JP2010252700A - New asticcacaulis excentricus strain, method for culturing microalgae by using the same, and method for producing hydrocarbon - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new Asticcacaulis excentricus strain enabling industrial mass culture of Botryococcus, to provide a method for culturing Botryococcus by using the strain, and to provide a method for producing a hydrocarbon. <P>SOLUTION: The new Asticcacaulis excentricus strain exhibits activity for promoting the proliferation of the Botryococcus of the microalgae. The method for culturing the Botryococcus of the microalgae includes adding the Asticcacaulis excentricus strain to the Botryococcus of the microalgae. The method for producing the hydrocarbon includes taking out the hydrocarbon from the Botryococcus of the microalgae cultured by the culturing method. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

The present invention relates to a novel Astica caulis extricus strain, a method for culturing microalgae using the same, and a method for producing hydrocarbons.

It is known that a green alga Botryococcus (hereinafter referred to as Botryococcus) belonging to microalgae generates carbon that can be used as a substitute for petroleum by fixing carbon dioxide by photosynthesis. Hydrocarbons obtained from Botryococcus have a low nitrogen and sulfur content and have a low environmental impact due to combustion. Further, since the hydrocarbon obtained from Botryococcus has high purity, it is expected to be used as a lubricating oil, solvent, cosmetics, medical products and the like.

However, since Botryococcus has a slow growth rate in an aseptic environment and growth is inhibited by contamination with other microorganisms in outdoor culture, it is difficult to continuously perform outdoor culture. I came.
In addition, mass breeding and dominant breeding of Botryococcus in the natural world such as dams and lakes around the world have been confirmed, but survey on characteristics of water quality, climate, weather etc. of lakes where mass breeding was confirmed However, the factors that induce mass breeding have not been identified so far.

  By the way, Chirac et al. (1985) reported the existence of bacteria that promoted the growth of Botryococcus and the production of hydrocarbons by co-culturing Botryococcus-free strain and five kinds of bacteria (non-patented). Reference 1).

Christian Chirac, Eliette Casadevall, Claude Largeau and Pierre Metzger, BACTERIAL INFLUENCE UPON GROWTH AND HYDROCARBON PRODUCTION OF THE GREEN ALGA BOTRYOCOCCUS BRAUNI, J. Phycol. 21, 380-387 (1985)

However, in the above method, it is unclear whether industrial mass culture can be performed by a method such as outdoor culture.
The present invention has been made in view of the above points, and a novel Astica caulis extricus strain that enables industrial large-scale cultivation of Botryococcus, a method of culturing Botryococcus using the same, and It aims at providing the manufacturing method of hydrocarbon.

(1) The Asticcacaulis excentricus strain of the invention according to claim 1 has an effect of promoting the growth of Botryococcus. Therefore, if this Astica caulis extricus strain is used, the growth of Botryococcus can be promoted and its cultivation can be performed efficiently.

  In addition, when the Astica caulis extricus strain of the present invention is used, growth inhibition by contaminating bacteria can be reduced when Botryococcus is cultured in an open outdoor system. That is, if the Astica caulis extricus strain of the present invention is used, industrial mass culture by outdoor culture becomes possible.

Examples of the Botryococcus include Botryococcus braunii strains.
The Astica caulis extricus strain of the present invention is deposited under the accession number NITE AP-704 at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology.
(2) The invention according to claim 2 is an Astica caulis extricus strain having a deposit number of NITE AP-704. The Astica caulis extricus strain of the present invention has an effect of promoting the growth of Botryococcus. Therefore, if this Astica caulis extricus strain is used, the growth of Botryococcus can be promoted and its cultivation can be performed efficiently.

In addition, when the Astica caulis extricus strain of the present invention is used, growth inhibition by contaminating bacteria can be reduced when Botryococcus is cultured in an open outdoor system. That is, if the Astica caulis extricus strain of the present invention is used, industrial mass culture by outdoor culture becomes possible.
(3) The culture method of the invention according to claim 3 is characterized in that the Astica caulis extricus strain according to claim 1 or 2 is added to a Botryococcus brownie strain. According to the culture method of the present invention, Botryococcus can be cultured efficiently.

  In addition, according to the culture method of the present invention, growth inhibition by contaminating bacteria can be reduced when botryococcus is cultured in an open outdoor system. That is, if the culture method of the present invention is used, industrial mass culture by outdoor culture becomes possible.

