposon transposition random mutagenesis (transposon-mediated random mutagenesis). Thereafter, Can contain 20 u g/ml kanamycin medium plate growth anti-cardStrains of natamycin but not erythromycin are successful mutants of transposition (transfer).
4. Screening of mutant strains promoting microalgae growth
Screening of mutant strains for promoting microalgae growth is performed by using a microalgae solid culture disc.
Firstly, preparing a solid culture plate with uniformly distributed microalgae. The preparation method of the solid culture plate with microalgae uniformly coated is as follows.
A microalgal medium with an agar content of 1.5% was plated on a culture dish and allowed to solidify to serve as a lower medium. The algal solution cultured to the stationary phase was mixed with a medium containing 1% agar at a ratio of 1:1 to form soft agar (soft agar), and the soft agar was poured uniformly onto the solidified lower medium to form a culture plate of double-layered agar.
The culture plate of the double-layer agar is placed in a microalgae incubator and is circularly cultured in an illumination/dark room for 12 hours at the temperature of 25 ℃ until the double-layer culture plate is bright green, which shows that the microalgae can grow on the double-layer culture plate, and the preparation of the solid culture plate with uniformly distributed microalgae is completed.
And then, inoculating the transposon mutant strain to the solid culture plate with the microalgae uniformly distributed. And after the bacterial colonies are generated, observing the green shade degree around each bacterial colony, wherein compared with the bacterial colony of the bacillus thuringiensis DSM29807, the bacterial colony with darker green around is the bacterial strain with the capability of promoting the growth of the microalgae. It was observed that a colony exhibited a darker green color than that of Bacillus thuringiensis DSM29807, and thus the colony was identified as a strain having the ability to promote the growth of microalgae, and the strain producing the colony was named Bacillus thuringiensis ITRI-BtM 101.
The Bacillus thuringiensis ITRI-BtM101 was deposited in the German Collection of microorganisms and strains in 2017 at 30.1.9 under the accession number DSM 32419.
5.16 sequencing of the S rRNA Gene
16S rRNA gene sequencing is carried out on the Bacillus thuringiensis ITRI-BtM101, and the sequence of the 16S rRNA gene is confirmed to be the sequence number: 1.
Example 2
Preparation of microalgae growth promoter
A strain of Bacillus thuringiensis ITRI-BtM101 was inoculated into nutrient broth (nutrient broth) (peptone)5g/L and yeast extract (yeast extract)3g/L), and cultured with shaking at 150rpm at 30 ℃ for 72 hours.
After completion of the culture, the culture solution of the cultured strain is centrifuged to remove the cells.
Then, 300mL of the culture solution was separated and purified by a reduced pressure heating procedure, and 100mL of a clear solution was obtained as the microalgae growth promoter. The conditions for heating under reduced pressure were as follows:
heating temperature: 50 deg.C
Pressure: 80mmHg
Time: for 2 hours.
Example 3
1. Growth promotion of haematococcus pluvialis
The haematococcus pluvialis culture medium is used for activating the haematococcus pluvialis to a green cell stage. The composition of the culture medium for Haematococcus pluvialis is shown in Table 1, and the composition of the trace metal solution in the culture medium for Haematococcus pluvialis is shown in Table 2.
TABLE 1 composition of Haematococcus pluvialis culture Medium
Figure BDA0001248779270000111
Composition of trace metal solution of Haematococcus pluvialis culture Medium in tables 2 and 1
Composition (I) Per liter content
H3BO3 2.86g
MnCl2·4H2O 1.81g
ZnSO4·7H2O 0.222g
NaMoO4·2H2O 0.39g
CuSO4·5H2O 0.079g
Co(NO3)2·6H2O 49.4mg
The different volume percentages of the accelerant (the volume of the accelerant added is larger than the total volume of the experimental group) are added into each experimental group to prepare the haematococcus pluvialis culture medium, and the sterile water is used for replacing the accelerant in the control group. The formulations of the control group and each experimental group are shown in table 3 below:
TABLE 3 preparation of control and Experimental groups
Figure BDA0001248779270000121
Then, the activated algae liquid was centrifuged to collect algae, and then inoculated into the above-mentioned culture medium of each group so that the initial culture concentration of the algae strain of each group was 1 × 105CFU/mL。
The experimental group and the control group are cultured, and the number of cells is counted by sampling at different culture time points. The above experiment was repeated 3 times to obtain the mean and standard deviation of the number of cells at each time point of the control group and each experimental group.
