WO2016010149A1

WO2016010149A1 - Method of acclimating algae belonging to aurantiochytrium sp. to low-salt conditions

Info

Publication number: WO2016010149A1
Application number: PCT/JP2015/070609
Authority: WO
Inventors: 真也福田; 石根鈴木; 信渡邉
Original assignee: 国立大学法人筑波大学
Priority date: 2014-07-18
Filing date: 2015-07-17
Publication date: 2016-01-21
Also published as: JPWO2016010149A1; JP6611715B2

Abstract

The present invention provides a method of producing an alga belonging to Aurantiochytrium sp. capable of being populated and producing a material under low salt conditions, a method for preparing hydrocarbon by using the alga, and a method for treating sewage by using nutrient anabolism of the alga.

Description

Method for acclimatizing low salinity conditions of Aurantiochytrium algae

The present invention relates to a method for producing an aurantiochytrium algae capable of growth and substance production under low salt concentration conditions, a hydrocarbon production method using the algae, and a sewage treatment method using the algae. About.

Biomass, especially biofuel, obtained from biological assimilation products has recently attracted attention due to problems such as global warming or depletion of reserve resources. Among these biomass, oils such as hydrocarbons and triacylglycerol produced by microorganisms do not compete with food, have high energy production efficiency, and can easily expand the production scale. It is desired.

Examples of microorganisms used for substance production include algae belonging to Labyrinthulomycetes. Labyrinthula algae have been reported to produce various hydrocarbons and fats and oils, and are attracting attention as promising materials for substance production technology using microorganisms. For example, a substance-producing labyrinthula algae having a property of accumulating a large amount of highly unsaturated fatty acids such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) (SR21 strain, Patent Document 1: Japanese Patent No. 2764572) And what produces squalene is known. (Non-patent document 1: G. Chen. Et al. New Biotechnology 27, 382-289 (2010); Non-patent document 2: Q. Li et al., J. Agric. Food Chem. 57 (10), 4267- 4272 (2009); Non-Patent Document 3: KW Fan et al., World J. Microbiol. Biotechnol. 26, 1303-1309 (2010)).

Among the Labyrinthula algae, the Aurantiochytrium algae defined in 2007 are heterotrophic algae that inhabit brackish waters, assimilate nutrients in water, produce lipids, Have the characteristics of accumulating. Hetani et al. Identified a squalene-producing auranthiochytrium algae tsukuba-3, and its production efficiency is a hydrocarbon that has been studied for industrial use such as Botryococcus braunii. It was found to be much better than the production algae (Non-patent Document 4: BioScience, Biotechnology, and Biochemistry 75, 2246-2248).

As for the culture conditions of the Aulanthiochytrium algae, diligent studies have been conducted. Usually, GTY medium (D (+) glucose 20 g / L, tryptone 10 g / L, yeast extract 5 g / L, artificial seawater 8. 5 g / L) is inoculated with an inoculum and cultured by stirring at room temperature (Patent Document 2: Table 2012/0777799). There has also been reported a technology for reducing the cost of medium supply and promoting purification of wastewater by using eutrophic wastewater such as dialysis drainage, food wastewater, and sewage instead of GTY medium as a culture medium (patent) Document 3: JP 2014-108101).

Here, since Aurantiochytrium algae are marine microalgae that inhabit brackish water, normal culture requires a salinity level close to that of seawater. Accordingly, the alga has a markedly reduced growth rate or substance production rate or is killed under low salt concentration conditions. Such algae culture can cause the following disadvantages.

Aulanthiochytrium algae are heterotrophic algae that do not perform photosynthesis, so when culturing in a large-scale plant, autotrophic that performs photosynthesis without requiring a large surface area culture tank considering photosynthesis efficiency. Compared to algae, there is an advantage that the location of the plant can be secured relatively easily. However, plant locations for such algae that require high salinity media are necessarily limited to coastal areas where seawater is readily available as a salinity source.