As conditions for carrying out the culture method of the present invention, the following conditions are suitable.
Temperature 25 ° C, pH 7, modified chu medium (modified chu medium composition: KNO ₂ 0.2g / L, K ₂ HPO ₄ 0.04g / L, MgSO ₄ · 7H ₂ O 0.1g / L, CaCl ₂ · 2H ₂ O 0.054g / L, Fe-EDTA 6.95μg / L, H ₃ BO ₃ 28.6μg / L, MnCl ₂・ 4H ₂ O 18.1μg / L, ZnSO ₄・ 7H ₂ O 2.22μg / L, Na ₂ MoO ₄・ 2H ₂ O 0.5μg / L, CuSO ₄・ 5H ₂ O 0.78μg / L, CoCl ₂ 0.8μg / L)
(4) Since the method for producing hydrocarbons of the invention according to claim 4 can use Botryococcus that has been efficiently cultured by the culture method according to claim 3, it is possible to produce hydrocarbons efficiently.
The hydrocarbon produced in the present invention can be used, for example, as a substitute for petroleum, and has a low nitrogen and sulfur content, so the burden on the environment due to combustion is small. Moreover, since the hydrocarbon manufactured by this invention has high purity, it is suitable as a lubricating oil, a solvent, cosmetics, a medical product, etc., for example.

Examples of the hydrocarbon include cyclohexene hydrocarbons such as C ₃₄ H ₅₈ . Examples of the method for extracting hydrocarbons from Botryococcus include a method for extracting hydrocarbons from Botryococcus using an organic solvent (for example, hexane or the like).

It is a graph showing the chlorophyll a fluorescence intensity after adding various microorganisms to Botryococcus Bot88-2 and culturing. It is a graph showing the chlorophyll a fluorescence intensity after adding various microorganisms to Botryococcus of Bot17 and culturing. It is a graph showing the chlorophyll a fluorescence intensity after adding various microorganisms to Botryococcus Bot142 and culturing. It is a graph showing the chlorophyll a fluorescence intensity after adding various microorganisms to Botryococcus of Bot124 and culturing. It is a graph showing transition of the culture solution dry weight after adding strain A44 to Botryococcus and culturing.

An embodiment of the present invention will be described.
1. Isolation of strain A44 that exerts the action of promoting the growth of Botryococcus (1) A colony of Botryococcus was collected from a dam in Japan where Botryococcus was mass-bred using a plankton net. The collected samples were stored frozen by adding a cryoprotectant immediately after collection. Dams and lakes where Botryococcus proliferates can be easily found in Japan.
(2) After thawing the sample collected in the above (1) and cryopreserved, Botryococcus colonies were separated one by one using a Pasteur pipette and washed with sterile water. Next, the washed Botryococcus colonies were cultured in an inorganic medium. The inorganic medium is a modified chu medium, and its composition is as shown below.

KNO ₂ 0.2g / L, K ₂ HPO ₄ 0.04g / L, MgSO ₄・ 7H ₂ O 0.1g / L, CaCl ₂・ 2H ₂ O 0.054g / L, Fe-EDTA 6.95μmg / L, H ₃ BO _{3 28.6μmg / L, MnCl 2 · 4H} 2 O 18.1μmg / L, ZnSO 4 · 7H 2 O 2.22μmg / L, Na 2 MoO 4 · 2H 2 O 0.5μmg / L, CuSO 4 · 5H 2 O 0.78μmg / L , CoCl ₂ 0.8μmg / L.
(3) Next, the inorganic medium in which the Botryococcus colonies were cultured was immersed in water and subjected to ultrasonic treatment. At this time, the microorganisms adhering to the Botryococcus colony were detached from the Botryococcus colony and transferred to water. Water containing microorganisms was serially diluted and seeded on three types of agar media (A: for actinomycetes, B: for general bacteria, C: for bacteria and yeast). In addition, the dilution rate in the serial dilution was a rate at which an appropriate number of colonies were obtained on the agar medium. Table 1 shows the composition of the three types of agar medium.

As a result, 50 microbial colonies having different shapes were obtained in three types of agar medium. Each of the 50 microbial colonies was isolated and cultured.
(4) Each well of a 48-well plate was seeded with Botryococcus free strain, and then 50 microbial colonies isolated and cultured in (3) above were inoculated one by one into one well. In addition, said Botryococcus non-strain can be obtained with reference to “Production method and purified strain of Botulinococcus genus (application number: patent application No. 6-63481 publication number: patent publication No. 7-265059)”. Is possible. That is, a mixture containing an algae belonging to the genus Botriococcus and a microorganism other than the algae is suspended in an aqueous solution of hypochlorous acid or a salt thereof, and the mixture is applied to a plate medium and cultured to cultivate the algae. A colony is formed, and a purified algal strain belonging to the genus Botriococcus can be collected from the colony formed on the plate medium under observation with a stereomicroscope. In addition, isolate Botryococcus washed with 1% hypochlorous acid with a Pasteur pipette under a microscope, and transfer to a 96-well plate with liquid medium instead of agar medium to obtain a strain-free strain You can also