The culture conditions were as follows:
the culture temperature is as follows: culturing at 24 deg.C;
introducing gas: 2% CO2；
Ventilation volume: 0.2 vvm;
stirring speed: 130 rpm;
illumination time: 24 hours (light intensity acceptance surface 16000 + -1000 lux; backlight surface 2600 + -1000 lux).
The results are shown in FIG. 2.
As can be seen from FIG. 2, the growth promoter for microalgae of the present invention can achieve the effect of promoting the growth of Haematococcus pluvialis when added in various volume percentages. When cultured up to day 4, the number of haematococcus pluvialis cells increased by more than 1-fold in the experimental groups to which 20% and 30% of the promoter was added, compared to the control group.
2. Growth promotion of Synechococcus
(1) Effect on the growth of Synechococcus algae cultivated on a basal Medium with Urea and ammonium Nitrogen as the principal Nitrogen Source
Inoculating the pre-cultured Synechococcus algal solution to 500mL Synechococcus culture Medium to obtain the inoculated algal solution OD685The value was 0.5, and the composition of the above-mentioned Synechococcus culture medium is shown in Table 4.
The experimental group contained 10% accelerator (volume of accelerator to total volume of experimental group) and the control group was replaced with an equal amount of sterile water.
The experimental group and the control group are cultured, and the number of cells is counted by sampling at different culture time points. The above experiment was repeated 3 times to obtain the mean and standard deviation of the number of cells at each time point of the control group and each experimental group.
The culture conditions were as follows:
the culture temperature is as follows: culturing at 24 deg.C;
introducing gas: 2% CO2；
Ventilation volume: 1.0 vvm;
stirring speed: 150 rpm;
illumination time: 24 hours (3500 lux).
TABLE 4 composition of Synechococcus culture Medium (Urea and ammonium Nitrogen as the main nitrogen sources)
Figure BDA0001248779270000131
The results are shown in FIG. 3A.
As can be seen from FIG. 3A, the microalgae growth promoter of the present invention can promote the growth of Synechococcus cultured in a basal medium containing urea and ammonium nitrogen as the main nitrogen sources.
(2) Effect on growth of Synechococcus sp cultured on a basal medium containing nitrate as the main nitrogen source.
Inoculating the pre-cultured Synechococcus algal solution to 500mL Synechococcus culture Medium to obtain the inoculated algal solution OD685The value was 0.5, and the composition of the above-mentioned chlorella culture medium is shown in Table 5, while the composition of the trace element solution in the chlorella culture medium is shown in Table 6.
The experimental group contained 10% accelerator (volume of accelerator to total volume of experimental group) and the control group was replaced with an equal amount of sterile water.
The experimental group and the control group are cultured, and the number of cells is counted by sampling at different culture time points. The above experiment was repeated 3 times to obtain the mean and standard deviation of the number of cells at each time point of the control group and each experimental group.
The culture conditions were as follows:
the culture temperature is as follows: culturing at 24 deg.C;
introducing gas: 2% CO2；
Ventilation volume: 1.0 vvm;
stirring speed: 150 rpm;
illumination time: 24 hours (3500 lux).
TABLE 5 composition of Synechococcus culture Medium (nitrate as the main nitrogen source)
Figure BDA0001248779270000141
The composition of the trace element solution of the Synechococcus culture Medium in tables 6 and 5
Composition (I) Per liter content
Na2EDTA·2H2O 4.4g
FeCl3·6H2O 3.2g
CuSO4·5H2O 0.01g
ZnSO4·7H2O 0.022g
CoCl2·6H2O 0.01g
MnCl2·4H2O 0.18g
Na2MoO4·2H2O 0.006g
The results are shown in FIG. 3B.
As can be seen from FIG. 3B, the microalgae growth promoter of the present invention can promote the growth of Synechococcus cultured in the basic medium containing nitrate as the main nitrogen source.
From the results shown in fig. 3A and fig. 3B, it can be seen that the microalgae growth promoter of the present invention can achieve the effect of promoting the growth of the nodulation algae for different nodulation algae culture mediums, for example, different nitrogen sources (such as urea and ammonium nitrogen, or nitrate) in the culture mediums.
3. Growth promotion of Chlorella
The pre-cultured chlorella solution was inoculated into 500mL of chlorella medium and the inoculated solution OD was adjusted685The value was 0.5, and the composition of the above-mentioned chlorella culture medium was the same as that of the haematococcus pluvialis culture medium shown in Table 1, while the composition of the trace element solution in the chlorella culture medium was the same as that of the trace metal solution in the haematococcus pluvialis culture medium shown in Table 2.
