JP6108331B2 - Hydrocarbon high-yield alga bodies and method for producing the same - Google Patents

Description

  The present invention relates to a hydrocarbon-producing alga-derived algal body derived from a hydrocarbon-producing algae having an intercellular matrix modified so as to have a property capable of recovering biosynthesized hydrocarbons in high yield, and a method for producing the same The present invention relates to a method for producing hydrocarbons.

  Oil is an indispensable energy resource in modern human society. However, with the burning of fossil fuels such as oil, global warming due to the greenhouse effect caused by carbon dioxide emitted into the atmosphere has become a major problem. In addition, international oil consumption is on the rise, and there are concerns about future oil depletion.

  For the above reasons, hydrocarbon-producing microorganisms that produce hydrocarbons have recently attracted attention as new energy resources that can replace petroleum. A hydrocarbon-producing microorganism is a microorganism having the ability to biosynthesize long-chain aliphatic hydrocarbons in a pathway that assimilates nutrient sources. Since it can be produced by culturing, it is not depleted, and hydrocarbons obtained from the microorganisms are circulating energy and do not increase the amount of carbon dioxide in the atmosphere. Furthermore, green algae having the ability to produce hydrocarbons represented by Botryococcus braunii can be linked to the reduction of greenhouse gases and the prevention of global warming due to photosynthesis.

  However, many problems to be solved remain in the practical use of hydrocarbon production using hydrocarbon-producing microorganisms. The biggest problem is the manufacturing cost. For example, B. braunii has low hydrocarbon production efficiency due to its slow growth rate. Moreover, in order to collect hydrocarbons from the algae, it is necessary to collect the hydrocarbons from the cells after concentrating the algae by filtering the culture solution, etc., but the moisture content of the algae itself is large and thick. Since it has a cell wall, the yield of hydrocarbons is extremely low in an extraction method using a normal organic solvent. Therefore, the manufacturing cost per unit amount is high and is not practical.

  Various attempts have been made to solve this problem. For example, Non-Patent Document 1 discloses a method for efficiently extracting hydrocarbons from an algal body using an organic solvent after dehydrating and drying after concentrating the algal body of B. braunii. Patent Document 1 discloses a method for efficiently extracting hydrocarbons from algal bodies by concentrating B. braunii algal bodies and then heating the aqueous slurry to recover oily substances. However, although these pretreatment methods can improve the yield of hydrocarbons, new capital investment is required due to the increase in production processes, and a great deal of energy must be invested in these processes. Therefore, it cannot be a fundamental solution for reducing manufacturing costs. In the practical use of hydrocarbon production derived from hydrocarbon-producing algae, it is important to reduce the amount of energy input necessary for recovery as much as possible and to recover hydrocarbons from alga bodies with high yield. Therefore, there is a need for a new technique for recovering hydrocarbons from algal bodies with a high yield without requiring the pretreatment.

JP2010-111865

Wake, L.V. et al., 1981, Australian Journal of Marine and Freshwater Research, 32-3: 353-367

  The present invention is a hydrocarbon in which algal bodies are modified so that hydrocarbons derived from hydrocarbon-producing algae have characteristics that allow easy and efficient recovery without passing through pretreatment steps such as drying and heat treatment. It is an object of the present invention to develop a production algae and to provide a method for producing hydrocarbons derived from a hydrocarbon-producing algae using the production algae.

In order to solve the above-mentioned problems, the present inventors conducted extensive research using hydrocarbon-producing algae, and as a result, using hydrocarbon-producing algae having an intercellular matrix, the osmotic pressure of the medium was adjusted and the algae was adjusted. By applying an osmotic shock to the body and culturing for a predetermined period, an algae body that has acquired properties that allow easy and high-capacity recovery of hydrocarbons without the need for drying or heat treatment is obtained. succeeded in. The present invention has been completed based on the newly developed technology, and provides the following.
(1) A method for producing a hydrocarbon high-yield algal body, wherein a hydrocarbon-producing alga having an intercellular matrix is treated for 38 hours or more in a medium having an osmotic pressure higher than the osmotic pressure of the main habitat area of the algae. The said manufacturing method including the process of culture | cultivating and providing an osmotic pressure impact to this hydrocarbon-producing algae.
(2) The production method according to (1), wherein a main inhabiting water area of the hydrocarbon-producing algae is a fresh water area.
(3) The production method according to (2), wherein the osmotic pressure of the medium is larger than 14 mOsm and not larger than 385 mOsm.
(4) The production method according to any one of (1) to (3), wherein the hydrocarbon-producing alga is a Botryococcus genus.
(5) A hydrocarbon-producing algae having an intercellular matrix is cultured for 38 hours or more in a medium having an osmotic pressure higher than the osmotic pressure of the main aquatic habitat of the algae, and an osmotic shock is imparted to the hydrocarbon-producing algae. The hydrocarbon high-yield alga body obtained by this.
(6) The algal body according to (5), wherein a main inhabiting water area of the hydrocarbon-producing algae is a fresh water area.
(7) The algal body according to (6), wherein the osmotic pressure of the medium is larger than 14 mOsm and not larger than 385 mOsm.
(8) The alga body according to any one of (5) to (7), wherein the hydrocarbon-producing algae is a Botryococcus genus.
(9) A method for producing hydrocarbons using hydrocarbon-producing algae having an intercellular matrix, wherein the hydrocarbon high-yield algal bodies according to (5) are converted into osmotic pressure in the main habitat of the algae. The said hydrocarbon manufacturing method including the culture | cultivation process which culture | cultivates in the culture medium which has higher osmotic pressure, and the extraction process which extracts a hydrocarbon from the algal body after the said culture | cultivation process with a physical method and / or a chemical method.
(10) The hydrocarbon production method according to (9), further comprising a concentration step of concentrating the algal bodies in the culture solution after the culture step.
(11) The production method according to (9) or (10), further including an algal body recovery step of recovering the hydrocarbon high-yield alga bodies after the extraction step for use in the culturing step.
(12) The hydrocarbon production method according to any one of (9) to (11), wherein a main inhabiting water area of the hydrocarbon-producing algae is a fresh water area.
(13) The hydrocarbon production method according to (12), wherein the osmotic pressure of the medium is larger than 14 mOsm and not larger than 385 mOsm.
(14) The hydrocarbon production method according to any one of (9) to (13), wherein the hydrocarbon-producing algae is a Botryococcus genus.
(15) The production method according to (14), wherein the hydrocarbon is a fatty acid-derived linear alkene or terpene.
(16) A hydrocarbon production system using a hydrocarbon-producing algae having an intercellular matrix, wherein the hydrocarbon high-yield alga body according to (5) is more than the osmotic pressure of the main habitat water area of the algae. The hydrocarbon production system comprising: a culture part that is cultured in a medium having a high osmotic pressure; and an extraction part that extracts hydrocarbons from the algal bodies after the culture process by a physical method and / or a chemical method.
(17) The hydrocarbon production system according to (16), further including a concentration unit that concentrates the algal bodies in the culture solution cultured in the culture unit.
(18) The method further includes an alga body recovery unit that recovers the hydrocarbon high-yield algal bodies after extracting the hydrocarbons in the extraction unit and supplies the algal bodies again to the culture unit. ) Manufacturing system.
(19) The culture unit includes osmotic pressure adjusting means for measuring an osmotic pressure in a culture solution and adjusting the culture unit to be in a predetermined osmotic pressure range. The described manufacturing system.
(20) A medium for producing a hydrocarbon high-yield algal body having an osmotic pressure higher than an osmotic pressure in a main inhabiting water area of a hydrocarbon-producing alga having an intercellular matrix.
(21) The medium according to (20), wherein a main inhabiting water area of the hydrocarbon-producing algae is a fresh water area, and the osmotic pressure is greater than 14 mOsm and not greater than 385 mOsm.
(22) The medium according to (21), which is a diluted solution of natural seawater or artificial seawater.
(23) The medium according to (20) or (21), comprising the hydrocarbon-producing algal synthetic medium and an osmotic pressure regulator.
(24) The medium according to (23), wherein the osmotic pressure regulator is a metal salt.
(25) The medium according to (23) or (24), wherein the synthetic medium is selected from the group consisting of Chu13 medium, AF-6 medium, and f / 2 medium.

  According to the hydrocarbon high-yield algal cells of the present invention or a method for producing the same, a pre-treatment step such as a drying treatment or a heat treatment is not required, and the carbonization has characteristics that allow easy and efficient recovery of hydrocarbons Hydrogen-producing algae can be provided. Moreover, the method of manufacturing the hydrocarbon derived from hydrocarbon production algae using it can be provided.

