TW201823445A - New mutant of bacillus thuringiensis and application thereof - Google Patents

Abstract

The present disclosure provides a new mutant of Bacillus thuringiensis which is deposited under Accession number BCRC 910754.

Description

 Novel Bacillus thuringiensis mutant strain and its application  

The present invention relates to a novel mutant strain of Bacillus thuringiensis and its use.

Because microalgae can effectively use light energy, carbon dioxide and inorganic salts to synthesize protein, fat, carbohydrate and a variety of high value-added biologically active substances, microalgae culture technology can be applied to raw fuel, health food, food additives. , feed and other chemicals.

In recent years, with the development of bioenergy technology, some oil-containing microalgae have high photosynthetic efficiency (about 20-60% of the dry weight of cells), high photosynthetic efficiency, short growth cycle, and can grow in different geographical environments. The advantages, and because it can absorb the carbon dioxide in the waste gas of the factory, while at the same time achieving the benefits of carbon reduction, all countries in the world have invested a lot of money and research and development energy to carry out research and development of related technologies.

However, large-scale, low-cost, high-efficiency access to micromass biomass is currently the biggest bottleneck in the industrialization of microalgae bioenergy. According to the analysis, the cost of obtaining the microalgae biomass quality accounts for about 60% of the overall biomass diesel production cost. Therefore, increasing the yield of microalgae and shortening the production time are the key technologies for improving the utilization of the microalgae industry.

Therefore, there is a need for a novel technique that promotes the growth of microalgae cells.

The present invention provides a novel mutant strain of Bacillus thuringiensis having the accession number BCRC 910754.

The present invention also provides a method for producing an accelerator for promoting microalgae cell growth, comprising: (a) inoculating a Bacillus thuringiensis mutant strain into a culture solution to obtain a bacterial suspension, wherein the Bacillus thuringiensis The Bacillus mutant strain has the accession number BCRC 910754; (b) the Bacillus thuringiensis mutant strain in the cell suspension is cultured to at least a stationary phase to obtain a cultured Bacillus thuringiensis mutant a culture solution in which the temperature of the Bacillus thuringiensis mutant strain is about 20-40 ° C; and (c) subjecting the culture solution of the Bacillus thuringiensis mutant strain to a reduced pressure heating program to obtain the promotion micro The promoter for growing algae cells, wherein the promoter for promoting the growth of the microalgae cells comprises an active substance for promoting the growth of the microalgae cells, wherein the heating temperature of the reduced pressure heating program is about 40-90 ° C, and the decompression The pressure of the heating procedure is about 10-400 mm Hg.

The present invention also provides an accelerator for promoting the growth of microalgae cells, which is produced by the above-described method for producing an accelerator for promoting growth of microalgae cells.

The invention further provides a method for promoting the growth of microalgae cells, comprising: 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.

The above and other objects, features, and advantages of the present invention will become more <

Figure 1 shows an embodiment of the present invention in Bacillus thuringiensis (registered at the Center for Biological Resource Conservation and Research of the Republic of China Food Industry Development Institute on October 22, 103, registration number BCRC 910651) The plastid with the transposon used in the transposon artificial mutagenesis process.

Figure 2 shows the effect of different concentrations of the microalgae cell growth promoter of the present invention 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 Chlorella vulgaris cultured from a basal medium containing urea and ammonium nitrogen as main nitrogen sources.

Fig. 3B shows the effect of the microalgae cell growth promoter of the present invention on the growth of Chlorella vulgaris cultured from a basal medium containing nitrate as a main nitrogen source.

Figure 4 shows the effect of the microalgae cell growth promoter of the present invention on the growth of chlorella.

Fig. 5 shows the effect of the microalgae cell growth promoter of the present invention on the growth of S. platensis.

Figure 6 is a graph showing the effect of the microalgae cell growth promoter of the present invention on the growth of Isochrysis.

Figure 7 is a graph showing the effect of the microalgae cell growth promoter of the present invention on the growth of Dunaliella.

In one aspect of the invention, the invention provides a novel mutant of Bacillus thuringiensis . The above mutant strain of Bacillus thuringiensis has the ability to promote the growth of microalgae cells, or to produce an active substance or a metabolite that promotes the growth of microalgae cells.

The microalgae cells described herein may be cells of single or multicellular microalgae of eukaryotic or prokaryotic cells, have cell walls, are feasible for photosynthesis, and can grow in fresh or salt water environment. Common microalgae include green algae and cyanobacteria. , brown algae, red algae, etc. Examples of the above microalgal cells may include, but are not limited to, Haematococcus pluvialis , Nannochloropsis sp. , Chlorella sp. , Tetraselmis sp. , etc. Isochrysis galbana and Dunaliella sp .