Thereafter, the relative amount of chlorophyll a contained in Botryococcus in each well was measured over time using a fluorescence microplate reader (excitation wavelength: 450 ± 25 nm, fluorescence wavelength: 685 ± 25 nm). Since the relative amount of chlorophyll a increases with the growth of Botryococcus, the growth of Botryococcus can be evaluated by the relative amount of chlorophyll a.
As a result, the relative amount of chlorophyll a was significantly increased in specific wells (proliferation of Botryococcus was promoted). That is, among the 50 types of microorganisms isolated and cultured in (2) above, the strain of the microorganism inoculated into the specific well exerted the action of promoting the growth of Botryococcus. Hereinafter, this strain is referred to as strain A44.

The strain A44 is deposited under the accession number NITE AP-704 at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology.
(5) A well plate different from that used in (4) above is divided into 4 regions, a, b, c, and d, and a Bot88-2 botryococcus-free strain is seeded in each well of the a region. Each well in the b region is seeded with a Bot17 botryococcus free strain, each well in the c region is seeded with a Bot142 botryococcus free strain, and each well in the d region is seeded with a Bot124 Botryococcus. The strain-free strain was seeded.

  Next, one type of microorganism shown in the horizontal axis of FIGS. 1 to 4 was added to each well in the region a. Some wells were controlled. Similarly, in the b to d regions, the same microorganisms as those added to the a region were added one by one to each well, and some wells were used as controls.

Thereafter, in the same manner as in (4) above, the relative amount of chlorophyll a contained in each well of Botryococcus was measured over time using a fluorescent microplate reader.
Fig. 1 shows the measurement results in the region a (seeded with Bot88-2 no strain of Botryococcus), and Fig. 2 shows the results of measurement in the region b (seeded with no Bot17 strain of Botryococcus). FIG. 3 shows the measurement results in the c region (seeding the Bot142-free strain of Botryococcus), and FIG. 4 shows the measurement results in the d-region (seeding the Bot124-free strain of Botryococcus). As shown in FIG. 1 to FIG. 4, in all the regions a to d, the relative amount of chlorophyll a is remarkable in the wells inoculated with the strain A44 as compared to the wells to which other microorganisms were added and the control wells. (Proliferation of Botryococcus was remarkably promoted).
From this result, it was confirmed that the strain A44 exerted the action of promoting the growth of all of the four strains (Bot88-2, Bot17, Bot142, Bot124) without Botryococcus.

2. Identification of isolated strain (1) For strain A44, a partial sequence (1236b) of 16S rDNA was identified using a known method. The partial sequence is as shown in the attached sequence listing. Then, this 16S rDNA partial sequence was compared with the 16S rDNA partial sequences of three strains of Astika caulis and extricus already registered in the database.
(i) Contrast with registered Astica caulis extricus strain (1): gi | 3551507 | dbj | AB016610.1 | Asticcacaulis excentricus 16S rRNA gene, partial sequence
Length = 1426
Comparison results: Score = 2411 bits (1216), Expect = 0.0
Identities = 1232/1236 (99%), Gaps = 1/1236 (0%)
Strand = Plus / Plus
(ii) Comparison with registered Astica caulis excentricus strain (2): gi | 8977595 | emb | AJ247194.1 | AEX247194 Asticcacaulis excentricus partial 16S rRNA gene for 16S ribosomal
RNA, strain DSM 4724 (T)
Length = 1416
Comparison results: Score = 2411 bits (1216), Expect = 0.0
Identities = 1232/1236 (99%), Gaps = 1/1236 (0%)
Strand = Plus / Plus
(iii) Contrast with registered Astica caulis extricus strain (Part 3): gi | 4580927 | gb | AF115499.1 | AF115499 Asticcacaulis excentricus 16S ribosomal RNA gene, partial sequence
Length = 1282
Comparison results: Score = 2135 bits (1077), Expect = 0.0
Identities = 1145/1157 (98%), Gaps = 8/1157 (0%)
Strand = Plus / Plus
As described above, the 16S rDNA partial sequence showed high homology between the strain A44 and the strain of Astica caulis extricus already registered in the database. From this, it was found that the strain A44 was Astica caulis extricus. Moreover, since homology was not 100%, it turned out that the strain A44 is a novel strain different from a well-known strain.
(2) The bacteriological properties (morphological properties, reproductive mode, physiological / chemical properties, etc.) of strain A44 were consistent with the type strain (reference strain) of Astica caulis excentricus. This also confirmed that the strain A44 was Astica caulis extricus.