It is a manufacturing flow of the hydrocarbon high-yield algal cells of the present invention. The solid line frame process indicates an essential process, and the broken line frame process indicates a selection process. It is a hydrocarbon production flow of the present invention. The solid line frame process indicates an essential process, and the broken line frame process indicates a selection process. It is a figure which shows the relationship between the osmotic pressure of a culture medium, and algal body growth rate. The osmotic pressure of the modified Chu13 medium is 14 mOsm, and the osmotic pressure of 1/4 artificial seawater is 192.87 mOsm (34 days) to 262.68 mOsm (178 days). It is a figure which shows the relationship between 1/4 artificial seawater and hydrocarbon content rate. It is a figure which shows the relationship between 1/4 artificial seawater and a hydrocarbon recovery rate. It is a figure which shows the relationship between a culture | cultivation period when a hydrocarbon high-yield alga body of this invention is continuously cultured without subculture in 1/4 artificial seawater, and a hydrocarbon recovery rate. It is a figure which shows the relationship between the osmotic pressure of a culture medium, and algal body growth rate. The osmotic pressure of the modified Chu13 medium is 14 mOsm, and the osmotic pressure of 1/4 natural seawater is 240.08 mOsm (33 days) and 304.62 mOsm (62 days). It is a figure which shows the relationship between 1/4 natural seawater and a hydrocarbon content rate. It is a figure which shows the relationship between the osmotic pressure of a culture medium, and algal body growth rate. The osmotic pressure of the modified Chu13 medium is 14 mOsm, and the osmotic pressures of the synthetic saline medium adjusted by adding NaCl to the modified Chu13 medium are 227.61 mOsm (33 days) and 288.59 mOsm (62 days). It is a figure which shows the relationship between a synthetic salt culture medium and a hydrocarbon content rate. It is a figure which shows the collection | recovery result of the hydrocarbon from the hydrocarbon high-yield alga body of this invention using the resin fat-and-oil adsorption sheet. A is a sheet before processing, and it indicates that the sheet has water repellency by dripping water onto the sheet. B is a sheet after the treatment, and it is understood that hydrocarbons are adsorbed as black spots on the sheet.

1. 1. Production method of hydrocarbon high-yield algal cells 1-1. Outline and Definition The first aspect of the present invention is a method for producing a hydrocarbon high-yield alga. According to this production method, a hydrocarbon high-yield alga body can be easily produced from hydrocarbon-producing algae having an intercellular matrix using existing culture equipment.

  In the present specification, the term “hydrocarbon high-yield alga body” means that a hydrocarbon-producing alga having an intercellular matrix has a statistically significant difference in biosynthesized hydrocarbons compared to wild-type algae. Algae that has acquired new properties that can be recovered at a high yield.

  “Hydrocarbon-producing algae” is a general term for microalgae that can biosynthesize long-chain aliphatic liquid hydrocarbons through a pathway that assimilate nutrient sources in photosynthesis. “Microalgae” refers to microalgae in which it is difficult to identify individual cells with the naked eye. The hydrocarbon-producing algae that are the subject of the invention in the present specification are unicellular algae having an intercellular matrix. The “intercellular matrix” here is a biopolymer composed of long-chain epoxides, aldehydes, and the like produced by some unicellular microalgae. Individual cells form cell clusters through this intercellular matrix. In general, hydrocarbon-producing algae having an intercellular matrix are known to accumulate in the intercellular matrix after secreting the hydrocarbons produced by photosynthesis (Largeau, C., 1980, phytochemistry, 19-6: 1043-1051). Accordingly, in the present specification, the “algae” means a hydrocarbon-producing algal individual having an intercellular matrix and a cell group composed of a plurality of hydrocarbon-producing algal individuals. Examples of the hydrocarbon-producing alga suitable for the present invention having such an intercellular matrix and forming a cell colony include species belonging to the genus Botryococcus. A typical example is B. braunii. In the present specification, the “hydrocarbon-producing algae having an intercellular matrix” are hereinafter often abbreviated as “hydrocarbon-producing algae” or “algae”.

  The “hydrocarbon” in the present specification refers to a hydrocarbon that is biosynthesized by hydrocarbon-producing algae and is liquid at normal temperature. The type of hydrocarbon that is biosynthesized generally varies depending on the type of hydrocarbon-producing algae, but is not particularly limited as long as it is derived from hydrocarbon-producing algae. For example, if the hydrocarbon-producing algae used is B. braunii, the hydrocarbons are linear alkenes derived from fatty acids such as alkadienes and alkatrienes, or triterpenes (including botriocossen and methylsqualene) and tetraterpenes (lycopadiene). Terpene hydrocarbon.

1-2. Configuration The manufacturing flow of this manufacturing method is shown in FIG. As shown in this figure, the production method includes an osmotic pressure application step (S0102) and a pre-culture step (S0101). Of these, the osmotic pressure application step is an essential step of the production method, and the pre-culture step is a selection step. Hereinafter, each process will be specifically described.

(1) Osmotic pressure application step "Osmotic pressure application step" (S0102) is a medium that has a higher osmotic pressure than hydrocarbon-producing algae having an intercellular matrix. In this step, the osmotic shock is applied to the algal bodies by culturing for a predetermined period or longer.

  As used herein, “applying an osmotic pressure impact” refers to applying an osmotic impact to an algal body during culture. In general, the term “culture” is accompanied by growth, but “culture” in the present specification is not necessarily accompanied by growth, and is used in a broader sense to survive algal bodies in the culture medium. Therefore, for example, even when the algal cells are survived and maintained for a time exceeding the normal doubling time in a state in which the doubling of the algal cells is suppressed, it corresponds to “culture” in the present specification.

  There are no particular limitations on the type of hydrocarbon-producing algae used in the present production method. What is necessary is just to select the hydrocarbon production algae which produces a desired hydrocarbon. Preferably, it is a hydrocarbon-producing algae with a clear main habitat.

  In this specification, the “main habitat area” is an area where the hydrocarbon-producing algae that are the subject of the present invention mainly inhabit, and can survive with the growth of the algae in the natural environment. A body of water with a higher number of colonies per unit capacity (habitat) than the body of water. A water area that can survive and / or grow but has a significantly lower survival rate and / or growth rate than other water areas does not fall within the main habitat area of the hydrocarbon-producing algae. For example, in hydrocarbon-producing algae distributed in both freshwater areas such as rivers and brackish waters near river mouths, the survival rate and / or growth rate in brackish water areas is statistically significant compared to that in freshwater areas. If low, the algae's main habitat will be freshwater rather than brackish. That is, the main habitat water area is the most preferable environment for survival and growth of the hydrocarbon-producing algae. Therefore, even if the main habitat area of hydrocarbon-producing algae is unknown, when the target hydrocarbon-producing algae are cultured in fresh water, brackish water and seawater, the water area with the highest survival rate and growth rate is the algae. It can be recognized as the main habitat area. On the other hand, in hydrocarbon-producing algae that inhabit or can live in different water areas, if there is no statistically significant difference in survival rate and growth rate in each water area, any water area becomes the main habitat area . In this case, in this specification, the water area with higher osmotic pressure is defined as the main habitat area of the algae. In the present specification, fresh water is a solution having an osmotic pressure of 14 mOsm (miliosmol) or less, brackish water is a solution having an osmotic pressure of greater than 14 mOsm and less than 1.2 Osm (osmol), and seawater is 1.2 Osm or more. The solution has an osmotic pressure.

  The hydrocarbon-producing algae in this specification are either freshwater algae whose main habitat is freshwater, brackish algae whose main habitat is seawater, and seawater algae whose main habitat is seawater. Also good. Preferably they are freshwater algae or brackish algae, more preferably freshwater algae. Specific examples of freshwater algae include the aforementioned B. braunii. Various strains are known for B. braunii, including the Showa strain (Nonomura M., 1988, J. Phycol., 36: 285-291). May be. In any of the above-described water areas, the water temperature is not particularly limited as long as the hydrocarbon-producing algae used in the production method of the present invention can survive. For example, it may be a water area having a higher temperature than the surrounding outdoor water temperature such as a hot spring. Preferably, the water temperature is optimal for the growth of the hydrocarbon-producing algae.

  The “medium” in this step is capable of survival and / or growth of the hydrocarbon-producing algae used in the production method of the present invention, and has an osmotic pressure higher than the osmotic pressure of the main habitat water area of the algae. It is characterized by.

  The component composition of the medium used in this step is not particularly limited as long as it is a medium containing nutrients essential for the survival and / or growth of the algae used in the present invention. The medium may be a natural medium, an artificial medium, a synthetic medium, or a combination thereof. In this specification, the “natural medium” is water derived from water bodies existing in nature (for example, lake water, river water or seawater) or a medium diluted with pure water or the like. For example, water derived from the main habitat of hydrocarbon-producing algae is suitable as a medium for this step because it is considered to contain all components necessary for the survival and / or growth of the algae. Further, in the present specification, the “artificial medium” refers to a pseudo natural medium prepared artificially so as to include a component equivalent to or similar to the component composition of the natural medium. For example, artificial seawater (for example, Daigo artificial seawater SP; Nippon Pharmaceutical Co., Ltd.) simulating the composition of natural seawater. Furthermore, the “synthetic medium” in the present specification is a medium prepared by preparing components so that the hydrocarbon-producing algae as a subject of the present invention can survive and / or grow. For example, if the hydrocarbon-producing alga is a freshwater hydrocarbon-producing alga such as B. braunii, Chu13 medium (SP Chu, The Journal of Ecology, 1942, 30 (2): 284-325), AF-6 medium (Kasai F., et al., 2004, NIES-Collection list of strains, 7th edn. National Institute for Environmental Studies, Tsukuba, p 49), or f / 2 medium (Guillard RR and Ryther JH, 1962, Gran. Can J Microbiol., 8: 229-239; Bazaes J., et al., 2012, J. Appl. Phycol., Published online: 08 February).