In one embodiment, the microalgae cell may be Haematococcus pluvialis. In another embodiment, the microalgal cell may be a Chlorella. In still another embodiment, the microalgal cell may be a chlorella. Further, in an embodiment, the microalgae cell may be a spirulina , and the spirulina may be Tetraselmis chui , but is not limited thereto. Further, in an embodiment, the microalgae cell may be Isochrysis. In addition, in an embodiment, the microalgae cell may be Dunaliella.

In one embodiment, the Bacillus thuringiensis mutant strain can be a mutant strain, which is mutated from Bacillus thuringiensis BCRC 910651 (on October 22, 103, deposited in the Center for Bioresource Conservation and Research of the Republic of China Food Industry Development Institute) , but not limited to this. The 16S rRNA gene of the above B. thuringiensis mutant strain may comprise a sequence having at least 95% sequence similarity to the sequence of sequence number: 1, but is not limited thereto.

In another embodiment, the B. thuringiensis mutant strain described above can be obtained via natural mutation or artificial mutagenesis. In a specific embodiment, the B. thuringiensis mutant strain is obtained by artificial transmutation of a transposon.

In a specific embodiment, the above-mentioned mutant strain of Bacillus thuringiensis can be deposited in the Biological Resource Preservation and Research Center of the Republic of China Food Industry Development Research Institute on November 23, 105, and the Bacillus thuringiensis mutant strain ITRI is registered as BCRC 910754. -BtM101. The above B. thuringiensis mutant ITRI-BtM101 can be obtained by transposon artificial mutagenesis of Bacillus thuringiensis BCRC 910651. Further, in this particular embodiment, the 16S rRNA gene of the B. thuringiensis mutant ITRI-BtM101 may be the sequence of sequence number: 1.

In another aspect of the invention, the invention provides a method of making an accelerator that promotes the growth of microalgal cells.

The microalgae cells described herein may be cells of single or multicellular microalgae of eukaryotic or prokaryotic cells, have cell walls, are feasible for photosynthesis, and can grow in fresh or salt water environment. Common microalgae include green algae and cyanobacteria. , brown algae, red algae, etc. Examples of the above microalgae cells may include, but are not limited to, Haematococcus pluvialis, Chlorella, Chlorella, cyanobacteria, Isochrysis, and Dunaliella.

In one embodiment, the microalgae cell may be Haematococcus pluvialis. In another embodiment, the microalgal cell may be a Chlorella. In still another embodiment, the microalgal cell may be a chlorella. Further, in an embodiment, the microalgae cell may be a spirulina, and the spirulina may be a kelp, but is not limited thereto. Further, in an embodiment, the microalgae cell may be Isochrysis. Further, in an embodiment, the microalgae cell may be Dunaliella.

The method for producing an accelerator for promoting microalgae cell growth of the present invention may include the following steps, but is not limited thereto.

First, a mutant strain of Bacillus thuringiensis is inoculated into a culture solution to obtain a bacterial suspension, and the mutant strain of Bacillus thuringiensis has the ability to promote the growth of microalgae cells, or can produce an active substance that promotes the growth of microalgae cells. Or metabolites.

In one embodiment, the B. thuringiensis mutant strain may be a mutant strain mutated from Bacillus thuringiensis BCRC 910651, but is not limited thereto. The 16S rRNA gene of the above B. thuringiensis mutant strain may comprise a sequence having at least 95% sequence similarity to the sequence of sequence number: 1, but is not limited thereto.

In another embodiment, the B. thuringiensis mutant strain described above can be obtained via natural mutation or artificial mutagenesis. In a specific embodiment, the B. thuringiensis mutant strain is obtained by artificial transmutation of a transposon.

In a specific embodiment, the above-mentioned mutant strain of Bacillus thuringiensis can be deposited in the Biological Resource Preservation and Research Center of the Republic of China Food Industry Development Research Institute on November 23, 105, and the Bacillus thuringiensis mutant strain ITRI is registered as BCRC 910754. -BtM101. The above B. thuringiensis mutant ITRI-BtM101 can be obtained by transposon artificial mutagenesis of Bacillus thuringiensis BCRC 910651. Further, in this particular embodiment, the 16S rRNA gene of the B. thuringiensis mutant ITRI-BtM101 may be the sequence of sequence number: 1.