The experimental group contained 10% accelerator (volume of accelerator to total volume of experimental group) and the control group was replaced with an equal amount of sterile water.
The experimental group and the control group are cultured, and sampling is carried out at different culture time points to measure OD685The value is obtained. The above experiment was repeated 3 times to obtain the OD of the control group and the experimental group at each time point685Mean and standard deviation of the values.
The culture conditions were as follows:
the culture temperature is as follows: culturing at 24 deg.C;
introducing gas: 2% CO2；
Ventilation volume: 1.0 vvm;
stirring speed: 150 rpm;
illumination time: 24 hours (10000 lux).
The results are shown in FIG. 4.
As can be seen from FIG. 4, the microalgae growth promoter of the present invention can promote the growth of chlorella.
4. Promotion of growth of Pantoea zhou
The pre-cultured Platymonas subcordiformis solution was inoculated into 500mL of Platymonas subcordiformis medium, and the inoculated solution OD was added685Value of 1.0, aboveThe composition of the periwinkle medium was the same as that of the coccoid medium shown in Table 5, and the composition of the trace element solution in the periwinkle medium was the same as that of the trace element solution in the coccoid medium shown in Table 6.
The experimental group contained 5% accelerator (volume of accelerator to total volume of experimental group) and the control group was replaced with an equal amount of sterile water.
The experimental group and the control group are cultured, and sampling is carried out at different culture time points to measure OD685The value is obtained. The above experiment was repeated 3 times to obtain the OD of the control group and the experimental group at each time point685Mean and standard deviation of the values.
The culture conditions were as follows:
the culture temperature is as follows: culturing at 24 deg.C;
introducing gas: 2% CO2；
Ventilation volume: 1.0 vvm;
stirring speed: 150 rpm;
illumination time: 24 hours (3500 lux).
The results are shown in FIG. 5.
As can be seen from FIG. 5, the microalgae growth promoter of the present invention can promote the growth of Pantoea zhouensis.
5. Growth promotion of Isochrysis galbana
Inoculating the pre-cultured Isochrysis galbana solution into 500mL Isochrysis galbana culture medium, and allowing the inoculated solution OD685The value was 1.0, and the composition of the above-mentioned isochrysis medium was the same as that of the coccoid medium shown in Table 5, while the composition of the trace element solution in the isochrysis medium was the same as that of the trace element solution in the coccoid medium shown in Table 6.
The experimental group contained 5% accelerator (volume of accelerator to total volume of experimental group) and the control group was replaced with an equal amount of sterile water.
The experimental group and the control group are cultured, and sampling is carried out at different culture time points to measure OD685The value is obtained. The above experiment was repeated 3 times to obtain the OD of the control group and the experimental group at each time point685Mean and standard deviation of the values.
The culture conditions were as follows:
the culture temperature is as follows: culturing at 24 deg.C;
introducing gas: 2% CO2；
Ventilation volume: 1.0 vvm;
stirring speed: 150 rpm;
illumination time: 24 hours (3500 lux).
The results are shown in FIG. 6.
As can be seen from FIG. 6, the microalgae growth promoter of the present invention can promote the growth of Isochrysis
6. Growth promotion of Dunaliella
The pre-cultured Dunaliella algae solution was inoculated into 500mL of the Dunaliella algae medium to OD685The value was 1.0, and the composition of the dunaliella culture medium was the same as that of the coccoid culture medium shown in Table 5, while the composition of the trace element solution in the dunaliella culture medium was the same as that of the trace element solution in the coccoid culture medium shown in Table 6.
The experimental group contained 5% accelerator (volume of accelerator to total volume of experimental group) and the control group was replaced with an equal amount of sterile water.
The experimental group and the control group are cultured, and sampling is carried out at different culture time points to measure OD685The value is obtained. The above experiment was repeated 3 times to obtain the OD of the control group and the experimental group at each time point685Mean and standard deviation of the values.
The culture conditions were as follows:
the culture temperature is as follows: culturing at 24 deg.C;
introducing gas: 2% CO2；
Ventilation volume: 1.0 vvm;
stirring speed: 150 rpm;
illumination time: 24 hours (3500 lux).
The results are shown in FIG. 7.
As can be seen from FIG. 7, the microalgae growth promoter of the present invention can promote the growth of Dunaliella.
