JP2019050731A - Method for promoting growth of organisms performing oxygen-generating photosynthesis - Google Patents

Abstract

To provide a method for promoting growth of organisms performing oxygen-generating photosynthesis, by an inexpensive, safe and easy process.SOLUTION: A culture promoter containing a pyrrole compound is added to a culture medium where organisms performing oxygen-generating photosynthesis are grown, and the culture medium is held in organism growth conditions. Here, the pyrrole compound preferably contains a tetrapyrrole compound, and as the organisms performing oxygen-generating photosynthesis, algae can be used.SELECTED DRAWING: None

Description

The present invention relates to a method for promoting the growth of an organism that performs oxygen-generating photosynthesis.

An organism that performs oxygen-generating photosynthesis excluding moss, fern, and seed plants (thus, an organism that performs oxygen-generating photosynthesis that exists in water) is called an algae, and it has a microscopic size (micron unit). Algae are called microalgae, and about 100,000 species live in diverse environments. In recent years, microalgae have a high ability to fix carbon dioxide by photosynthesis, a high ability to produce useful substances (eg, lipids) as raw materials for foods, pharmaceuticals, and biofuels, and can be harvested throughout the year. For these reasons, it is attracting attention as a biomass resource.

Here, the production amount of useful substances by microalgae is proportional to the product of the content of useful substances contained in each microalgae and the number of individuals of microalgae. However, there are problems that the growth rate of microalgae is slow under natural conditions and the production efficiency of useful substances is low. For this reason, it is necessary to artificially improve the production efficiency of useful substances using microalgae and the growth rate of microalgae (mass culture) for the practical use (use on a commercial basis) of the production of useful substances using microalgae. In particular, increasing the efficiency of mass culture of microalgae (that is, improving the economic efficiency of the culture process) is a major issue.

Various approaches such as screening of microalgae and genetic recombination of microalgae have been reported in Japan and overseas for the practical application of the production of useful substances by mass culture of microalgae. However, there is a problem that it takes a long time and a lot of money to select promising species of microalgae having a high growth rate by screening. On the other hand, when a genetically modified strain is cultured outdoors, there are various limitations, and there are many unclear points regarding the influence on the environment when the genetically modified strain is scattered outside the culture site.
Therefore, using microalgae that are expected to have high production efficiency of useful substances, add chemical substances that promote growth to the culture medium, and cultivate microalgae in large quantities to achieve practical production of useful substances. Has been proposed (see, for example, Non-Patent Documents 1 to 3).

Reijiro Nogami, Minato Wakisaka, “Promotion of Spirulina Growth by Addition of Alginate Oligomer”, 17th Annual Meeting of the Marine Biotechnology Society, May 30, 2015, p. 109 Reijiro Nogami, Minato Wakisaka, “Promoting Euglena Growth by Addition of Alginate Oligomers”, Abstracts of the 16th Annual Meeting of the Marine Biotechnology Society, May 31, 2014, p. 61 Reijiro Nogami, Minato Wakisaka, CHE MAAIL Che Mohd Hakiman “Promoting Botryococcus Growth by Addition of Alginate Oligomers”, Proceedings of Annual Conference of Ecological Engineering Society 2014, p. 83-84

In order to commercialize useful substances by mass cultivation of microalgae, it is said that the growth rate of microalgae needs to be improved at least 2 to 3 times the current growth rate. The methods described in 1 to 3 have not led to further improvement of the growth of microalgae. For this reason, when culture conditions that can maintain a high growth rate of microalgae and can be stably cultured have been found, an inexpensive and safe effect that further promotes the growth rate of microalgae (impact on the environment is affected). There is a need for new chemical substances that are easy to use.

The present invention has been made in view of such circumstances, and an object of the present invention is to provide a method for promoting the growth of a living body that performs oxygen-generating photosynthesis that can be achieved by an inexpensive, safe and simple technique.

The present inventors have found that chlorophyll chlorophyll responsible for oxygen-generating photosynthesis has a tetrapyrrole ring structure in which four five-membered ring structures (pyrrole ring structures) of the molecular formula C ₄ H ₅ N are bonded with one carbon atom in between. Since it is a compound having one chlorin ring, it is hypothesized that if microalgae are cultured in a medium containing a pyrrole compound as a raw material for chlorophyll chlorophyll, photosynthesis is promoted to promote microalgae growth. As a result of various studies based on the above, as a result of adding a pyrrole compound containing a tetrapyrrole compound obtained by bioproduction by Escherichia coli described in WO2009 / 069806 to a microalgae culture medium, the promotion of microalgae growth is inexpensive. The inventors have found that this can be achieved by a safe and simple technique and have completed the present invention.
That is, in the method for promoting the growth of a living organism that performs oxygen-generating photosynthesis according to the present invention, a culture promoter containing a pyrrole compound is added to a medium in which a living organism that performs oxygen-generating photosynthesis grows, and the medium is propagated. This is done by maintaining the conditions.