In addition, if a medium with a high salinity concentration is used for culturing the above algae, wastes such as waste liquids and residues with a high salinity concentration will be generated during the cultivation process. Equipment and costs are required, which is a burden in commercialization. In addition, since the high concentration of salt contained in the medium deteriorates the plant structure, the culture plant requires surface treatment such as rust prevention to have salt tolerance, and the construction costs and maintenance costs of the plant are excessive. There is also a risk of becoming.

Furthermore, when various eutrophic wastewaters are to be used for algae culture following the above prior art, a large amount of salt is added to the wastewater for culturing, and the added salt from the wastewater after cultivation is processed at a cost. And the process becomes redundant. Although the use of eutrophic wastewater for algae culture also serves as wastewater purification, if wastewater that needs to be treated is newly produced, it will be overturned.

Therefore, the technology that enables the cultivation of marine algae, Aulanthiochytrium algae, at a low salinity level that does not cause the above-mentioned adverse effects, has been developed with an eye toward industrial use of hydrocarbon production technology by culturing such algae. The demand in this technical field is extremely large.

There are known techniques for adapting microorganisms to specific environments. For example, Osaka University's group includes high-temperature acclimated strains of Escherichia coli (Non-patent Document 5: Kishimoto et al 2010, PLoS Genetics., 6 (10), e1001164) and ethanol-resistant strains (Non-patent Document 6: Horinouchi et al. 2010, BMC Genomics., 11 (579), e579) has been reported. Both documents describe that an environmentally-adapted E. coli strain was obtained by applying stepwise environmental stresses over a period of several hundred days.

However, the environmental adaptation process in E. coli cannot be discussed in the same way as eukaryotic microorganisms. In the study of artificial evolution (laboratory evolution), the generation time of the starting microorganism significantly affects the success or failure of environmental adaptation. That is, since the environmental resistance of microorganisms is acquired by repeating many generational changes, the frequency of environmental adaptation increases as the frequency of generational changes increases (the generation time decreases). Here, the generation time of Escherichia coli used in the research of the Osaka University group is about 20 minutes, that is, 72 generations are changed within one day. On the other hand, the generational change of microalgae is remarkably slower than that of Escherichia coli. For example, the generation time of Aulanthiochytrium algae used in the present invention is 2.86 hours, that is, the number of generational changes per day is only 8.4. This shows that environmental adaptation occurs only at a rate of 8.6 times that of E. coli. Therefore, if it is assumed that algae acquire resistance to environmental stress through the same number of generations as E. coli, acclimatization for a long period of several years to 10 years is required to obtain environmentally compliant strains. End up.

Accordingly, even if a person skilled in the art attempts to adapt the environment of the Aulanthiochytrium algae with reference to the above prior art, the time and labor required to obtain the desired strain will be enormous. Considering that it is uncertain whether the target stock will be obtained or not, no matter how much time is taken.

: Japanese Patent No. 2764572 : Table 2012-1277799 gazette : JP-A-2014-108101

Hetani et al. Established the Aurantiochytrium tsukuba-3 strain with high squalene production ability, and are conducting research and development of various culture techniques using the strain. However, as mentioned above, the marine algae strain requires a high concentration of salinity in culture, and such characteristics significantly impede the industrial utilization of hydrocarbon production technology using the strain. And there is a risk of damaging its value.

As a result, the inventors have intensively studied the method for obtaining auranthiochytrium algae acclimated to a low salinity condition that can be cultured at a salinity lower than that in a normal growth environment. In addition, by gradually reducing the salt concentration in the algae culture medium, it is possible to obtain an acclimatized strain that can finally grow in a medium with a salt concentration of several thousandths (~ 10 ppm) of seawater. It has been found. Even more surprisingly, the low algal acclimation process of the algae in the present invention has been shown to be achieved in a much shorter period of time than expected from the prior art, about 100 days.

In addition, the inventors examined the acquisition of a low-salinity-resistant strain of auranthiochytrium algae by mutagenesis using a mutagen under various conditions, but in the end, a desired resistant strain could not be acquired. There wasn't.