  The osmotic pressure of the medium used in this step is higher than the osmotic pressure of the main aquatic habitat of the algae used in the present invention and is within a range where the algae can survive. For example, if the hydrocarbon-producing alga used is a freshwater algae such as B. braunii, the osmotic pressure of the medium used in this step is larger than 14 mOsm, not more than 385 mOsm, preferably not less than 50 mOs and not more than 360 mOsm, more preferably It may be 100 mOs or more and 340 mOsm or less, more preferably 150 mOs or more and 330 mOsm or less, and still more preferably 180 mOs or more and 320 mOsm or less. Any known method may be used to adjust the osmotic pressure of the medium used in this step. For example, when the algae to be used is a freshwater algae such as B. braunii, as a method of adjusting the osmotic pressure of the medium to be greater than 14 mOsm and 385 mOsm or less, seawater that is a natural medium or artificial seawater that is an artificial medium A method of diluting with pure water or the like so as to be within the osmotic pressure range, a method of adjusting to the osmotic pressure range by adding a metal salt or the like to a river or lake water or a synthetic medium for freshwater algae as a main habitat water area Or the method of using the water derived from the brackish water area whose osmotic pressure is in the said osmotic pressure range is mentioned. The metal salt added to the medium for adjusting the osmotic pressure is not particularly limited as long as it is harmful to the survival and / or growth of hydrocarbon-producing algae used in a small amount. Preferred are metal salts generally used for osmotic pressure adjustment, such as sodium chloride, calcium chloride, magnesium chloride, calcium chloride.

  Furthermore, this step is characterized in that the hydrocarbon-producing algae are cultured in a medium having an osmotic pressure higher than the osmotic pressure of the main aquatic habitat of the algae for a predetermined time or more. The culture time is 38 hours or more, preferably 2 days or more, 3 days or more, 5 days or more, 10 days or more, 15 days or more, 20 days or more, 25 days or more, or 30 days or more. In addition, as described above, this step only needs to give an osmotic shock to the algal bodies for a predetermined time or more, and does not necessarily involve proliferation, so the culture time does not need to span multiple generations, It may be within a single generation time under normal growth conditions, that is, within a doubling time. The upper limit of the culture time is not limited. Even when the subculture (culture by planting) is continued, the hydrocarbon-producing algae of the present invention maintain the properties of the hydrocarbon high-yield algal bodies and achieve the object of the present invention. Because you get. However, if the hydrocarbon high-yield algal cells are cultured for a long time in the same medium without subculture, and the culture is a culture with growth, the algal cells in the culture solution The culture medium that has reached the late logarithmic phase or the early or middle stationary phase is preferably planted in a new medium periodically because it enters the death phase after a certain period.

As for a specific method for culturing hydrocarbon-producing algae including various conditions such as culture temperature, CO ₂ concentration, light irradiation amount and light irradiation time, it is known in the art for hydrocarbon-producing algae to be subjected to the osmotic pressure application step. All culture methods can be used. Usually, the culture may be performed under conditions suitable for culturing the hydrocarbon-producing algae used, preferably under optimum conditions. For example, if the hydrocarbon-producing algae is B. braunii, Tanoi T., et al., 2010, J. Appl. Phycol., Published online: 10 June; Okada S., et al., 1997, Applied Biochemistry and Biotechnology, 67-1: 79-86; Casadevall E., et al., 1985, Biotechnology and Bioengineering, 27-3, 286-295; Wolf FR, et al., 1983, Applied Biochemistry and Biotechnology, 8-3: The culture method described in 249-260 may be referred to.

(2) Pre-culture process The "pre-culture process" (S0101) is a culture process involving proliferation that is performed prior to the osmotic pressure application process. The purpose of this step is to increase the number of algal bodies of the hydrocarbon-producing algae used in the osmotic pressure applying step to a predetermined amount in advance. Since this step is a selection step as described above, it is not necessarily a required step when the number of hydrocarbon-producing algae used in the osmotic pressure application step is sufficiently large from the beginning. Therefore, it may be appropriately performed according to the situation and necessity.

  The medium used in this step is not particularly limited as long as it is a medium in which hydrocarbon-producing algae used in the osmotic pressure application step can grow. Any of a natural medium, an artificial medium, or a combination thereof may be used. Preferably, water derived from the main habitat of the hydrocarbon-producing algae, or a solution having an equivalent or similar component composition and osmotic pressure, or an optimal component composition and osmotic pressure so that the hydrocarbon-producing algae can suitably grow Any solution may be used. For example, if the hydrocarbon-producing algae is B. braunii, the above-mentioned Chu13 medium, AF-6 medium, f / 2 medium, etc. can be used.

  The specific method for culturing the hydrocarbon-producing algae may be the same as the culturing method described in the osmotic pressure application step.

  The culture time is not particularly limited as long as the number of hydrocarbon-producing algae individuals (or the number of cell populations) in the culture solution reaches a desired value by growth, but if the purpose of this step is taken into consideration It is preferable to culture until the hydrocarbon-producing algae in the liquid reach the late logarithmic growth phase or the stationary phase.

  When inoculating the hydrocarbon-producing algae after the pre-culture process in the medium of the osmotic shock application process, all or part of the culture solution after the pre-culture process may be added to the medium of the osmotic shock application process as it is. Then, after centrifuging the culture solution after the pre-culture step, all or part of the algal body slurry or pellet obtained by removing the supernatant may be added to the medium in the osmotic pressure application step. The amount of planting when the cultured hydrocarbon-producing algae is added to the medium in the osmotic pressure application step is the amount of hydrocarbon-producing algae in the medium after planting corresponding to the number of individuals corresponding to the period after the induction period to the beginning of the stationary period Is preferred. For example, there is a method in which the culture medium cultured until reaching the stationary phase in the pre-culture step is added at 1/10 volume to 1/4 volume of the medium in the osmotic pressure application step.

1-3. Effect According to the method for producing a hydrocarbon high-yield alga body of the present invention, a hydrocarbon high-yield alga body capable of recovering hydrocarbons from a hydrocarbon-producing alga having an intercellular matrix in a high yield It can be easily manufactured.

  According to the method for producing a hydrocarbon high-yield alga body of the present invention, the object of the invention is achieved only by culturing a hydrocarbon-producing alga in a medium with controlled osmotic pressure for a predetermined time or more and applying an osmotic pressure impact. Therefore, it is only necessary to have an existing culture facility for hydrocarbon-producing algae. Therefore, it can be implemented without requiring new capital investment.

2. 2. High-yield hydrocarbon alga body 2-1. Outline | summary The 2nd aspect of this invention is a hydrocarbon high-yield alga.

  Conventionally, in order to recover hydrocarbons from hydrocarbon-producing algae having an intercellular matrix, is it possible to extract the hydrocarbons from the intracellular or intercellular matrix by concentrating the algal bodies and then physically destroying them by pressing or the like? Alternatively, a chemical extraction method using an organic solvent has been employed. However, these methods are difficult to recover efficiently due to the characteristics of hydrocarbon-producing algae having a high water content and a thick cell wall. Therefore, it has become a big obstacle for practical use. However, according to the hydrocarbon high-yield alga body of the present invention, it does not require a pretreatment step such as a drying treatment or a heat treatment, which is an essential step in the chemical recovery method for hydrocarbons, and is a physical method. Even so, hydrocarbons can be recovered in high yield.

2-2. Configuration As described above, the hydrocarbon high-yield alga body of the present invention is obtained by applying a hydrocarbon-producing alga having an intercellular matrix in a medium having an osmotic pressure higher than the osmotic pressure of the main habitat area of the algae for 38 hours. Or more, preferably 2 days or more, 3 days or more, 5 days or more, 10 days or more, 15 days or more, 20 days or more, 25 days or more, or 30 days or more This is an algal body that has acquired a new property capable of recovering a biosynthesized hydrocarbon in a statistically significantly higher yield as compared with a wild-type homologous algae. “Statistically significant” means that there is a significant difference when the difference in hydrocarbon yield between the two is treated statistically. As a test method for statistical processing, a known test method capable of determining the presence or absence of significance may be appropriately used. For example, Student's t test or multiple comparison test can be used. Specifically, for example, the risk rate (significance level) is less than 5%, 1%, or 0.1%. That is, the hydrocarbon high-yield alga body of this aspect is an alga body that has acquired high yield of hydrocarbons by the treatment according to the production method of the first aspect.

  By subjecting the algae with a hydrocarbon-producing algae having an intercellular matrix to osmotic shock for a predetermined time or more at an osmotic pressure higher than the osmotic pressure of the main inhabiting water area, any change occurs in the algal bodies, resulting in biosynthesis. It is not clear about the specific mechanism of obtaining the property of recovering the produced hydrocarbon in high yield. However, one hypothesis is that osmotic shock suppresses the production of the viscous polysaccharides that make up the intercellular matrix and increases the permeability of the intercellular matrix. There is also a possibility that leakage into the water will increase. This new property acquired by hydrocarbon-producing algae is not a transient property, but is continued by culturing in a medium having an osmotic pressure higher than that of the main habitat of the algae or by cryopreserving it. Can be maintained.

2-3. Effect According to the hydrocarbon high-yield alga body of the present invention, the hydrocarbon produced by the hydrocarbon-producing algae can be recovered without going through a pretreatment step such as a drying treatment or a heat treatment.