In the method for promoting the growth of an organism that performs oxygen-generating photosynthesis according to the present invention, the pyrrole compound contained in the culture promoter is safe because it is part of the structure of the chlorolin ring of chlorophyll chlorophyll (it exists in nature). This has no impact on the environment. Then, the culture promoter is simply added to the culture medium of the organism that performs oxygen-generating photosynthesis, and the culture medium to which the culture promoter is added is kept under the growth conditions of the organism that performs oxygen-generated photosynthesis. Growth of organisms that perform developmental photosynthesis can be performed. As a result, it is possible to carry out mass growth of organisms that perform oxygen-generating photosynthesis by an inexpensive, safe and simple method.

It is explanatory drawing of the culture result of Euglena in Example 1. It is explanatory drawing of the culture result of Botryococcus in Example 2. It is explanatory drawing of the culture result of Spirulina in Example 3.

The method for promoting the growth of a living organism that performs oxygen-generating photosynthesis according to an embodiment of the present invention includes, for example, using a pyrrole compound obtained by bioproduction using Escherichia coli in which the gene ypjD (b2611) cannot be expressed due to a mutation, It is added to a medium in which microalgae, which are examples of organisms that perform type photosynthesis, grow, and the medium is maintained under the microalgae growth conditions.
In addition, since the pyrrole compound can be obtained in a large amount at a low cost in the bioproduction using Escherichia coli, the culture promoter can be produced at a low cost.

The microalgae cultured by the method of the present invention preferably carry out oxygen-generating photosynthesis and preferably contain chlorophyll as a main photosynthesis pigment. For example, prokaryotic cyanobacteria, gray plants in eukaryotic supergroups , Green plants, crypto algae, unequal hairy plants, hapto algae, dinoflagellates, euglena algae, chloracarnion algae, chromella algae and the like.
The growth conditions for microalgae may be general growth conditions that have been conventionally recommended for each microalgae (conditions that allow stable growth while maintaining a high growth rate).

E. coli is cultured in an oligotrophic medium. At this time, Escherichia coli is precultured in a suitable medium other than the oligotrophic medium, for example, a medium such as LB medium (after culturing at a temperature of 15 to 40 ° C. for 6 to 24 hours), and the obtained preculture is obtained. It is preferable to inoculate an oligotrophic medium and perform main culture (culture at a temperature of 20 ° C. to 40 ° C. for 12 hours to 96 hours). The culture conditions for E. coli may be general conditions for E. coli.
Incidentally, as a poor nutrient medium, e.g., deionized water in 1 liter, a _{KH 2 PO 4 9g, K 2} HPO 4 to _21g, 2 g of _{(NH 4)} 2 SO _4, citric acid dihydrate 1g, An aqueous solution in which 3.6 g of glucose and 100 mg of MgSO ₄ are dissolved can be used.

During the cultivation of E. coli, pyrrole compounds (cultured products) such as tetrapyrrole compounds and tetrapyrrole compound precursors are discharged out of the cells from the E. coli, and the discharged pyrrole compounds are mixed in the poor nutrient medium. For this reason, the oligotrophic medium after the cultivation of E. coli is centrifuged to precipitate the suspension (aggregates of E. coli cells), and the resulting supernatant is filtered through a filter (pore size 0.22 μm). A culture promoter containing a pyrrole compound from which E. coli has been removed can be obtained.
In addition, the density | concentration of the pyrrole compound in a culture promotion agent can be adjusted to arbitrary values by adjusting the moisture content in a culture promotion agent. Thereby, the content of the pyrrole compound in the culture medium (more specifically, the content of the nitrogen compound containing the pyrrole compound) when the culture promoter is added to the culture medium can be easily adjusted.

Example 1
Euglena (NIES-48 strain) was cultured. For cultivation, add Euglena with a constant weight to 10 ml of medium injected into a 30 ml screw cap test tube, leave the screw cap test tube in an incubator, and adjust the temperature to 22 to 26 ° C. while ventilating air. The test was carried out for 35 days under conditions of a light-dark cycle using a white fluorescent lamp with an illuminance of 6000 to 6500 Lux, and a white fluorescent lamp having a turn-on time and a turn-off time of 12 hours, respectively. The screw cap test tube was shaken every other day.