In the acclimation culture, the algae are subcultured in a relatively short cycle of 120 hours, and the culture temperature is set to 20 ° C., which is 5 ° C. lower than the optimum temperature condition of 25 ° C., so that the high growth potential is long. The period is maintained. Maintaining the growth activity of the algae at a high level has led to a drastically accelerated acquisition of resistance to low salinity stress.

Accordingly, the present application provides the following inventions.
1. A method for producing an aurantiochytrium algae capable of growth and substance production under low salinity conditions, comprising the following steps:
(I) a step of providing auranthiochytrium algae cultured in a culture medium to which a salt content corresponding to 10% (v / v) or more of seawater is added;
(Ii) culturing the algae in a culture medium in which the amount of salt added is less than that in the above step, and substituting the algae until the growth rate of the algae reaches a reference value; and (iii) the above step (Ii) is repeated at a lower salt addition amount to obtain an auranthiochytrium algae capable of growth and substance production under desired salt concentration conditions;
Including the production method.
2. Item 2. The production method according to Item 1, wherein the Aulanthiochytrium algae is Aulanthiochytrium tsukuba-3 strain (Accession No. FERM BP-11442).
3. Item 3. The production method according to any one of

Items

1 and 2, wherein the decrease in the amount of salt added is reduced to 1/2 to 1/50 of the amount added so far.
4). Item 4. The production method according to any one of Items 1 to 3, wherein the desired salt concentration is 100 ppm or less.
5. Item 5. The production method according to any one of Items 1 to 4, wherein the culture medium contains 20 g / L of D (+) glucose, 10 g / L of tryptone, and 5 g / L of yeast extract.
6). In addition, the following steps:
(Iv) The algae obtained in the above step (iii) is cultured in a culture medium in which the addition amount of one or more components other than salt is reduced, and the culture is continued until the growth rate of the algae reaches a reference value. And (v) repeating the above step (iv) by further reducing the amount of the component added to obtain alanthiochytrium algae that can be cultured and produced under desired nutritional conditions. Process;
Item 6. The production method according to any one of Items 1 to 5, comprising
7). Item 7. The production method according to Item 6, wherein the decrease in the amount of the one or more components added to the culture medium is reduced to 80% to 90% of the content of the components until then.
8). Item 8. The production method according to any one of Items 6 and 7, wherein the one or more components are one or more of D (+) glucose, tryptone, and yeast extract.
9. Item 9. The production method according to any one of Items 6 to 8, wherein the desired nutrient condition is 70% or less of the nutrient component at the beginning of culture.
10. A method for producing a hydrocarbon, comprising culturing an auranthiochytrium algae produced by the method according to any one of Items 1 to 9 in a medium having a low salinity, and in the algal cells. Separating and purifying the hydrocarbon accumulated in the process.
11. Item 11. The production method according to Item 10, wherein the hydrocarbon is squalene.
12 Item 12. The method according to any one of Items 10 or 11, wherein the medium contains sewage.
13 Item 13. The method according to Item 12, wherein the sewage contains a salt content of 10 ppm or more.
14 Any of paragraphs 12 and 13, wherein any one or more of carbohydrates, organic acids, inorganic acids, organic bases, inorganic bases, vitamins, amino acids, peptides, proteins, and minerals are added to the sewage to adjust the components. The production method according to claim 1.
15. Item 15. The method for purifying sewage by using a culture medium containing sewage and assimilating nutrients in the sewage to algae in the production method according to Item 12-14.

Surprisingly, the marine microalgae, Aulanthiochytrium algae, can be reduced to about 50 to 100 days by gradually decreasing the salinity of the medium while being subcultured at a frequency that can maintain the logarithmic growth phase. It was found that acclimatization to low salinity can be achieved in a short period of time. Acclimatized strains obtained by the method of the present invention show inferior levels of growth and substance production rates compared to culturing in seawater. Therefore, by using the acclimatized strain obtained by the method of the present invention as a material for hydrocarbon production technology, it is possible to avoid the above-mentioned various disadvantages that are normally expected when the algae require a high salt concentration. I can do it.