Although the present invention has been described with respect to the preferred embodiments, it is not intended to limit the present invention, and variations and modifications may be made by one skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Biological material preservation
1. Bacillus thuringiensis ITRI-G1
The preservation unit: deposited in the German Collection of microorganisms
Preservation time: 11 days 12 months 2014
The preservation number is: DSM29807
2. Bacillus thuringiensis mutant strain ITRI-BtM101
The preservation unit: deposited in the German Collection of microorganisms
Preservation time: 30 days 1 month in 2017
The preservation number is: DSM32419
Figure BDA0001248779270000191
Figure BDA0001248779270000201
Figure BDA0001248779270000211
[ sequence listing ]
<110> institute of Industrial and technology of financial group legal
<120> novel Bacillus thuringiensis mutant strain and application thereof
<130> TWGQ548-17P1
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1504
<212> DNA
<213> Bacillus thuringiensis (Bacillus thuringiensis)
<400> 1
gatgaacgct ggcggcgtgc ctaatacatg caagtcgagc gaatggatta agagcttgct 60
cttatgaagt tagcggcgga cgggtgagta acacgtgggt aacctgccca taagactggg 120
ataactccgg gaaaccgggg ctaataccgg ataacatttt gaaccgcatg gttcgaaatt 180
gaaaggcggc ttcggctgtc acttatggat ggacccgcgt cgcattagct agttggtgag 240
gtaacggctc accaaggcaa cgatgcgtag ccgacctgag agggtgatcg gccacactgg 300
gactgagaca cggcccagac tcctacggga ggcagcagta gggaatcttc cgcaatggac 360
gaaagtctga cggagcaacg ccgcgtgagt gatgaaggct ttcgggtcgt aaaactctgt 420
tgttagggaa gaacaagtgc tagttgaata agctggcacc ttgacggtac ctaaccagaa 480
agccacggct aactacgtgc cagcagccgc ggtaatacgt aggtggcaag cgttatccgg 540
aattattggg cgtaaagcgc gcgcaggtgg tttcttaagt ctgatgtgaa agcccacggc 600
tcaaccgtgg agggtcattg gaaactggga gacttgagtg cagaagagga aagtggaatt 660
ccatgtgtag cggtgaaatg cgtagagata tggaggaaca ccagtggcga aggcgacttt 720
ctggtctgta actgacactg aggcgcgaaa gcgtggggag caaacaggat tagataccct 780
ggtagtccac gccgtaaacg atgagtgcta agtgttagag ggtttccgcc ctttagtgct 840
gaagttaacg cattaagcac tccgcctggg gagtacggcc gcaaggctga aactcaaagg 900
aattgacggg ggcccgcaca agcggtggag catgtggttt aattcgaagc aacgcgaaga 960
accttaccag gtcttgacat cctctgaaaa ccctagagat agggcttctc cttcgggagc 1020
agagtgacag gtggtgcatg gttgtcgtca gctcgtgtcg tgagatgttg ggttaagtcc 1080
cgcaacgagc gcaacccttg atcttagttg ccatcattaa gttgggcact ctaaggtgac 1140
tgccggtgac aaaccggagg aaggtgggga tgacgtcaaa tcatcatgcc ccttatgacc 1200
tgggctacac acgtgctaca atggacggta caaagagctg caagaccgcg aggtggagct 1260
aatctcataa aaccgttctc agttcggatt gtaggctgca actcgcctac atgaagctgg 1320
aatcgctagt aatcgcggat cagcatgccg cggtgaatac gttcccgggc cttgtacaca 1380
ccgcccgtca caccacgaga gtttgtaaca cccgaagtcg gtggggtaac ctttttggag 1440
ccagccgcct aaggtgggac agatgattgg ggtgaagtcg taacaaggta gccgtatcgg 1500
aagg 1504

Claims (19)

1. A novel Bacillus thuringiensis mutant strain deposited at the German Collection of microorganisms and strains with the accession number DSM 32419.
2. A method for preparing a promoter for promoting growth of microalgae cells comprises the following steps:
(a) inoculating a bacillus thuringiensis mutant strain into a culture solution to obtain a strain suspension, wherein the bacillus thuringiensis mutant strain has a preservation number of DSM 32419;
(b) culturing the mutant strain of Bacillus thuringiensis in the bacterial suspension at least until the growth stationary phase to obtain a culture solution for culturing the mutant strain of Bacillus thuringiensis, wherein the temperature for culturing the mutant strain of Bacillus thuringiensis is 20-40 ℃; and
(c) and (2) carrying out a reduced pressure heating procedure on the culture solution for culturing the bacillus thuringiensis mutant strain to obtain the promoter for promoting the growth of the microalgae cells, wherein the promoter for promoting the growth of the microalgae cells contains active substances for promoting the growth of the microalgae cells, the heating temperature of the reduced pressure heating procedure is 40-90 ℃, and the pressure of the reduced pressure heating procedure is 10-400 mmHg.