  If the hydrocarbon high-yield alga body of the present invention is used, hydrocarbons can be recovered at a very high yield as compared with wild-type algae just by suspending in an organic solvent. The yield is 5% or less in the same strain of wild type algae subjected to the same recovery operation, whereas it is 85% or more in the hydrocarbon high-yield alga body of the present invention.

  According to the hydrocarbon high-yield alga body of the present invention, the alga body can survive after the hydrocarbon recovery, because the hydrocarbon can be recovered without destroying the alga body by drying treatment or heat treatment. The collected algal bodies can be reused for the production of hydrocarbons.

  According to the hydrocarbon high-yield alga body of the present invention, storage is possible in a state where the hydrocarbon high-yield characteristic is acquired. Therefore, it can be provided as a hydrocarbon high-yield algal body.

3. 3. Hydrocarbon production method 3-1. Outline | summary The 3rd aspect of this invention is a hydrocarbon manufacturing method. According to this production method, hydrocarbons derived from hydrocarbon-producing algae can be efficiently produced at low cost using hydrocarbon high-yield alga bodies.

3-2. Configuration The manufacturing flow of the manufacturing method of this aspect is shown in FIG. As shown in this figure, the production method of this embodiment includes a culture step (S0201), a concentration step (S0202), an extraction step (S0203), and an algal body recovery step (S0204). Among these, a culture process and an extraction process are essential processes of the manufacturing method of this aspect, and a concentration process and an algal body collection process are selection processes. Hereinafter, each step will be specifically described.

(1) Cultivation process “Cultivation process” (S0201) is the cultivation of hydrocarbon high-yield alga bodies for a specified period of time while applying an osmotic shock to a medium having an osmotic pressure higher than that of its main habitat. It is a process to do. The purpose of this step is to culture a hydrocarbon high-yield alga body and biosynthesize a sufficient amount of hydrocarbons in the alga body.

  The hydrocarbon high-yield alga body used in the production method of the present aspect is the hydrocarbon high-yield algal body according to the second aspect, which has acquired the high-yield property of hydrocarbons by osmotic pressure impact. Osmotic pressure impacts even on hydrocarbon high-yield algal bodies derived from two or more different hydrocarbon-producing algae or hydrocarbon high-yielding algal bodies derived from two or more different types of hydrocarbon-producing algae If the osmotic pressures of the added media are within the same range, they can be used simultaneously.

  This step may be basically performed by the same method as the osmotic pressure imparting step (S0102) in the method for producing a hydrocarbon high-yield algal body of the first aspect. In this step, if the hydrocarbon high-yield algal bodies in the culture medium can survive and the hydrocarbon can be sufficiently biosynthesized, the hydrocarbon high-yield property is the same as in the osmotic pressure imparting step (S0102). Algal growth is not always necessary. However, in order to increase the yield of hydrocarbons obtained in a single production process, it is desirable to obtain a large number of hydrocarbon high-yield algal bodies in the culture solution by growth. In addition, since the hydrocarbon high-yield alga body used in the production method of this embodiment is an alga body that has already acquired the property of recovering hydrocarbons at high yield, at least the hydrocarbon high-yield alga body It is sufficient if the culture time is sufficient to biosynthesize hydrocarbons, and the ordinary hydrocarbon-producing algae are necessary for acquiring the above properties as in the method for producing hydrocarbon high-yield alga bodies of the first aspect. Incubation time is not required. As in the osmotic pressure imparting step (S0102) of the first aspect, when culturing with long-term growth is performed, if the culture is continued in the same medium, the hydrocarbon-producing algae in the culture solution enter the death phase. For example, when the hydrocarbon-producing algae in the culture solution reach the late logarithmic growth phase or the early or middle stationary phase, the whole or a part of the culture solution, or all of the hydrocarbon-producing algal slurry recovered from the culture solution or It is desirable to plant a part in a new medium.

  By combining the osmotic pressure applying step (S0102) of the first aspect and the culture step (0201) of the present aspect, the first aspect and the third aspect can be performed consistently. For example, after obtaining a hydrocarbon high-yield alga body in the osmotic pressure imparting step (0102) of the hydrocarbon high-yield alga body production method of the first aspect, the hydrocarbon high-yield alga is obtained from the culture solution. You may culture | cultivate as it is as the culture | cultivation process (0201) of this process, providing an osmotic pressure impact, without isolate | separating a body.

(2) Concentration step The “concentration step” (S0202) is a step of concentrating the hydrocarbon high-yield alga bodies in the culture solution after the culturing step. In this step, in the next extraction step (S0203), in order to efficiently extract hydrocarbons from hydrocarbon high-yield alga bodies, unnecessary liquid components are removed or reduced in advance from the culture solution after the culture step. The purpose of this is to increase the number of hydrocarbon high-yield alga bodies per unit volume. As described above, this step is a selection step and may be performed as necessary.

  The concentration method is not particularly limited as long as it can concentrate the hydrocarbons produced by the hydrocarbon high-yield algal cells by oxidation or the like, and any known method can be used. It can be concentrated. For example, after centrifuging the culture solution for a suitable time with a centrifuge, the centrifugal concentration method for collecting the alga body-containing phase, removing the culture solution using a suitable filter having a pore size smaller than the alga body size, Filtration concentration method for recovering algal body slurry, evaporation concentration method for concentrating the alga body by evaporating the culture solution using an evaporator, air-dry concentration method for concentrating the alga body by evaporating the culture solution by blowing air, etc. A combination etc. are mentioned. Usually, a centrifugal concentration method or a filtration concentration method is preferably applied. The concentration rate is not limited, but it is preferable to concentrate to the extent that all or most of the culture solution can be removed, that is, to the extent that the algal bodies become a pellet or slurry.

  The algal bodies after the concentration step can be washed once or more as necessary. Washing may be performed using pure water (including sterilized water, ion-exchanged water, and ultrapure water), the medium used in the culturing step, or a buffer or physiological saline having an osmotic pressure comparable to that of the medium.

(3) Extraction Step The “extraction step” (S0203) is a step of extracting hydrocarbons from the algal bodies after the culturing step or the concentration step by a physical method and / or a chemical method. The purpose of this step is to biosynthesize hydrocarbon high-yield alga bodies and extract the accumulated hydrocarbons from algal bodies.

  As a method for recovering hydrocarbons from hydrocarbon-producing algae having an intercellular matrix, conventionally, a method in which an organic solvent such as hexane is mixed and the hydrocarbon is dissolved in the organic solvent is mainly used. . However, hydrocarbons can hardly be recovered by simply mixing hydrocarbon-producing algae cultured in a normal medium with an organic solvent. Therefore, in order to efficiently recover hydrocarbons from the alga bodies, it is necessary to destroy the algal bodies by a physical method, or to perform a drying treatment or a heat treatment as a pretreatment before the extraction step. On the other hand, if the hydrocarbon high-yield alga bodies of the present invention are used, hydrocarbons can be recovered from the alga bodies very efficiently only by mixing with an organic solvent in the extraction step. Therefore, according to the hydrocarbon production method of the present embodiment, unlike conventional methods, it is possible to efficiently recover hydrocarbons under mild conditions that do not destroy algal bodies with fewer steps. Therefore, as described in detail in the next algal body recovery step (S0204), the hydrocarbon high-yield algal bodies can be recovered in a viable state after this step.

  The hydrocarbon extraction method used in this step may be performed according to any physical method and / or chemical method known in the art for extracting hydrocarbons from hydrocarbon-producing algae. As described above, in the hydrocarbon production method of this aspect, pretreatment such as destruction of the algal bodies by a physical method or drying treatment or heat treatment is not necessary in this step, but it may be performed if desired.

  Examples of the physical method include extraction methods by pressing, crushing, ultrasonic crushing, or a combination thereof. As a specific example of the extraction method by pressing, the algal body after the culturing step or after the concentration step is brought into contact with the fat and oil adsorbent, and an appropriate pressure is applied so as to increase the contact area between the two. The method of adsorb | sucking to a fats and oils adsorbent and collect | recovering is mentioned. As a specific example of the extraction method by crushing, the algae bodies after the culturing step or the concentration step are physically ground using glass beads, plastic beads, grind mills, etc., and then the protein components derived from the algae are denatured and removed. Then, the remaining solution is brought into contact with an oil and fat adsorbent to recover hydrocarbons, or the solution is recovered as it is or after adding an organic solvent or the like and centrifuging it. As a specific example of the extraction method by ultrasonic crushing, the algal bodies after the culturing step or the concentrating step are placed in an ultrasonic crushing device (sonicator), the algal bodies are crushed with an appropriate oscillation frequency and output, Examples include a method in which protein components derived from the body are denatured and removed, and hydrocarbons are recovered from the remaining solution in the same manner as the extraction method by crushing.