FIG. 1 shows that the auranthiochytrium algae low salinity acclimated strain produced according to the present invention retains squalene production capacity. FIG. 2 shows that the auranthiochytrium algae low salinity acclimated strain produced according to the present invention is superior in growth ability compared to the wild strain in a low salinity medium prepared based on primary treated water of sewage. Indicates that FIG. 3 shows that an lanthanum chitolium algae low salinity acclimated strain produced according to the present invention is derived from sewage in a culture medium prepared using primary treated water of sewage and acid saccharified material derived from dried dehydrated sludge. It can be grown using the nutrients. FIG. 4 shows that an lanthanum chitolium algae low salinity acclimated strain produced according to the present invention is used in a culture medium prepared using sewage primary treated water and acid-dehydrated sludge derived from dried dehydrated sludge. It shows that it can accumulate lipid.

1. Aulanthiochytrium algae The microorganisms subjected to freshwater acclimation in the present invention may be any microorganisms belonging to the genus Aurantiochytrium. Specifically, for example, the above-mentioned Aurantiochytrium / tsukuba-3 strain can be used, but not limited to this, any strain having substantially the same mycological properties as the strain can be used. Can also be used.

The alanthiochytrium algae that are acclimated to fresh water in the present invention are microalgae belonging to Labyrinthulomycetes, are heterotrophic, and accumulate a large amount of hydrocarbons in cells. Labyrinthula belongs to the oomycete, but unlike other fungi, it is closely related to unequal hairy plants such as brown algae and diatoms. It constitutes the system.

The auranthiochytrium algae are marine algae newly defined in 2007 and have the characteristic of producing lipids and accumulating in cells. For example, Aurantiochytrium tsukuba-3 strain was collected and isolated from mangrove leaves inhabited by Okaya et al. The algae has been found to have superior hydrocarbon production capacity and growth compared to other microalgae such as Botriococcus brownies that were previously known to produce hydrocarbons. It is expected to be used for hydrocarbon production technology by algae culture. Aurantiochytrium tsukuba-3 shares have been deposited on December 9, 2010 to the National Institute of Advanced Industrial Science and Technology (address: 1st, 1st East, 1-chome, Tsukuba, Ibaraki, Japan) FERM BP-11442).

2. Normal Algal Culture Conditions The method for culturing the above Aulanthiochytrium algae is established in the art. That is, normal maintenance culture is carried out according to a conventional method after seeding in an appropriate medium prepared with components. Any known medium can be used as the medium. Examples of the carbon source include saccharides such as glucose, fructose, and saccharose. These carbon sources are used, for example, at a concentration of 20 to 120 g per liter of medium. Nitrogen sources include organic nitrogen such as sodium glutamate and urea, or inorganic nitrogen such as ammonium acetate, ammonium sulfate, ammonium chloride, sodium nitrate, and ammonium nitrate, or yeast extracts, corn steep liquor, polypeptone, peptone, tryptone, and other biological sources There are digestions. Moreover, the said culture medium can also contain vitamins suitably. The medium is prepared with artificial seawater with an appropriate salinity. Preferably, the medium has a final salinity greater than or equal to about 10% (v / v) or about 10% (v / v) to about 100 of seawater (salt concentration 3.4% (w / v)). % (V / v), such as a salt concentration of about 1.0 to 3.0% (w / v). More preferably, the medium has a final salinity of about 20% (v / v) to 80% (v / v), about 30% (v / v) to 70% (v / v) of seawater, It is prepared to be about 40% (v / v) to 60% (v / v), or about 50%.

In the present specification, “salt” means a mixture of main salts contained in seawater, that is, sodium chloride, magnesium chloride, magnesium sulfate, calcium sulfate, and potassium chloride, in the same composition ratio as seawater. In another embodiment, “salt content” means a mixture of 75% (w / w) or more of sodium chloride and one or more main salts contained in the seawater other than sodium chloride, or sodium chloride alone. To do.