3. The method according to claim 2, further comprising removing the microbial cells from the culture medium in which the Bacillus thuringiensis mutant strain is cultured, between the step (b) and the step (c).
4. The method for producing a promoter for promoting the growth of microalgae according to claim 3, wherein the means for removing the microbial cells from the culture solution in which the Bacillus thuringiensis mutant strain is cultured comprises centrifuging or filtering the culture solution in which the Bacillus thuringiensis mutant strain is cultured.
5. The method of claim 2, wherein the culture medium comprises peptone and yeast extract.
6. The method for producing an accelerator for accelerating the growth of microalgae cells according to claim 2, wherein the mutant strain of Bacillus thuringiensis is cultured in the cell suspension for 12 to 72 hours at least until the stationary phase of growth.
7. The method of preparing an enhancer for promoting growth of microalgae cells according to claim 2, wherein the heating temperature of the reduced-pressure heating process is 50 ℃ and the pressure of the reduced-pressure heating process is 80 mmHg.
8. An enhancer for promoting the growth of microalgae cells, which is prepared by the method for preparing the enhancer for promoting the growth of microalgae cells according to claim 2.
9. The promoter for promoting the growth of microalgae according to claim 8, further comprising removing thallus from the culture solution in which the Bacillus thuringiensis mutant strain is cultured between the step (b) and the step (c).
10. The promoter for promoting the growth of microalgae according to claim 8, wherein the means for removing the biomass from the culture solution in which the mutant Bacillus thuringiensis strain is cultured comprises centrifuging or filtering the culture solution in which the mutant Bacillus thuringiensis strain is cultured.
11. The promoter for promoting the growth of microalgae cells according to claim 8, wherein the components of the culture solution include peptone and yeast extract.
12. An enhancer for promoting growth of microalgae cells as claimed in claim 8, wherein the mutant strain of Bacillus thuringiensis is cultured in the bacterial suspension for 12-72 hours to at least the stationary phase of growth.
13. An enhancer for promoting the growth of microalgae cells according to claim 8, wherein the heating temperature of the reduced-pressure heating process is 50 ℃ and the pressure of the reduced-pressure heating process is 80 mmHg.
14. A method of promoting growth of a microalgae cell, comprising:
culturing the microalgae cell in the presence of the promoter for promoting the growth of the microalgae cell of claim 8 to promote the growth of the microalgae cell.
15. The method of promoting the growth of microalgae cells according to claim 14, wherein the culturing the microalgae cells in the presence of the promoter for promoting the growth of microalgae cells comprises adding the promoter for promoting the growth of microalgae cells to a solution or a culture medium containing microalgae cells to form a mixture, and culturing.
16. The method for promoting the growth of microalgae cells of claim 15, wherein the promoter for promoting the growth of microalgae cells comprises 1-60% (v/v) of the mixed solution.
17. The method for promoting the growth of microalgae cells of claim 14, wherein the microalgae cells are eukaryotic or prokaryotic cells of unicellular or multicellular microalgae, and the microalgae cells have cell walls and are capable of photosynthesis.
18. The method of promoting growth of a microalgae cell of claim 17 wherein the microalgae cell comprises Haematococcus pluvialis (Haematococcus pluvialis), coccidioides (Nannochloropsis sp.), Chlorella vulgaris (Chlorella sp.), Tetraselmis galbana (Tetraselmis sp.), Isochrysis galbana (Isochrysis galbana), or Dunaliella salina (Dunaliella sp.).
19. The method for promoting growth of microalgae cells of claim 18, wherein the Tetraselmis periwinia (Tetraselmis chui).
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Publication number Priority date Publication date Assignee Title
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Publication number Priority date Publication date Assignee Title
CN105985986A (en) * 2014-12-18 2016-10-05 财团法人工业技术研究院 Active substance for inducing autolysis of microalgae cell and method for producing same

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Title
溶藻细菌MS7的分类鉴定及其溶藻活性的初步研究;黄晶晶等;《海洋与湖沼》;20130531;第44卷(第3期);第632-637页 *

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