  Moreover, as a chemical method, the extraction method by the elution of the hydrocarbon using an organic solvent or melt | dissolution of the alga body using an enzyme is mentioned, for example. The organic solvent used in the hydrocarbon elution method using an organic solvent may be a low-polarity or nonpolar water-immiscible medium, and may be n-hexane (hereinafter also referred to as “hexane”) or n-hexane. C6-10 aliphatic or alicyclic hydrocarbons such as heptane or C6-10 aromatic hydrocarbons such as benzene are preferred, and hexane or n-heptane is more preferred. As described above, the hydrocarbon elution method using an organic solvent can recover the hydrocarbon dissolved in the organic phase simply by mixing the algal cells after the incubation step or the concentration step with an appropriate organic solvent. it can. The method for recovering or purifying hydrocarbons from the organic solvent used may be any method known in the art and is not particularly limited. Examples thereof include concentration by distillation under reduced pressure. Examples of the enzyme used in the extraction method by lysis of algal bodies using an enzyme include cellulase and pectinase. In the case of enzyme treatment, there is a method of treating algal bodies under the optimum conditions of the enzyme used, then denaturing and removing the protein components derived from algal bodies, and recovering from the remaining solution in the same manner as the extraction method by crushing. It is done.

(4) Algae recovery step The “algae recovery step” (S0204) is a step of recovering the hydrocarbon high-yield alga bodies after the extraction step (S0203) for use in the culture step (S0201). . The purpose of this step is to reuse the algal bodies after the extraction step for the culture step of the second and subsequent production cycles.

  This step is a case where, in the extraction step (S0203), hydrocarbons are extracted by a method that does not kill all hydrocarbon high-yield alga bodies, and the production method of this embodiment is continuously performed for 2 cycles or more. This is a selection process that is performed only when it is performed.

  Although this step is a selection step, it is the most characteristic and unique step in the hydrocarbon production method of this embodiment that has been made possible for the first time by using the hydrocarbon high-yield alga bodies of the second embodiment. . As described above, in order to efficiently recover hydrocarbons from hydrocarbon-producing algae having an intercellular matrix, conventionally, it has been necessary to perform a drying treatment or a heat treatment as a pretreatment before the extraction step. However, when such pretreatment was performed, the algal bodies used for the extraction of hydrocarbons were almost completely killed. Therefore, when the production cycle is performed continuously, a hydrocarbon high-yield alga body for the cultivation process must be newly prepared each time. For example, it takes time for the algal bodies to reach the logarithmic growth phase, which has been a major obstacle to the large-scale production of hydrocarbons using hydrocarbon-producing algae. On the other hand, the hydrocarbon production method of this embodiment uses the hydrocarbon high-yield alga bodies of the second embodiment, so that it is not necessary to go through a pretreatment step such as a drying treatment or a heat treatment in the extraction step, and mild extraction is performed. Even with this method, it is possible to recover hydrocarbons in a high yield. Therefore, the algal bodies after the extraction process are viable, and can be collected and reused as seeding alga bodies in the culture process. As a result, it is not necessary to prepare a new hydrocarbon high-yield alga body even when mass-culturing in the culturing process, and the number of alga bodies to be planted can be increased, thereby reaching the logarithmic growth phase. It is possible to solve the above-mentioned obstacles, such as shortening the time required.

  Any method known in the art that can recover hydrocarbon-producing algae can be used as the method for recovering hydrocarbon-producing alga bodies from the culture solution after the extraction step. A method similar to the concentration method described may be used. For example, the solution containing the aqueous phase and the intermediate phase after separating the oil phase containing hydrocarbons from the solution after the extraction step may be centrifuged for an appropriate time to collect alga bodies as a slurry, or the alga body size The solution may be filtered using a suitable filter having a smaller pore size to form alga bodies. It is not necessary for all the collected algal bodies to be alive.

  The collected algal bodies can be washed once or more as necessary. Washing may be performed using the medium used in the culturing step or a buffer or physiological saline having an osmotic pressure comparable to that of the medium.

  The collected alga bodies can be used as hydrocarbon high-yield alga bodies in the culturing step of the second and subsequent production cycles. The planting amount of the hydrocarbon high-yield algal bodies when added to the culture medium in the culturing step is preferably such that the algal bodies in the medium after seeding have an individual number corresponding to the early logarithmic growth phase.

3-3. Effect According to the hydrocarbon production method of the present invention, by using the hydrocarbon high-yield alga bodies of the second aspect, it is possible to produce hydrocarbons in a high number of steps with fewer steps than the conventional method. . As a result, production costs can be reduced, and hydrocarbons can be provided at low cost.

  Moreover, according to the hydrocarbon production method of the present invention, unlike the conventional method, the alga body having the property can be used to produce hydrocarbons because it can recover the hydrocarbon without destroying the alga body. Algae can be reused. This makes it possible to shorten the growth time of the high-yield hydrocarbon-producing alga bodies in the culturing process after the second production cycle.

4). Hydrocarbon production system 4-1. Outline A fourth aspect of the present invention is a hydrocarbon production system. The hydrocarbon production system of the present invention is a systemization of the hydrocarbon production method of the third aspect, and aims to produce hydrocarbons on a large scale from hydrocarbon-producing algae having an intercellular matrix. . According to the hydrocarbon production system of the present invention, hydrocarbons can be efficiently produced at low cost using the hydrocarbon high-yield alga bodies of the second aspect.

4-2. Configuration The hydrocarbon production system of the present invention includes a culture section and an extraction section. Furthermore, a concentration part and an algal body collection | recovery part can be included as needed. Hereinafter, each part will be described.

(1) Culture part "Culture part" is a part which performs the culture | cultivation process in the hydrocarbon manufacturing method of the said 3rd aspect. That is, in the culture unit, the hydrocarbon high-yield algal bodies derived from the hydrocarbon-producing algae having the intercellular matrix described in the second aspect are infiltrated in a culture tank higher than the osmotic pressure of the main aquatic waters of the algae. Culture in medium with pressure. The specific method for culturing hydrocarbon high-yield algal cells including the medium composition and the osmotic pressure of the medium in the main culture part can be performed according to the method described in the culturing step in the hydrocarbon production method of the third aspect. Good.

  The culture unit can include osmotic pressure adjusting means, light irradiation means, carbon dioxide supply means, stirring means, or a combination thereof as necessary.

  The “osmotic pressure adjusting means” is a means for measuring the osmotic pressure in the culture solution in the culture part and adjusting the culture part to be in a predetermined osmotic pressure range. The osmotic pressure adjusting means is configured to include, for example, an osmotic pressure measuring device, an osmotic pressure increasing device, an osmotic pressure decreasing device, and an osmotic pressure controller. The osmotic pressure measuring device can measure the osmotic pressure of the culture solution in the culture tank when necessary. What is necessary is just to utilize an osmotic pressure measuring device well-known in the said field | area as an osmotic pressure measuring device. The osmotic pressure increaser is a device for adding an osmotic pressure increasing agent to the culture tank in order to increase the osmotic pressure of the culture solution when the osmotic pressure in the culture solution falls below a predetermined osmotic pressure range. Examples of the osmotic pressure increasing agent include a metal salt or a solution thereof. The metal salt described in the first aspect may be used. The osmotic pressure reducer is a device for adding water to the culture tank in order to reduce the osmotic pressure of the culture solution when the osmotic pressure in the culture solution exceeds a predetermined osmotic pressure range. Based on the measurement result of the osmotic pressure measuring device, the osmotic pressure controller controls so that the osmotic pressure increasing agent is put into the culture tank from the osmotic pressure increasing device when the measured value is lower than the predetermined osmotic pressure range. In addition, when the measured value is higher than the predetermined osmotic pressure range, control can be performed so that water is introduced into the culture tank from the osmotic pressure reducer.

  The “light irradiating means” is means for artificially irradiating a culture tank with light necessary for photosynthesis of hydrocarbon high-yield algal bodies for a predetermined time. Any method known in the art can be used as the means for irradiating light. For example, LED, fluorescent lamp, mercury lamp, HID, light bulb and the like can be mentioned. In addition, when installing a culture tank outdoors, natural light (sunlight) may be sufficient normally.

  The “carbon dioxide supply means” is means for supplying carbon dioxide necessary for photosynthesis of hydrocarbon high-yield algal bodies into the culture tank. It has a carbon dioxide cylinder, an open / close valve, and a supply pipe. In order to increase the solubility of carbon dioxide in the culture solution, the partial pressure of carbon dioxide may be increased by increasing the airtightness in the culture tank.

  The “stirring means” is a means for stirring the culture solution being cultured. The stirring means is not particularly limited as long as it can efficiently stir so that the culture solution becomes uniform. For example, the culture solution may be stirred by a stirring rod or a rotating blade installed in the culture tank, or the culture tank may be stirred by rotating, reciprocating, reversing, or a combination thereof.

(2) Concentration part The "concentration part" is a part that concentrates the algal bodies in the culture solution cultured in the culture part, and is a part that performs the concentration step in the hydrocarbon production method of the third aspect. As described above, the enrichment unit may be added to the hydrocarbon production system of this embodiment as necessary. What is necessary is just to perform the specific concentration method of the hydrocarbon high-yield alga body in a culture solution in this concentration part according to the method as described in the concentration process in the hydrocarbon manufacturing method of the said 3rd aspect. In the concentration section, the concentrated algal bodies can be washed once or more as necessary.