Artificial seawater is a mixture of salts that can constitute various ionic compositions close to seawater by dissolving in water. Preferably, the artificial seawater contains sodium chloride, magnesium chloride, magnesium sulfate, calcium sulfate, potassium chloride, trace metals, organic matter, and the like at an appropriate ratio so as to imitate seawater. In the present specification, as a component of the medium of the present invention, sodium chloride in an amount constituting an aqueous solution isotonic with seawater may be used instead of artificial seawater. Alternatively, natural seawater or seawater concentrate may be used instead of artificial seawater.

Preferably, the medium is a 50% (v / v) artificial seawater supplemented with 20 g / L D (+) glucose, 10 g / L tryptone, and 5 g / L yeast extract (GTY medium).

The pH of the medium can be adjusted appropriately by adding an appropriate acid or base after preparation. The pH of the culture medium is pH 2.0-11.0, preferably pH 3.0-10.0, more preferably pH 4.0-9.0, more preferably pH 4.5-9.0, most preferably Use pH 6.5.

The above medium may be sterilized by an autoclave before seeding with the Aulanthiochytrium algae.

Cultivation is performed at a culture temperature of 5 to 40 ° C., preferably 10 to 35 ° C., more preferably 10 to 30 ° C. Passaging is usually performed every 1 to 10 days, preferably every 3 to 7 days. The culture can be carried out by aeration and agitation culture, shaking culture or stationary culture, but is preferably cultured by aeration and agitation culture or shaking culture.

Usually, in the maintenance culture of Auranthiochytrium algae, which are marine microalgae, it is essential to add salt to the culture solution at the same concentration as seawater to about 10% (v / v). The growth activity of the algae is remarkably reduced depending on the decrease of the salinity concentration in the maintenance medium, and the algae stop growing or die in the medium prepared without adding the salt.

3. Acclimatization Conditions Acclimatization culture of alanthiochytrium algae is carried out by reducing the salinity of the culture medium used for the normal maintenance culture. Preferably, the composition of the medium used for acclimatization to the low salinity is the same as the composition of the medium used for maintenance culture except that the salinity is changed. Alternatively, the composition of the maintenance medium may be appropriately changed in order to maintain the growth activity of the algae at a high level depending on the type, state, and implementation environment of the specific algae used in the method of the present invention.

The reduction of the salinity of the medium used for the low salinity acclimation culture is carried out by reducing the salinity of the fresh medium used for the passage of algae. The extent of the decrease in salinity is 2 to 1/100, and is selected depending on the specific type of algae used in the method of the present invention, the state, the operating environment, and the level of acclimatization desired. The success rate of acclimatization decreases as the range of decrease increases, and the time required for acclimatization increases. On the other hand, if the range of decrease is reduced, the success rate of acclimatization increases and the time required for acclimatization decreases.

If the desired level of acclimatization cannot be achieved by one time of acclimatization culture (such as 1/1000), by dividing the acclimatization culture into two or more stages, that is, by gradually reducing the salt concentration. Do. For example, when acquiring a strain that has been acclimatized to a salinity of 1/1000 of the normal salinity, first obtain a strain that has been acclimatized to 1/50 of the salinity, and further reduce the salinity to 1/20 By carrying out acclimatization by lowering, a strain having a desired acclimatization level can be obtained.

The passage of the algae in the low salinity acclimation culture is performed at a time point before the algal culture enters the stationary phase, preferably at the time of the logarithmic growth phase. Growth phase of algal culture, the absorbance measurement of _{OD 660,} can be easily determined by methods commonly used in the art. The stage of growth of the algae culture may be derived by actually sampling the algae culture and measuring OD ₆₆₀ , or refer to the growth curve generated under the same conditions in the algae. It may be estimated. For example, in the examples of the present invention, the Aulanthiochytrium tsukuba-3 strain is estimated to be in the logarithmic growth phase at 120 hours after seeding in a fresh medium so that the OD ₆₆₀ is 0.05. Then, the passage was performed at a cycle of 120 hours.