Furthermore, the composition of the above culture solution is not particularly limited. In an embodiment, the components of the culture solution may include, but are not limited to, peptone, yeast extract, and the like. The concentration of the peptone in the culture solution may be about 0.5 to 10 g/L, and the concentration of the yeast extract in the culture solution may be about 0.1 to 5 g/L, but is not limited thereto. In a particular embodiment, the components of the culture fluid can include 5 g/L peptone and 3 g/L yeast extract.

Next, the B. thuringiensis mutant strain in the cell suspension is cultured at least to a stationary phase to obtain a culture solution in which the Bacillus thuringiensis mutant strain is cultured. In one embodiment, when the OD ₆₀₀ value of the bacterial cell suspension reaches 2.0 or more, the mutant strain of Bacillus thuringiensis in the bacterial suspension can be regarded as reaching a growth stable period. Further, in one embodiment, the colony of the B. thuringiensis mutant is cultured in the cell suspension for about 12-72 hours to allow the Bacillus thuringiensis mutant to grow to a stationary phase. In a specific embodiment, the Bacillus thuringiensis mutant strain is cultured for about 18 hours to allow the B. thuringiensis mutant to grow to a stationary phase.

Further, the temperature at which the B. thuringiensis mutant strain is cultured is about 20 to 40 ° C, but is not limited thereto. In one embodiment, the B. thuringiensis mutant is cultured at 30 °C.

Then, the above culture medium for culturing the B. thuringiensis mutant strain is subjected to a reduced pressure heating process to obtain an accelerator for promoting microalgae cell growth, wherein the promoter for promoting microalgae cell growth contains a microalgae promoting cell Active substance for growth.

The heating temperature of the above-described reduced pressure heating program may be about 40 to 90 ° C, but is not limited thereto. In one embodiment, the heating temperature can be about 40 ° C, about 50 ° C, about 60 ° C, about 75 ° C, or about 90 ° C. In a particular embodiment, the heating temperature of the reduced pressure heating program can be about 50 °C. Further, the pressure of the above-described reduced pressure heating program may be about 10 to 400 mmHg, but is not limited thereto. In one embodiment, the pressure can be about 10-50 mm Hg, about 50-100 mm Hg, about 100-200 mm Hg, about 200-300 mm Hg, or about 300-400 mm Hg. In a particular embodiment, the pressure of the reduced pressure heating program can be about 80 mm Hg.

In one embodiment, the heating temperature of the reduced pressure heating program may be about 50 ° C, and the pressure of the reduced pressure heating program may be about 80 mm Hg.

Furthermore, in one embodiment, the method for producing a promoter for promoting microalgae cell growth of the present invention, the step of obtaining a culture solution for culturing the mutant strain of Bacillus thuringiensis, and the culturing the above-mentioned cultured Bacillus thuringiensis The step of performing the step of heating and cooling the culture solution of the mutant strain may further include the step of removing the cells in the culture solution of the mutant strain of Bacillus thuringiensis.

The manner of removing the cells in the culture solution of the mutant strain of Bacillus thuringiensis is not particularly limited as long as the cells can be removed from the culture solution of the mutant strain of Bacillus thuringiensis, and the damage is not impaired. The component of the culture solution of the B. thuringiensis mutant strain may be cultured, for example, by centrifugation, filtration, or the like, but is not limited thereto.

In another embodiment, the invention also provides an enhancer that promotes the growth of microalgae cells. The accelerating agent for promoting the growth of microalgae cells of the present invention can be obtained by any of the above-described methods for producing a promoter for promoting microalgae cell growth of the present invention.

In yet another embodiment, the invention also provides a method of promoting the growth of microalgal cells. The method for promoting the growth of microalgae cells of the present invention described herein may include the following steps, but is not limited thereto.

First, the microalgal cells are cultured in the presence of any of the above-described promoters for promoting the growth of microalgae cells of the present invention to promote the growth of microalgae cells.

The microalgae cells described herein may be cells of single or multicellular microalgae of eukaryotic or prokaryotic cells, have cell walls, are feasible for photosynthesis, and can grow in fresh or salt water environment. Common microalgae include green algae and cyanobacteria. , brown algae, red algae, etc. Examples of the above microalgae cells may include, but are not limited to, Haematococcus pluvialis, Chlorella, Chlorella, Spirulina, Isochrysis, and Dunaliella.