(3) Extraction unit The “extraction unit” is a physical unit for hydrocarbons contained in the algal body from the cultured algal body in the culture solution from the culture unit or from the concentrated cultured algal body from the concentration unit. It is a part which performs the extraction process in the hydrocarbon manufacturing method of the said 3rd aspect in the part extracted by a general method and / or a chemical method. What is necessary is just to perform the specific extraction method of the hydrocarbon from the hydrocarbon high-yield alga body in an extraction part according to the method as described in the extraction process in the hydrocarbon manufacturing method of the said 3rd aspect. The means of the extraction unit is appropriately determined according to the extraction method. For example, when physically extracting by pressing the algal bodies, for example, when pressing and adsorbing means having the oil and fat adsorbing material according to the third aspect is physically extracted by crushing the algal bodies, When the algal body is physically extracted by ultrasonic crushing, the grind mill may be provided with, for example, an ultrasonic crusher. In addition, when a hydrocarbon is chemically extracted using an organic solvent such as hexane, it has an organic solvent charging means for charging the solvent into the extraction tank, and a stirring means for mixing the algal bodies and the organic solvent. You can do it. In the extraction unit, when hydrocarbons are extracted from the cultured algal cells by a method that does not kill all hydrocarbon high-yield algal cells, the hydrocarbon production system according to this aspect includes the following algal cell recovery unit. Can be provided.

(4) Algae body recovery part The "algae body recovery part" is a part that recovers hydrocarbon high-yield alga bodies after the extraction of hydrocarbons in the extraction part and supplies the algal bodies again to the culture part. And a part for performing an algal body recovery step in the hydrocarbon production method of the third aspect. As described above, the alga body recovery unit is a case where the extraction unit extracts hydrocarbons by a method that does not kill all hydrocarbon high-yield alga bodies, and the production method of this embodiment is performed for two cycles or more. It is a part that is provided only when performing continuously. In the alga body recovery part, the method of recovering the hydrocarbon high-yield alga bodies after hydrocarbon extraction in the extraction part is performed according to the method described in the alga body recovery step in the hydrocarbon production method of the third aspect. Good. That is, basically, a method similar to the concentration method of the concentration unit may be used. In the alga body collecting part, the collected alga bodies can be washed once or more as needed. The collected alga bodies can be used as hydrocarbon high-yield alga bodies to which a new medium is added in the culture section for use in the second and subsequent production cycles.

5. 5. Medium for producing hydrocarbon high-yield algal cells 5-1. Outline | summary The 5th aspect of this invention is a culture medium for hydrocarbon high-yield alga bodies manufacture. The medium of this aspect is a medium for producing the hydrocarbon high-yield algal bodies of the second aspect. According to the culture medium of this embodiment, hydrocarbons can be easily and efficiently recovered from wild-type hydrocarbon-producing algae having an intercellular matrix without requiring a pretreatment step such as drying or heat treatment. The hydrocarbon high-yield alga body of the 2nd aspect which acquired the property can be manufactured.

5-2. Configuration The medium of this embodiment is intended for hydrocarbon-producing algae having an intercellular matrix. Since the hydrocarbon-producing algae having an intercellular matrix are described in detail in the first embodiment, the description thereof is omitted here.

  The composition of the medium of this aspect may be basically the same as the medium composition described in the osmotic pressure imparting step of the first aspect. That is, the medium is a medium in which the hydrocarbon-producing algae to be manufactured can survive and / or grow, and it may be adjusted so as to have an osmotic pressure higher than the osmotic pressure in the main habitat area of the algae. . The medium can be a natural medium, an artificial medium, a synthetic medium, or a combination thereof. As a specific example, if the hydrocarbon-producing algae is a freshwater hydrocarbon-producing algae whose main habitat is a freshwater area such as B. braunii, the osmotic pressure of the medium is greater than 14 mOsm, not more than 385 mOsm, preferably not less than 50 mOs. 360 mOsm or less, more preferably 100 mOs or more and 350 mOsm or less, further preferably 150 mOs or more and 330 mOsm, and further preferably 180 mOs or more and 320 mOsm. In this case, if the culture medium is natural seawater, artificial seawater, or a combination thereof, it may be diluted with a solvent such as water so as to be within the osmotic pressure range. In addition, when the medium is a synthetic medium such as Chu13 medium, AF-6 medium, or f / 2 medium, an osmotic pressure regulator is added to the synthetic medium so as to be within the above osmotic pressure range. Good. As used herein, the term “osmotic pressure regulator” is a drug for regulating the osmotic pressure of a medium. There is no particular limitation as long as the drug can adjust the osmotic pressure of the solution and has no or little influence on the survival or growth of hydrocarbon-producing algae. A metal salt generally used as an osmotic pressure regulator is also suitable as an osmotic pressure regulator in the medium of this embodiment. Examples of such metal salts include sodium chloride, calcium chloride, magnesium chloride, and calcium chloride.

  The medium of this embodiment may be in a liquid state or a solid state (including a powder state and a granular state). In the solid state, it is dissolved in an appropriate solvent such as water before use. In addition, the medium of this embodiment is a kit containing a synthetic medium and an osmotic pressure regulator separately, and a necessary amount of osmotic pressure regulator is added to the synthetic medium before use so that the medium has a predetermined osmotic pressure. It can also be configured as described above. In this case, by adjusting the amount of the osmotic pressure regulator added, it is possible to provide a medium for producing hydrocarbon high-yield alga bodies corresponding to hydrocarbon-producing algae in various main habitat water areas. The kit may be accompanied by instructions describing the type of hydrocarbon-producing algae to be used and the amount of osmotic pressure and / or osmotic pressure regulator added to the medium of this embodiment applied to the kit.

  The medium of this embodiment can also be used as a medium for survival or growth of the produced hydrocarbon high-yield algal cells.

5-3. Effect According to the medium for producing a hydrocarbon high-yield alga body of the present invention, the hydrocarbon high-yield alga body according to the second aspect is easily produced from a hydrocarbon-producing alga having an intercellular matrix. Can do.

<Example 1: Preparation of medium for producing hydrocarbon high-yield alga bodies and production of hydrocarbon high-yield alga bodies (1)>
(the purpose)
The conditions and the like of the medium for producing hydrocarbon high-yield alga bodies for efficiently producing hydrocarbon high-yield alga bodies from hydrocarbon-producing algae having an intercellular matrix were verified.

(material)
In all Examples, B. braunii Showa strains having a main inhabiting water area as a fresh water area were used as hydrocarbon-producing algae having an intercellular matrix.

As the medium, an artificial seawater medium and a modified Chu13 medium were used.
Artificial seawater medium is MgCl ₂・ 6H ₂ O (9.474 mg / L), CaCl ₂・ 2H ₂ O (1.326 mg / L), Na ₂ SO ₄ (3.505 mg / L), KCl (597 mg / L), NaHCO ₃ (171 mg / L), KBr (85 mg / L), Na ₂ B ₄ O ₇ / 10H ₂ O (34 mg / L), SrCl ₂ (12 mg / L), NaF (3 mg / L), LiCl (1 mg / L ), KCl (0.07mg / L), CoCl ₂・ 6H ₂ 0 (0.0002mg / L), AlCl ₃・ 6H ₂ 0 (0.008mg / L), FeCl ₃・ 6H ₂ O (0.005mg / L), Na ₂ WO ₄・ 2H ₂ O (0.0002 mg / L), (NH ₄ ) ₆ Mo ₇ O ₂₄・ 4H ₂ O (0.02 mg / L), MnCl ₂・ 4H ₂ O (0.0008 mg / L) and NaCl (20.747 mg / L) Daigo artificial seawater SP was adjusted to 1/2 and 1/4, respectively, KNO ₃ (600 mg / L), K ₂ HPO ₄ / 3H ₂ O (52 mg / L), FeNaEDTA (10 mg / L) L) was added to the solution which was dissolved in pure water, H ₃ BO ₃ as a trace element _{(572mg / L), MnSO 4} · H 2 O (308mg / L), ZnSO 4 · 7H 2 O (44mg / L), _{CuSO 4 · 5H 2 O (16mg} / L), a solution of _{Na 2 MoO 4 · 2H 2 O} (12mg / L) and _{CoSO 4 · 7H 2 O (18mg} / L) solution prepared was added 5 mL. The osmotic pressure is 1/2 dilution of the artificial seawater medium with pure water (hereinafter referred to as “1/2 diluted artificial seawater”) and 1/4 diluted (hereinafter referred to as “1/4 diluted artificial seawater”). Was used).

In addition, the modified Chu13 medium includes KNO ₃ (600 mg / L), MgSO ₄ 7H ₂ O (100 mg / L), K ₂ HPO ₄ 3H ₂ O (52 mg / L), CaCl ₂ 2H ₂ O (54 mg / L). L) and FeNaEDTA (10 mg / L) dissolved in pure water, H ₃ BO ₃ (572 mg / L), MnSO ₄ · H ₂ O (308 mg / L), ZnSO ₄ · 7H ₂ O as trace elements _{(44mg / L), CuSO 4} · 5H 2 O (16mg / L), Na 2 MoO 4 · 2H 2 O (12mg / L) and CoSO ₄ · 7H ₂ O those prepared (18mg / L) 5mL added Solution.

The pH of each medium was adjusted with dilute sulfuric acid so as to be in the range of 7.2 to 7.5.
The osmotic pressure of the 1/2 diluted artificial seawater is 600 mOsm, the osmotic pressure of the 1/4 diluted artificial seawater is 300 mOsm, and the osmotic pressure of the modified Chu13 medium is 14 mOsm.