Medium is A1 (10 ml of AF-6 medium), A2 (mixture of 9 ml of AF-6 medium and 1 ml of culture accelerator), A3 (mixture of 5 ml of AF-6 medium and 5 ml of culture accelerator), And A4 (10 ml of culture accelerator). The composition per liter of AF-6 medium is as follows: NaNO ₃ is 140 mg, NH ₄ NO ₃ is 22 mg, MgSO ₄ .7H ₂ O is 30 mg, KH ₂ PO ₄ is 10 mg, K ₂ HPO ₄ is 5 mg, CaCl ₂ 2H ₂ O 10 mg, Fe-citrate 2 mg, Citric acid 2 mg, Biotin 2 μg, Thiamine HCl 2 μg, Vitamin B ₆ 10 μg, Vitamin B ₁₂ 1 μg, Na ₂ EDTA · 2H ₂ O 5 mg, FeCl ₆ 0.98 mg 6H ₂ O, 180 μg MnCl ₂ .4H ₂ O, 52 μg ZnCl ₂ , 20 μg CoCl ₂ .6H ₂ O, 12.5 μg Na ₂ MoO ₄ .2H ₂ O, 1 liter Distilled Water is there. In addition, the content of the nitrogen compound including the pyrrole compound in the culture promoter is 560 mg / liter, which includes pyrrole compound having a porphyrin ring structure of 16 mg / liter.

5 ml of each cell suspension from A1 to A4 after culturing was filtered using a φ25 GF / C filter inserted in a Sweeness filter holder, and the used filter paper taken out from the GF / C filter after filtration was removed from the petri dish. It was put in and it was made to dry for 5 hours with a 105 degreeC drying cabinet. After drying, the petri dish containing the used filter paper was transferred into a desiccator and cooled, and the weight of the petri dish containing the used filter paper after cooling was measured. Then, the dry weight of Euglena present in the 5 ml cell suspension was calculated by subtracting the dry weight of the filter paper and the dry weight of the petri dish previously obtained from the weight of the petri dish containing the used filter paper. . FIG. 1 shows the dry weight of Euglena contained in each 5 ml cell suspension collected from A1 to A4.

As shown in FIG. 1, it can be confirmed that all the media A2 to A4 to which a culture accelerator is added show a large growth rate compared to the growth rate when growing on A1 (AF-6 medium only). In the medium (A2) diluted 10-fold with the promoter, it was doubled, in the medium (A3) diluted twice with the culture promoter, it was 7.7 times, and in the medium (A4) with only the culture promoter, it was 6.7 times. .
In addition, FIG. 1 shows that there is an optimum value for the dilution rate of the culture promoter contained in the medium that increases the growth rate of Euglena (content of nitrogen compound including pyrrole compound). It is not preferable to add an excessive amount of the nitrogen compound containing, which leads to an increase in the cost of the culture promoter.

(Example 2)
Botryococcus (NIES-2199 strain) was cultured. For cultivation, a certain weight of Botryococcus was added to 10 ml of medium injected into a 30 ml screw cap test tube, the screw cap test tube was left still in an incubator, and the temperature was adjusted to 22 to 26 while ventilating air. It was performed for 65 days under the conditions of a light-dark cycle in which the illuminance was 6000 to 6500 Lux using a white fluorescent lamp and the lighting time and extinguishing time of the white fluorescent lamp were 12 hours each. The screw cap test tube was shaken every other day.

There are four types of culture media: B1 (BG11 medium is 10 ml), B3 (5 ml of BG11 medium and 5 ml culture accelerator), and B4 (10 ml culture accelerator). The composition per liter of BG11 medium is 1500 mg NaNO ₃ , 40 mg KH ₂ PO ₄ .3H ₂ O, 75 mg MgSO ₄ .7H ₂ O, 36 mg CaCl ₂ .2H ₂ O, and 6 mg Ferric ammonium citrate. , Citric acid 6 mg, Na ₂ EDTA-Mg 1 mg, Na ₂ CO ₃ 20 mg, H ₃ BO ₃ 2.86 mg, MnCl ₂ .4H ₂ O 1.81 mg, ZnSO ₄ .7H ₂ O 0. 222 mg, Na ₂ MoO ₄ .2H ₂ O is 0.39 mg, CuSO ₄ .5H ₂ O is 79 μg, Co (NO ₃ ) ₂ .6H ₂ O is 49 μg, and Distilled Water is 1 liter. In addition, the content of the nitrogen compound including the pyrrole compound in the culture promoter is 560 mg / liter, which includes pyrrole compound having a porphyrin ring structure of 16 mg / liter.

5 ml cell suspensions from each of B1, B3, and B4 after culturing were filtered using a φ25 GF / C filter inserted into a Sweeness filter holder, and 5 ml of cell suspension was filtered in the same manner as in Example 1. The dry weight of Botryococcus present in the cell suspension was calculated. FIG. 2 shows the dry weight of Botryococcus contained in 5 ml of cell suspension collected from B1, B3, and B4.