In one embodiment, the microalgae cell may be Haematococcus pluvialis. In another embodiment, the microalgal cell may be a Chlorella. In still another embodiment, the microalgal cell may be a chlorella. Further, in an embodiment, the microalgae cell may be a spirulina, and the spirulina may be a kelp, but is not limited thereto. Further, in an embodiment, the microalgae cell may be Isochrysis. In addition, in an embodiment, the microalgae cell may be Dunaliella.

The manner in which the microalgae cells are cultured 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 only necessary to grow microalgae cells in the presence of a promoter for promoting the growth of microalgae cells or to cause The microalgae cells may be in contact with an accelerating agent that promotes the growth of microalgae cells. For example, an accelerator that promotes the growth of microalgae cells may be directly added to a solution or medium containing microalgae cells and cultured.

In one embodiment, the manner in which the microalgal cells are cultured in the presence of any of the above-described promoters for promoting the growth of microalgae cells of the present invention may include, but is not limited to, promoting the growth of the microalgae cells of the present invention. The agent is added to a solution or medium containing microalgae cells to form a mixed solution, and cultured.

The amount of the promoter for promoting the growth of the microalgae cells of the present invention is not particularly limited. For example, the promoter for promoting the growth of microalgae cells of the present invention may constitute 1-60% (v/v) of the above mixture, for example, 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 55%, 60%, but is not limited thereto.

In one embodiment, the method of the invention for promoting the growth of microalgal cells can increase the number of microalgae cells by at least about 5%. In one embodiment, the method of the invention for promoting the growth of microalgae cells can increase the number of microalgae cells by more than 50%.

Example

Example 1

Obtainment of mutant strain

Transposon artificial mutagenesis

The mutant strain of the present invention is translocated by Bacillus thuringiensis (registered at the Center for Biological Resource Conservation and Research of the Republic of China Food Industry Development Institute, registered number BCRC 910651 on October 22, 103). Transposon) obtained by artificial mutagenesis.

Transposons, also known as "jumping genes," are small pieces of DNA that can be transferred between molecules of DNA and can be transferred from plastid DNA to chromosomal DNA. The transfer behavior of transposons between genes often leads to the recombination of genes and the variability of cell functions.

The steps of artificial transmutation of transposons performed in this experiment are as follows:

1. Make competent cells

A single colony of Bacillus thuringiensis BCRC 910651 was selected and inoculated into LB medium and incubated overnight at 30 ° C with shaking at 150 rpm.

Next, the cultured bacterial liquid was transferred to fresh LB medium at a ratio of 1/200, and cultured at 30 ° C with shaking at 150 rpm until the OD ₅₅₀ value was 0.2, and then the bacterial liquid was centrifuged to collect the bacterial cells.

The obtained cells were resuspended in an electroporation buffer (400 mM sucrose, 1 mM MgCl ₂ , 7 mM phosphate buffer, pH 6.0) to wash the cells. The foregoing washing step was repeated twice, and the washing process was carried out at 4 ° C. Finally, the cells were resuspended in an electroporation buffer to be a competent cell of Bacillus thuringiensis.

2. Transposition of plastids

Transfer of Bacillus thuringiensis BCRC 910651 to transgenic plastids was performed according to the method described in the literature for DNA transfer of Bacillus thuringiensis via electroporation (Walter Schurter et. al, 1989).

The plastid with the transposon used in this experiment is shown in Figure 1. The plastids have antibiotics ampicillin and erythromycin resistance genes Amp ^R and Erm ^R , which can be used to screen for successful transgenic strains. Further, a resistance gene kan ^R having a kanamycin on the transposon can be used to screen a mutant strain in which transposition is completed.

The prepared Bacillus thuringiensis BCRC 910651 competent cells were mixed with the transposon plastids and then placed on ice for 10 minutes. Next, the mixture of competent cells and transposon plastids was transferred to a pre-cooled electroporation tube (electroporation cuvette), and the cells were electrically pulsed at a voltage of 1.3-2.0 kV, a capacitance of 25 μF, and a resistance of 100 Ω.

The cells subjected to the electric pulse were placed on ice for 10 minutes, and then LB medium was added to shake culture to repair and replicate the cells. After culturing for 2 hours, the cells were collected and uniformly spread on a medium plate containing 5 μg/ml of erythromycin, and cultured overnight to select a successful colonization strain.