(Method)
As a preculture, the B. braunii Showa strain was cultured in a modified Chu13 medium for 19 days. 1/2 diluted artificial seawater or 1/4 diluted artificial seawater: Inoculated so that the culture solution reached the first half of the stationary phase = 1: 0.4, followed by culturing. After 34 days, measure the algal growth rate, hydrocarbon content rate and hydrocarbon recovery rate in each culture solution, and add new 1/2 diluted artificial seawater or 1/4 diluted artificial seawater, a part of the culture solution. Planting was continued so that the liquid volume ratio was 1: 0.4, and the culture was performed again. Similarly, after 74 days, 137 days, and 178 days after the first planting, the growth rate of algal bodies, hydrocarbon content, and hydrocarbon recovery rate in the culture solution are measured, and transplantation is continued at the above liquid ratio. did. Therefore, the actual osmotic pressure of each medium at 34 days, 35-74 days, 75-137 days and 138-178 days after seeding is 385.74 mOsm (34 days after seeding) in 1/2 diluted artificial seawater, 1/4 dilution artificial seawater becomes 192.87mOsm, 243.93mOsm, 258.52mOsm and 262.68mOsm, respectively. In addition, when 1/2 dilution artificial seawater was used, since the alga body died 34 days after planting as will be described later, no further culture was performed. The modified Chu13 medium is a medium commonly used in the culture of B. braunii, and the B. braunii Showa strain used in this example is an alga body subcultured for several years in the modified Chu13 medium. is there. Therefore, it is considered that there is no significant change in algal body growth rate, hydrocarbon content rate and hydrocarbon recovery rate by subculture, so algal body growth rate and hydrocarbon content in the culture solution at any time point It is sufficient to measure the rate and hydrocarbon recovery. Therefore, in this example, the algal growth rate, hydrocarbon content rate, and hydrocarbon recovery rate were measured only for the culture solution on the 19th day of pre-culture. That is, the algal cells cultured in the modified Chu13 medium are not hydrocarbon high-yield algal cells, but a wild-type B. braunii Showa strain for control.

All cultures were performed in a roux-type culture bottle (volume: 1000 mL, Tokyo Glass Instrument Co., Ltd.) with a culture temperature (liquid temperature) of 25 ° C., light irradiation conditions of photon flux density of 40-60 μmol / m ² / s and light / dark 12 hours. The cycle was carried out in static culture with a continuous supply of sterile air with a carbon dioxide concentration of 1.0%.

  The algal body growth rate was calculated as a value (g / L / week) obtained by subtracting the algal body concentration (g / L) at the time of seeding from the algal body concentration (g / L) at the time of recovery. Algae concentration is determined by dropping the algae onto glass fiber filter paper (particle retention capacity 1.6μm, GE Healthcare Japan Ltd.), washing the algae with a sufficient amount of pure water, drying at 105 ° C, and then drying It was measured by measuring the weight.

  The total mass of the hydrocarbon was measured by the following procedure. First, a predetermined amount of the aqueous slurry obtained in the concentration step was freeze-dried (FDU-2200 type, Tokyo Rika Kikai Co., Ltd., cooling temperature -80 ° C., degree of vacuum 25 Pa). This lyophilized product was put into a glass flask, and a sufficient amount of hexane was added to extract hydrocarbons. “Extracting hydrocarbons by adding a sufficient amount of hexane” means that extraction is performed by adding hexane to the lyophilized product until pigment components are no longer extracted even if additional hexane is added to the lyophilized product. It means repeating the operation. Under this extraction condition, the total amount of hydrocarbons accumulated in the intercellular matrix of the microalgae is extracted. The pigment component was removed from the obtained hexane extract by silica gel column chromatography (developing solvent: hexane) to obtain a purified hydrocarbon fraction. The solvent was evaporated from the purified fraction using a rotary evaporator, the obtained residue was dried under reduced pressure, and the dry mass was measured. This dry mass was taken as the total mass of hydrocarbons.

  200 mL of the culture broth was placed in a separatory funnel with 200 mL of hexane, stirred for 30 seconds, and then the organic phase was recovered. After recovering the intermediate layer of algae, it was mixed with 100 mL of fresh hexane and stirred again for 30 seconds. Later, the organic phase was recovered. After the collected organic phases were mixed, the pigment component was removed from the obtained hexane extract by silica gel column chromatography (developing solvent: hexane) to obtain a purified hydrocarbon fraction. The solvent was evaporated from the purified fraction using a rotary evaporator, the obtained residue was dried under reduced pressure, and the dry mass was measured. This dry mass was taken as the amount of recovered hydrocarbon. The hydrocarbon recovery rate was calculated by dividing the recovered amount by the total mass.

(result)
(1) Relationship between osmotic pressure and algal body growth rate The results are shown in FIG. However, growth rate data on the 34th day after seeding of 1/2 artificial seawater are not shown. This is because yellowing of the culture solution was confirmed at the medium osmotic pressure (385.74 mOsm) during this culture period. Yellowing indicates chlorophyll fading and subsequent algal body death. This means that in the case of hydrocarbon-producing algae whose main inhabiting waters are freshwater such as B. braunii, it is impossible to survive if the osmotic pressure of the medium is 385.74 mOsm or more. On the other hand, in the case of 1/4 dilution artificial seawater having an osmotic pressure of 192.87 mOsm to 262.68 mOsm, B. braunii survived and survived for a long period of 178 days, although a slight decrease was observed by subculture. It was revealed that growth was possible.

(2) Relationship between osmotic pressure and hydrocarbon content Figure 4 shows the results. Since B. braunii died in 1/2 diluted artificial seawater as described above, only the results of modified Chu13 medium and 1/4 diluted artificial seawater are shown here. As shown in this figure, when B. braunii was cultured in 1/4 diluted artificial seawater, the hydrocarbon content in the algal bodies increased compared to when cultured in the modified Chu13 medium in any culture period. This is considered because the hydrocarbon content was improved by reducing the viscous component of the intercellular matrix.

(3) Relationship between osmotic pressure and hydrocarbon recovery rate Fig. 5 shows the results. As described above, in the 1/2 dilution artificial seawater with an osmotic pressure of 385.74 mOsm, B. braunii died, so here, only the results of the modified Chu13 medium and 1/4 dilution artificial seawater are shown. When B. braunii was subcultured in 1/4 dilution artificial seawater, the recovery rate (diamond spot) reached 85% or more after 35 to 74 days corresponding to the second passage. On the other hand, in the culture using the modified Chu13 medium of B. braunii, the recovery rate was 5% or less (specifically, 3.1%) in the recovery method using only mixing with organic solvent and stirring. On the other hand, the modified Chu13 medium showed only the recovery rate (round spot) on the 19th day. Only 3.1% of hydrocarbons could be recovered from B. braunii, which is not a hydrocarbon high-yield alga body passaged on modified Chu13 medium. From this result, by culturing B. braunii in 1/4 dilution artificial seawater, extraction of hydrocarbons from algal cells only requires mixing with hexane and stirring, and the length of the incubation period of hydrocarbons As a result, the recovery rate dramatically increased, reaching 85% or more around 70 days of culture.

<Example 2: Relationship between continuous culture and hydrocarbon recovery rate in medium for producing hydrocarbon high-yield alga bodies>
(the purpose)
In Example 1, when a 1/4 diluted artificial seawater was used, it was possible to achieve a high hydrocarbon recovery rate with the passage of culture time by culturing by culturing in a medium in which the osmotic pressure gradually increased. Therefore, it was verified whether the same effect can be obtained even when continuous culture is performed for a long time under the same osmotic pressure.

(Method)
The basic method was performed according to the method of Example 1. However, in this example, the culture solution of B. braunii Showa strain cultured for 19 days in a modified Chu13 medium as a preculture was added to 1/4 diluted artificial seawater to 1/4 diluted artificial seawater: culture solution = 1: 0.4. The seedlings were inoculated and continuously cultured without grafting. After recovering the culture solution on the 34th and 81st days after seeding, the hydrocarbon recovery rate was calculated in the same manner as in Example 1 from the algae in each culture solution.

(result)
The results are shown in FIG. As shown in this figure, even in the case of continuous culture under the same osmotic pressure (here, 192.87 mOsm), the recovery rate of hydrocarbons from algal bodies increased with the passage of culture time, and about 30 after planting. On day, 30% or more equivalent to about 10 times that of the modified Chu13 medium reached 95% or more about 32 times that of the modified Chu13 medium about 80 days after seeding. From this result, according to the medium for producing hydrocarbon high-yield alga bodies and the method for producing hydrocarbon high-yield alga bodies according to the present invention, the osmotic pressure of the culture medium used for each planting of the alga body and each planting joint It became clear that hydrocarbon high-yield alga bodies can be produced regardless of the increase.

<Example 3: Preparation of medium for producing hydrocarbon high-yield alga bodies and production of hydrocarbon high-yield alga bodies (2)>
(the purpose)
In Example 1, although the dilution liquid of the artificial seawater culture medium was used as a medium for producing hydrocarbon high-yield alga bodies, it was verified whether the same effect could be obtained even with the dilution liquid of the natural seawater culture medium.

(Method)
The same procedure as described in Example 1 was performed, except that the medium for producing hydrocarbon high-yield algal bodies was changed from an artificial seawater dilution to a natural seawater dilution. Specifically, it is as follows.