As shown in FIG. 2, it can be confirmed that the mediums B3 and B4 to which the culture promoter is added have a large growth rate compared to the growth rate in the case of growing on B1 (BG11 medium only). It was 23 times in the medium (B3) diluted twice, and 5 times in the medium (B4) containing only the culture accelerator.
FIG. 2 also shows that there is an optimum value for the dilution ratio of the culture promoter that increases the growth rate of Botryococcus (content of nitrogen compound including pyrrole compound). Adding an excessive amount of the compound is not preferable because it leads to an increase in the cost of the culture promoter.

(Example 3)
Spirulina (NIES-39 strain) was cultured. The culture is performed by adding a constant weight of Spirulina to 10 ml of medium injected into a 30 ml screw cap test tube, leaving the screw cap test tube still in the incubator, and keeping the temperature at 22-26 ° C. while ventilating air. Using a white fluorescent lamp, the illuminance was 6000 to 6500 Lux, and the white fluorescent lamp was turned on and off for 12 hours. The screw cap test tube was shaken every other day.

Medium is S1 (10 ml of SOT medium), S2 (mixture of 9 ml of SOT medium and 1 ml of culture promoter), S3 (mixture of 5 ml of SOT medium and 5 ml of culture promoter), and S4 (culture promoter) 10 milliliters). The composition per liter of the SOT medium is NaHCO ₃ 16800 mg, K ₂ HPO ₃ 500 mg, NaNO ₃ 2500 mg, K ₂ SO ₄ 1000 mg, NaCl 1000 mg, MgSO ₄ .7H ₂ O 200 mg, CaCl ₂ 2H ₂ O 40 mg, FeSO ₄ · 7H ₂ O 10 mg, Na ₂ EDTA · 2H ₂ O 80 mg, H ₃ BO ₃ 2.86 mg, MnSO ₄ · 5H ₂ O 2.5 mg, ZnSO ₄ · 7H ₂ O is 0.22 mg, CuSO ₄ .5H ₂ O is 80 μg, Na ₂ MoO ₄ .2H ₂ O is 20 μg, and Distilled Water is 1 liter. In addition, the content of the nitrogen compound including the pyrrole compound in the culture promoter is 560 mg / liter, which includes pyrrole compound having a porphyrin ring structure of 16 mg / liter.

After 14 days from the start of culturing, 5 ml of each cell suspension from S1 to S4 was filtered using a φ25 GF / C filter inserted into a Sweeness filter holder, and the same method as in Example 1 was used. The dry weight of Spirulina present in 5 ml of cell suspension was calculated. FIG. 3 shows the dry weight of Spirulina contained in each 5 ml cell suspension collected from S1 to S4.

As shown in FIG. 3, with respect to the growth rate when growing in S1 (SOT medium only),
In the medium (S2) in which the culture promoter was diluted 10-fold, the ratio was 1.04 times, and in the medium (S3) in which the culture accelerator was diluted 2-fold, it was 1.7 times. There was no growth and a decrease in population occurred.
This indicates that when adding pyrrole compounds to the medium to increase the growth rate of spirulina, the optimum value of the dilution ratio of the culture promoter (content of nitrogen compounds including pyrrole compounds) is in a fairly narrow range It was.

As described above, the present invention has been described with reference to the embodiment. However, the present invention is not limited to the configuration described in the above-described embodiment, and the matters described in the scope of claims. Other embodiments and modifications conceivable within the scope are also included.
For example, in this embodiment, the culture accelerator containing a pyrrole compound was prepared using an extract from an E. coli medium, but after removing E. coli from the medium after the cultivation of E. coli was completed, the water content was increased. It can also be produced by adjusting.

Claims (4)

A growth promoter containing a pyrrole compound is added to a medium in which an organism that performs oxygen-generating photosynthesis grows, and the medium is maintained under the growth conditions of the organism, and the growth promotion of an organism that performs oxygen-generating photosynthesis is characterized by Method. The method for promoting the growth of an organism performing oxygen-generating photosynthesis according to claim 1, wherein the pyrrole compound contains a tetrapyrrole compound. 3. The method for promoting the growth of a living organism that performs oxygen-generating photosynthesis according to claim 1, wherein the living organism is an algae. 4. The method for promoting the growth of an organism performing oxygen-generating photosynthesis according to claim 3, wherein the concentration of the nitrogen compound containing the pyrrole compound contained in the medium is adjusted to 16 to 560 milligrams / liter. A method for promoting the growth of organisms that perform photosynthesis.

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