3. Screening of transposon mutants

Since the transposon on the transposon plastid carries the antibiotic kanamycin resistance gene (kan ^R ), when the translocation occurs, the transposon carries the resistance gene of kansin (kan ^R ) Moves and inserts into the genomic DNA of the strain, and can subsequently screen for a strain that has been successfully transposon mutagenesis using kantromycin. Therefore, in order to screen the transposon mutant, the above-mentioned successful transgenic strain was spread on a medium plate containing 20 μg/ml of oxytetracycline, and cultured at 45 ° C to induce transposon-mediated random mutagenesis. Thereafter, the strain capable of growing on the medium plate containing 20 μg/ml of oxytetracycline but not against erythromycin was a mutant which was successfully transposed.

4. Screening of mutant strains that promote microalgae growth

Screening of mutant strains that promote microalgae growth using a microalgae solid culture plate.

First, a solid culture plate in which microalgae are uniformly distributed is prepared. The preparation method of the solid culture plate in which the microalgae is uniformly coated is as follows.

A microalgae medium having a gelatin content of 1.5% was plated on a culture plate and to be coagulated to serve as an underlying medium. In addition, the algae solution cultured to a stable phase is mixed with a medium having a 1% vegetable gum content to form a soft agar in a ratio of 1:1, and then uniformly poured onto the previously solidified medium to form A double layer of acacia gelatin culture plate.

The culture plate of the double layer of acacia gum was placed in a microalgae incubator, and cultured at a temperature of 25 ° C and a light/dark room for 12 hours until the double-layer culture plate showed a bright green color, indicating that the microalgae could be double layered. The growth of the culture plate was carried out, and the preparation of the solid culture plate in which the microalgae was uniformly distributed was completed.

Thereafter, the transposon mutant was inoculated to the aforementioned solid culture plate in which the microalgae were uniformly distributed. After the colony was formed, the degree of greenness around each colony was observed. Compared with the colony of Bacillus thuringiensis BCRC 910651, the surrounding green showed dark colonies, which is a strain having the ability to promote the growth of microalgae. Upon observation, it was found that a colony was darker than the colony of Bacillus thuringiensis BCRC 910651, so it was determined that the colony should be a strain capable of promoting the growth of microalgae, and the strain producing the colony was named as Suyun Golden Spore. Bacillus ITRI-BtM101.

The Bacillus thuringiensis ITRI-BtM101 was deposited in the Bioresource Conservation and Research Center of the Republic of China Food Industry Development Institute on November 23, 105, and the registration number is BCRC 910754.

5. Sequence of 16S rRNA gene

The sequence of the 16S rRNA gene was sequenced by B. thuringiensis ITRI-BtM101, and the sequence of the 16S rRNA gene was confirmed to be the sequence of sequence number: 1.

Example 2

Preparation of microalgae growth promoter

The strain of Bacillus thuringiensis ITRI-BtM101 was inoculated into a nutrient broth (peptone 5 g/L and yeast extract 3 g/L), and cultured at 150 rpm at 30 ° C. 72 hours.

After the completion of the culture, the culture solution of the cultured strain is centrifuged to remove the cells.

Next, 300 mL of the culture solution was separated and purified by a reduced pressure heating procedure, and 100 mL of a transparent solution was obtained as a microalgae growth promoter. The conditions for heating under reduced pressure are as follows: heating temperature: 50 ° C

Pressure: 80mmHg

Time: 2 hours.

Example 3

1. Promotion of the growth of Haematococcus pluvialis

The Haematococcus pluvialis was first activated to the green cell stage by a Haematococcus pluvialis culture medium. The composition of the above Haematococcus pluvialis culture medium is shown in Table 1, and the composition of the trace metal solution in the Haematococcus pluvialis culture medium is shown in Table 2.

In each experimental group, Haematococcus pluvialis culture medium was prepared by adding different volume percentage accelerators (the volume of the added accelerator was larger than the total volume of the experimental group), and the control group replaced the accelerator with sterile water. The control group and each experimental group are prepared as shown in Table 3 below:

Thereafter, the activated algae liquid was centrifuged to collect the algal bodies, and then inoculated to each of the above-mentioned groups of culture medium so that the initial culture concentration of each group of algae strains was 1×10 ⁵ CFU/mL.

The experimental group and the control group were cultured, and the number of cells was sampled and counted at different culture time points. The above experiment was repeated 3 times to obtain the mean and standard deviation of the number of cells of the control group and each experimental group at each time point.

The culture conditions were as follows: culture temperature: constant temperature culture at 24 ° C; gas introduction: 2% CO ₂ ; aeration rate: 0.2 vvm; stirring rate: 130 rpm; illumination time: 24 hours (light intensity receiving surface 16000 ± 1000 lux; backlight) Face 2600 ± 1000 lux).