The 1/4 diluted natural seawater medium used in this example was obtained by collecting seawater off the Izu-Nanashima island and diluting it to 1/4 with pure water, then KNO ₃ (600 mg / L), K ₂ HPO ₄ · 3H ₂ O (52 mg / L), FeNaEDTA (10 mg / L), was added, further H ₃ BO ₃ as a trace element _{(572mg / L), MnSO 4} · H 2 O (308mg / L), ZnSO 4 · 7H ₂ O (44 mg / L), CuSO ₄・ 5H ₂ O (16 mg / L), Na ₂ MoO ₄・ 2H ₂ O (12 mg / L) and CoSO ₄・ 7H ₂ O (18 mg / L) were prepared. A solution prepared by adding 5 mL.

  B. braunii Showa strain cultured for 19 days in a modified Chu13 medium as a preculture was inoculated so that the 1/4 diluted natural seawater: the culture solution reached the first period of stationary phase = 1: 0.4, followed by culture Went. After 33 days, while measuring the algal body growth rate and hydrocarbon recovery rate in each culture solution, a new 1 / 2-dilution artificial seawater or 1 / 4-dilution artificial seawater, a part of the culture solution with a liquid volume ratio of 1 : Planted to 0.4, and cultured again. Similarly, the growth rate of algal bodies and the hydrocarbon recovery rate in the culture broth were measured 62 days after the first seeding. The actual osmotic pressure of each medium on the 33rd day and 34-62 days after seeding is 240.08 mOsm and 304.62 mOsm, respectively.

(result)
The results are shown in FIGS. From the results shown in FIG. 7, in the subculture in the 1/4 natural seawater medium, the algal growth rate was slightly reduced as compared with the 1/4 artificial seawater medium, but even in the culture for 62 days. Survival and growth have been shown to be possible. In addition, from the results shown in FIG. 8, the recovery rate of hydrocarbons from alga bodies increased with the passage of culture time even in 1/4 natural seawater medium, and about 30 days after seeding, the modified Chu13 medium (3.1%) More than 16%, which is more than 5 times, and more than 80%, which is more than 25 times that of the modified Chu13 medium, reached about 62 days after seeding. From this result, regardless of whether the medium used by adjusting the osmotic pressure of the medium to be used within a predetermined range and culturing the hydrocarbon-producing algae is artificial seawater or natural seawater, the high yield of hydrocarbons of the present invention can be obtained. It was demonstrated that algal bodies can be produced.

<Example 4: Preparation of medium for producing hydrocarbon high-yield alga bodies and production of hydrocarbon high-yield algal bodies (3)>
(the purpose)
In Examples 1 to 3, the culture medium was a dilution based on an artificial or natural seawater culture medium. Therefore, it was verified whether a synthetic salt medium in which a osmotic pressure was adjusted to a predetermined value with a osmotic pressure adjusting agent in a medium based on a synthetic medium could obtain the same effect as a diluted solution of a seawater medium.

(Method)
The same procedure as described in Example 1 was performed, except that the medium for producing hydrocarbon high-yield algal bodies was changed from an artificial seawater dilution to a synthetic saline medium. Specifically, it is as follows.

  The synthetic saline medium used in this example was a solution prepared by adding NaCl (8.80 g / L) to the modified Chu13 medium described in Example 1 above to an osmotic pressure comparable to 1/4 artificial seawater ( Modified Chu13 + NaCl medium).

  The culture was carried out by inoculating the B. braunii Showa strain, which had been cultured for 19 days in a modified Chu13 medium as a preculture, so that the above-mentioned synthetic saline medium: the culture solution reached the first period of stationary phase = 1: 0.4, followed by culturing. . After 33 days, while measuring the algal body growth rate and hydrocarbon recovery rate in each culture solution, a new 1 / 2-dilution artificial seawater or 1 / 4-dilution artificial seawater, a part of the culture solution with a liquid volume ratio of 1 : Planted to 0.4, and cultured again. 62 days after the first planting, the algal growth rate and the hydrocarbon recovery rate in the culture were measured in the same manner. The actual osmotic pressure of each medium on the 33rd and 34-62 days after seeding is 227.61 mOsm and 288.59 mOsm, respectively.

(result)
The results are shown in FIGS. From the results shown in FIG. 9, in the subculture in the synthetic saline medium (modified Chu13 + NaCl medium), the algal body growth rate was decreased as compared with the modified Chu13 medium, similarly to the 1/4 artificial seawater medium. However, it was shown that it can grow even in 62 days of culture. In addition, from the results shown in FIG. 10, the recovery rate of hydrocarbons from algal bodies increased with the passage of culture time even in the synthetic saline medium (modified Chu13 + NaCl medium), and about 30 days after seeding, the modified Chu13 medium ( It reached 71% or more equivalent to 20 times more than 3.1%), and reached 98% or more equivalent to 32 times or more of the modified Chu13 medium about 62 days after seeding. From this result, regardless of whether the medium used by culturing hydrocarbon-producing algae by adjusting the osmotic pressure of the medium to be used within a predetermined range, the high yield of the hydrocarbon of the present invention It has been proved that algae bodies can be produced.

<Example 5: Recovery of hydrocarbons using resinous fat and oil adsorption sheet from hydrocarbon high-yield alga bodies>
(the purpose)
In order to recover hydrocarbons from the conventional algal bodies of hydrocarbon-producing algae, after pretreatment such as drying treatment and heat treatment as described above, it can be efficiently recovered unless extracted with an organic solvent. could not. In this example, it was verified that the hydrocarbon high-yield algal bodies of the present invention can recover hydrocarbons from algal bodies without performing a pretreatment such as a drying treatment or a heat treatment, and without using an organic solvent.

(Method)
Filtered high yielding algal bodies of hydrocarbons cultured in 1/4 artificial seawater for 70 days through a mesh with a pore size of 20μm, and sandwiched between resin oil and fat adsorption sheets (GATSBY OIL CLEAR SHEET, Mandom Co., Ltd.), 40-50kPa Pressed with weight. After pressing, the algal bodies on the sheet were removed by washing, and the hydrocarbon adsorbed on the sheet was visually observed.

(result)
The results are shown in FIG. A shows the resin fat-and-oil adsorbing sheet before pressing used in this example, and B shows the resin-made fat and oil adsorbing sheet washed after pressing the algal bodies. As indicated by A, the resin-made fat-and-oil adsorbing sheet has a water-repellent effect, so even if water adheres, it does not penetrate into the sheet and forms water droplets. On the other hand, squalene has a high adsorptivity to this resinous fat and oil adsorption sheet. The genus Botryococcus is known to produce squalene such as methyl squalene. However, even if it is cultured in a normal medium (for example, Chu13 medium), the alga body is sandwiched between the resin fat and oil adsorption sheet and pressed. Almost no adsorption (data not shown). However, when the hydrocarbon high-yield algal cells of the present invention were sandwiched and pressed between the resin-made fat and oil-absorbing sheets, the fats and oils that seemed to be squalene were adsorbed on the sheets as shown in B (those with black spots) ). From this result, the hydrocarbon high-yield alga body of the present invention is changed to a property in which the alga body itself easily collects hydrocarbons by culturing the hydrocarbon-producing alga in a medium having a predetermined osmotic pressure, As a result, it was shown that hydrocarbons can be recovered without performing pretreatment such as drying treatment and heat treatment, and without using an organic solvent.

Claims (10)

A method for producing a hydrocarbon high-yield algal body,
Culturing Botryococcus genus algae having an intercellular matrix for 38 hours or more in a medium having a viable osmotic pressure higher than the osmotic pressure of the main habitat of the algae, and applying an osmotic shock to the algae. The said manufacturing method containing.   The production method according to claim 1, wherein a main habitat water area of the algae is a fresh water area.   The manufacturing method of Claim 2 whose osmotic pressure of the said culture medium is larger than 14mOsm and is 385mOsm or less. A method for producing hydrocarbons using Botryococcus algae having an intercellular matrix,
Culturing for 38 hours or more in a medium having an osmotic pressure higher than the osmotic pressure of the main aquatic habitat of the algae, and applying an osmotic shock to the algae, and the algae after the culturing step The said hydrocarbon manufacturing method including the extraction process of extracting a hydrocarbon from an algal body by a physical method and / or a chemical method. The hydrocarbon production method according to claim 4 , further comprising a concentration step of concentrating the algal bodies in the culture solution after the culturing step. The hydrocarbon production method according to claim 4 or 5 , further comprising an algal body recovery step of recovering the hydrocarbon high-yield alga bodies after the extraction step for use in the culturing step. The hydrocarbon production method according to any one of claims 4 to 6 , wherein the osmotic pressure of the medium is larger than 14 mOsm and not larger than 385 mOsm. The hydrocarbon production method according to claim 7 , wherein the hydrocarbon is a linear alkene derived from a fatty acid or a terpene. A hydrocarbon production system using Botryococcus algae having an intercellular matrix,
Culturing for 38 hours or more in a medium having a viable osmotic pressure higher than the osmotic pressure of the main aquatic habitat of the algae, and a culture part for imparting an osmotic shock to the algae, The said hydrocarbon production system containing the extraction part which extracts a hydrocarbon from a physical method and / or a chemical method. The hydrocarbon production according to claim 9 , further comprising an algal body recovery unit that recovers the hydrocarbon high-yield algal bodies after hydrocarbon extraction in the extraction unit, and supplies the algal bodies to the culture unit again. system.

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