3. Botryococcus culture method (1) BOT144 strain (sterile), one of the strains of Botryococcus, was inoculated with strain A44, and flask culture was performed in a flask containing the culture solution. The culture conditions were as follows.

Temperature 25 ° C, pH 7, 200ml modified chu medium, aeration (about 30ml / second) (modified chu medium composition: KNO ₂ 0.2g / L, K ₂ HPO ₄ 0.04g / L, MgSO ₄ · 7H ₂ O 0.1g / L , CaCl ₂・ 2H ₂ O 0.054g / L, Fe-EDTA 6.95μg / L, H ₃ BO ₃ 28.6μg / L, MnCl ₂・ 4H ₂ O 18.1μg / L, ZnSO ₄・ 7H ₂ O 2.22μg / L _{, Na 2 MoO 4 · 2H 2} O 0.5μg / L, CuSO 4 · 5H 2 O 0.78μg / L, CoCl 2 0.8μg / L)
Further, control was cultured in the same manner except that the strain A44 was not inoculated. In addition, as shown in FIG. 5, a plurality of culture periods were set in a range of a maximum of 33 days, and an experiment was performed for each.

  After culturing for a predetermined period, hydrocarbons were extracted from Botryococcus as follows. First, the culture solution was freeze-dried and the dry weight was measured. Hexane was added to this and allowed to stand for 30 minutes. Thereafter, ultrasonic waves were applied to extract hydrocarbons into hexane, and the hexane layer was transferred to a test tube whose weight was measured in advance. Hexane was added to the residue again, hexane was extracted in the same manner as above, and the hexane layer was added to the same test tube as above. This operation was repeated until the hexane layer added to the residue became transparent even after applying ultrasonic waves. Thereafter, hexane in the test tube was removed using a centrifugal evaporator, and the weight of the remaining hydrocarbon was measured.

  The dry weight of the culture solution before extracting with hexane is shown in FIG. 5 for each of the case where the strain A44 was inoculated and the case where the strain A44 was not inoculated. When the culture period was the maximum of 33 days, the dry weight of the culture solution was increased by 39% when the strain A44 was inoculated, compared to when the strain A44 was not inoculated.

Moreover, about the amount of hydrocarbon, when the culture | cultivation period was the maximum 33 days, the direction in the case of inoculating the strain A44 increased 30% compared with the case where it did not inoculate the strain A44.
(2) Botryococcus Bot144 and strain A44 were added to 250 ml of modified Chu medium, and cultured with aeration in a 500 ml glass flask. Incubation was performed outdoors (in a greenhouse). Further, control was cultured in the same manner except that the strain A44 was not added.

  On the 6th day of culture, Botryococcus of control (no addition of strain A44) was slightly yellowed and sank to the bottom. This is thought to be because, in the case of no addition of strain A44, the influence of contamination (contamination) due to contaminating bacteria appears significantly, and the cell state of Botryococcus has deteriorated. On the other hand, Botryococcus added with strain A44 was kept dark green, and it was confirmed that colonies were floating. This is probably because the culture of Botryococcus is in a better state.

Detailed culture conditions were as follows.
Temperature 25 ° C, pH 7, 200ml modified chu medium, aeration (about 30ml / second) (modified chu medium composition: KNO ₂ 0.2g / L, K ₂ HPO ₄ 0.04g / L, MgSO ₄ · 7H ₂ O 0.1g / L , CaCl ₂・ 2H ₂ O 0.054g / L, Fe-EDTA 6.95μg / L, H ₃ BO ₃ 28.6μg / L, MnCl ₂・ 4H ₂ O 18.1μg / L, ZnSO ₄・ 7H ₂ O 2.22μg / L _{, Na 2 MoO 4 · 2H 2} O 0.5μg / L, CuSO 4 · 5H 2 O 0.78μg / L, CoCl 2 0.8μg / L)
As described above, by adding the strain A44 to Botryococcus, the growth of Botryococcus can be promoted, and the growth inhibition by the mixed bacteria can be reduced.

  In addition, this invention is not limited to the said embodiment at all, and it cannot be overemphasized that it can implement with a various aspect in the range which does not deviate from this invention.

Claims (4)

  An Astica caulis excentricus strain that acts to promote the growth of the microalga Botryococcus.   Astica caulis extricus strain having a deposit number of NITE AP-704.   A method for culturing a microalga Botryococcus, comprising adding the Astica caulis extricus strain according to claim 1 or 2 to the microalga Botryococcus.   A method for producing hydrocarbons, comprising extracting hydrocarbons from the microalga Botryococcus cultured by the culture method according to claim 3.