The result is shown in Figure 2.

According to Fig. 2, the microalgae growth promoter of the present invention can be added in various volume percentages to promote the growth of Haematococcus pluvialis. Further, compared with the control group, the number of cells of Haematococcus pluvialis was more than doubled in the experimental group to which 20% and 30% of the promoter were added after the fourth day of culture.

2. Promotion of the growth of Chlorella

(1) Effect on the growth of Chlorella vulgaris cultured in a basal medium containing urea and ammonium nitrogen as main nitrogen sources.

The precultured Pseudomonas algae solution was inoculated into 500 mL of the Chlorella algae culture medium to have an OD ₆₈₅ value of 0.5 after inoculation, and the composition of the above Pseudococci medium was as shown in Table 4.

The experimental group contained 10% of the accelerator (the volume of the accelerator was larger than the total volume of the experimental group), and the control group was replaced with an equal amount of sterile water.

The experimental group and the control group were cultured, and the number of cells was sampled and counted at different culture time points. The above experiment was repeated 3 times to obtain the mean and standard deviation of the number of cells of the control group and the experimental group at each time point.

The culture conditions were as follows: culture temperature: constant temperature culture at 24 ° C; gas introduction: 2% CO ₂ ; aeration rate: 1.0 vvm; stirring rate: 150 rpm; illumination time: 24 hours (3500 lux).

The result is shown in Figure 3A.

According to Fig. 3A, the microalgae growth promoter of the present invention can promote the growth of Chlorella vulgaris cultured in a basal medium containing urea and ammonium nitrogen as main nitrogen sources.

(2) Effect on the growth of Chlorella vulgaris cultured from a basal medium containing nitrate as a main nitrogen source.

The pre-cultured Pseudomonas aeruginosa was inoculated into 500 mL of Chlorella algae culture medium to make the OD ₆₈₅ value of the algae liquid after inoculation 0.5, the composition of the above-mentioned Chlorella culture medium is shown in Table 5, and the trace amount in the algae culture medium The composition of the element solution is shown in Table 6.

The experimental group contained 10% of the accelerator (the volume of the accelerator was larger than the total volume of the experimental group), and the control group was replaced with an equal amount of sterile water.

The experimental group and the control group were cultured, and the number of cells was sampled and counted at different culture time points. The above experiment was repeated 3 times to obtain the mean and standard deviation of the number of cells of the control group and the experimental group at each time point.

The culture conditions were as follows: culture temperature: constant temperature culture at 24 ° C; gas introduction: 2% CO ₂ ; aeration rate: 1.0 vvm; stirring rate: 150 rpm; illumination time: 24 hours (3500 lux).

The result is shown in Figure 3B.

According to Fig. 3B, the microalgae growth promoter of the present invention can promote the growth of Chlorella vulgaris cultured in a basal medium containing nitrate as a main nitrogen source.

According to the results of the 3A and 3B, the microalgae growth promoter of the present invention is different for different Chlorella culture media, for example, the nitrogen source in the culture medium (for example, the nitrogen source is urea and ammonium nitrogen, or the nitrogen source). For nitrates, it can achieve the effect of promoting the growth of Chlorella.

3. Promotion of chlorella growth

The pre-cultured chlorella algae solution was inoculated into 500 mL of Chlorella culture medium to have an OD ₆₈₅ value of 0.5 after inoculation, and the composition of the above chlorella culture medium was the same as that of the Haematococcus pluvialis medium shown in Table 1. The composition of the trace element solution in the chlorella culture medium is the same as that of the trace metal solution of the Haematococcus pluvialis medium shown in Table 2.

The experimental group contained 10% of the accelerator (the volume of the accelerator was larger than the total volume of the experimental group), and the control group was replaced with an equal amount of sterile water.

The experimental group and the control group were cultured, and the OD ₆₈₅ value was measured at different culture time points. The above experiment was repeated 3 times to obtain the mean and standard deviation of the OD ₆₈₅ values of the control group and the experimental group at each time point.

The culture conditions were as follows: culture temperature: constant temperature culture at 24 ° C; gas introduction: 2% CO ₂ ; aeration rate: 1.0 vvm; stirring rate: 150 rpm; illumination time: 24 hours (10000 lux).

The result is shown in Figure 4.

According to Fig. 4, the microalgae growth promoter of the present invention can promote the growth of chlorella.

4. Promotion of the growth of cyanobacteria

The pre-cultured Cyanophyta algae solution was inoculated into 500 mL of the Cyanobacteria culture medium, and the OD ₆₈₅ value of the algae liquid after inoculation was 1.0, and the components of the above-mentioned Cyanobacteria culture medium and the components of the pseudocystis culture medium shown in Table 5 were prepared. Similarly, the components of the trace element solution in the Cyanobacteria culture medium are the same as those of the trace element solution of the Chlorella culture medium shown in Table 6.

The experimental group contained 5% of the accelerator (the volume of the accelerator was larger than the total volume of the experimental group), and the control group was replaced with an equal amount of sterile water.

The experimental group and the control group were cultured, and the OD ₆₈₅ value was measured at different culture time points. The above experiment was repeated 3 times to obtain the mean and standard deviation of the OD ₆₈₅ values of the control group and the experimental group at each time point.

The culture conditions were as follows: culture temperature: constant temperature culture at 24 ° C; gas introduction: 2% CO ₂ ; aeration rate: 1.0 vvm; stirring rate: 150 rpm; illumination time: 24 hours (3500 lux).

The result is shown in Figure 5.

According to Fig. 5, the microalgae growth promoter of the present invention can promote the growth of S. cerevisiae.

5. Promotion of growth of Isochrysis

The pre-cultured Isochrysis galbana solution was inoculated into 500 mL of Isochrysis algae culture medium, and the OD ₆₈₅ value of the algae liquid after inoculation was 1.0, and the composition of the isochrysis medium was the same as that of the Chlorella culture medium shown in Table 5. The composition of the trace element solution in the culture of Isochrysis is the same as that of the trace element solution of the Chlorella culture medium shown in Table 6.

The experimental group contained 5% of the accelerator (the volume of the accelerator was larger than the total volume of the experimental group), and the control group was replaced with an equal amount of sterile water.

The experimental group and the control group were cultured, and the OD ₆₈₅ value was measured at different culture time points. The above experiment was repeated 3 times to obtain the mean and standard deviation of the OD ₆₈₅ values of the control group and the experimental group at each time point.

The culture conditions were as follows: culture temperature: constant temperature culture at 24 ° C; gas introduction: 2% CO ₂ ; aeration rate: 1.0 vvm; stirring rate: 150 rpm; illumination time: 24 hours (3500 lux).

The result is shown in Figure 6.

According to Fig. 6, the microalgae growth promoter of the present invention can promote the growth of Isochrysis.

6. Promotion of growth of Dunaliella

The pre-cultured Dunaliella algae solution was inoculated into 500 mL of Dunaliella culture medium, and the OD ₆₈₅ value of the algae liquid after inoculation was 1.0, and the composition of the Dunaliella culture medium was the same as that of the Chlorella culture medium shown in Table 5, and the Dunaliella culture medium was used. The composition of the trace element solution in the medium is the same as that of the trace element solution of the rhodococcus medium shown in Table 6.

The experimental group contained 5% of the accelerator (the volume of the accelerator was larger than the total volume of the experimental group), and the control group was replaced with an equal amount of sterile water.

The experimental group and the control group were cultured, and the OD ₆₈₅ value was measured at different culture time points. The above experiment was repeated 3 times to obtain the mean and standard deviation of the OD ₆₈₅ values of the control group and the experimental group at each time point.

The culture conditions were as follows: culture temperature: constant temperature culture at 24 ° C; gas introduction: 2% CO ₂ ; aeration rate: 1.0 vvm; stirring rate: 150 rpm; illumination time: 24 hours (3500 lux).

The result is shown in Figure 7.

According to Fig. 7, the microalgae growth promoter of the present invention can promote the growth of Dunaliella.

While the present invention has been described in its preferred embodiments, the present invention is not intended to limit the invention, and the present invention may be modified and modified without departing from the spirit and scope of the invention. The scope of protection is subject to the definition of the scope of the patent application.

 【Biomaterial Storage】    Domestic registration information [please note according to the registration authority, date, number order]  

1. Bacillus thuringiensis ITRI-G1

Bioresource Conservation and Research Center of the Republic of China Food Industry Development Institute

October 22, 103, Republic of China

BCRC 910651

2. Bacillus thuringiensis mutant ITRI-BtM101

Bioresource Conservation and Research Center of the Republic of China Food Industry Development Institute

November 23, 105, Republic of China

BCRC 910754

<110> Institute of Industrial Technology

<120> Novel Bacillus thuringiensis mutant strain and its application

<130>

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 1504

<212> DNA

<213> Bacillus thuringiensis

<400> 1

Claims (19)

A novel mutant strain of Bacillus thuringiensis having the accession number BCRC 910754. A method for producing an accelerator for promoting microalgae cell growth, comprising: (a) inoculating a mutant strain of Bacillus thuringiensis into a culture solution to obtain a bacterial suspension, wherein the Bacillus thuringiensis mutant strain The storage number is BCRC 910754; (b) the Bacillus thuringiensis mutant strain in the cell suspension is cultured to at least one growth stable phase to obtain a culture solution in which the Bacillus thuringiensis mutant strain is cultured, wherein the Su Yunjin is cultured The temperature of the Bacillus mutant is about 20-40 ° C; and (c) the culture solution of the Bacillus thuringiensis mutant strain is subjected to a reduced pressure heating process to obtain the promoter for promoting the growth of the microalgae cell, wherein The promoter for promoting the growth of the microalgae cells comprises an active substance for promoting the growth of the microalgae cells, wherein the heating temperature of the reduced pressure heating program is about 40-90 ° C, and the pressure of the reduced pressure heating program is about 10 - 400mmHg.  The method for producing a promoter for promoting the growth of microalgae cells according to the second aspect of the invention, wherein the step (b) and the step (c) further comprises removing the cultured strain of the Bacillus thuringiensis mutant. The cells in the culture solution.    The method for producing a promoter for promoting the growth of microalgae cells according to the third aspect of the invention, wherein the method of removing the cells in the culture solution of the mutant strain of Bacillus thuringiensis comprises cultivating the Su Yunjin The culture solution of the Bacillus mutant strain is centrifuged or filtered.    A method for producing a promoter for promoting growth of microalgae cells according to claim 2, wherein the composition of the culture solution comprises peptone and yeast extract.    The method for producing a promoter for promoting microalgae cell growth according to the second aspect of the invention, wherein the strain of the Bacillus thuringiensis mutant in the cell suspension is cultured for at least one to about one hour to grow at least one to one. stable period.    The method for producing a promoter for promoting microalgae cell growth according to the second aspect of the invention, wherein the heating temperature of the reduced pressure heating program is about 50 ° C, and the pressure of the reduced pressure heating program is about 80 mmHg.    An accelerator for promoting the growth of microalgae cells is produced by the method for producing an accelerator for promoting microalgae cell growth as described in claim 2 of the patent application.    In the step (b) and the step (c), the step of removing the culture medium of the mutant strain of Bacillus thuringiensis is further included in the step (b) and the step (c). The bacteria.    The promoter for promoting the growth of microalgae cells according to claim 8, wherein the manner of removing the cells in the culture solution of the mutant strain of Bacillus thuringiensis comprises culturing the Bacillus thuringiensis mutant The culture solution of the strain is centrifuged or filtered.    The promoter for promoting the growth of microalgae cells according to claim 8, wherein the composition of the culture solution comprises peptone and yeast extract.    The promoter for promoting the growth of microalgae cells according to claim 8, wherein the strain of the Bacillus thuringiensis mutant in the cell suspension is cultured for about 12-72 hours to at least one growth stable phase.    An accelerator for promoting growth of microalgae cells according to the invention of claim 8, wherein the heating temperature of the reduced pressure heating program is about 50 ° C, and the pressure of the reduced pressure heating program is about 80 mmHg.    A method for promoting growth of microalgae cells, comprising: culturing microalgae cells in the presence of a promoter for promoting microalgae cell growth as described in claim 8 to promote microalgae cell growth.    The method for promoting the growth of microalgae cells according to claim 14, wherein the microalgae cells are cultured in the presence of the promoter for promoting microalgae cell growth, comprising: promoting the growth of the microalgae cells. The promoter is added to a solution or medium containing microalgae cells to form a mixed solution, and cultured.    The method for promoting the growth of microalgae cells according to claim 15, wherein the promoter for promoting the growth of the microalgae cells accounts for 1-60% (v/v) of the mixture.    The method for promoting the growth of microalgae cells according to claim 14, wherein the microalgae cell is a cell of a single cell or a multicellular microalgae of eukaryotic or prokaryotic cells, and the microalgal cell has a cell wall and has a line The ability to photosynthesis.   The method for promoting the growth of microalgae cells according to claim 17, wherein the microalgae cells include Haematococcus pluvialis , Nannochloropsis sp. , Chlorella sp. ), Tetraselmis sp. , Isochrysis galbana or Dunaliella sp . The method for promoting the growth of microalgae cells according to claim 18, wherein the spirulina is Tetraselmis chui .