In the low salinity acclimation culture, various culture environments such as temperature, humidity, lighting, stirring speed, aeration rate, culture equipment configuration, etc. are appropriately adjusted so as to maintain the algal growth activity at a high level. Alternatively, when the growth rate is excessive and burdens the practitioner in terms of cost and labor, the culture environment is appropriately adjusted so as to maintain the growth activity of the algae at an appropriate level. For example, in the examples of the present invention, the auranthiochytrium tsukuba-3 strain was expected to have an excessive growth rate at the optimal culture temperature of 25 ° C., making it difficult to acclimate for a long time. Incubation was performed at 0 ° C.

In one embodiment, whether or not the algae has acclimatized to a certain salt concentration is determined by the growth rate of the algae reaching a reference value. That is, an acceptable level compared to the culture rate of the algae before reducing the salinity concentration (when trying to acclimatize an algae acclimatized to a certain salinity concentration to a lower salinity concentration, before attempting further acclimatization) When the growth rate is reached, it is judged that the salt concentration has been acclimatized. Preferably, the acceptable level is 50% or more relative to the culture rate of the algae before acclimatization. In this determination, the algal growth rate may be derived based on the rate at which the culture OD ₆₆₀ increases. In another aspect, whether or not the algae has acclimatized to a certain salt concentration may be determined by a substance production rate such as hydrocarbons reaching a reference value instead of a growth rate.

The time required to obtain the desired low salinity-adapted algal strain in the present invention is the type of algae used in the method of the present invention, the state, the implementation environment, the desired acclimatization level, and the decrease in salinity. Although it varies depending on the width, it can be acclimatized to a level of 1/1000 or less of a normal culture medium salinity (50% (v / v) artificial seawater) in several months. It takes several hundred days to acquire an environmental stress-resistant strain even in E. coli that far exceeds algae at a growth rate (Non-patent Document 5: Kishimoto et al 2010, PLoS Genetics., 6 (10), e1001164; Non-Patent Document 6 : Horinouchi et al. 2010, BMC Genomics., 11 (579), this is a shorter period than expected.

By applying the method of the present invention for obtaining a low salinity-adapted algal strain, auranthiochytrium algae can be acclimatized to conditions under which the amount of medium components other than salt is reduced. . In the acclimatization, the Aulanthiochytrium algae are cultured in a medium in which any one or more components are reduced from the algal culture medium.

The conditions used when acclimatizing to a medium in which any component has been reduced should be considered by those skilled in the art depending on the type of component to be reduced, It may be based on specified conditions.

For example, when using a GTY medium (50% (v / v) artificial seawater with D (+) glucose 20 g / L, tryptone 10 g / L, yeast extract 5 g / L) for algae culture, In the crystallization medium, the addition of one or more of glucose, tryptone and yeast extract is reduced. Preferably, in the conditioned medium, the addition amount of glucose, tryptone and yeast extract is reduced to 70% to 90% of the addition amount of normal culture medium.

Since Aulanthiochytrium algae flexibly change the growth rate according to the nutrient content in the medium, reducing the nutrient content of the GTY medium itself does not cause environmental stress. However, by reducing the nutrient components present in the medium at a relatively high concentration, the osmotic pressure of the medium is remarkably lowered, which becomes a stress on the algal cells. Actually, when the nutrient content of the GTY medium is decreased, the growth rate of the algae decreases. However, if the subculture is continued in the medium in the same manner as the low salinity acclimation procedure, the growth rate passes through a certain period. Slightly increases. This suggests that algae have acclimatized to the medium with reduced osmotic pressure by reducing the nutrient components of the GTY medium.

4). Characteristics of low salinity-adapted algae strains Auranthiochytrium algae are extremely promising materials for hydrocarbon production technology by algae culture, but they require high concentrations of salinity for cultivation. When considering the use, there were concerns about disadvantages such as plant location restrictions and increased costs. However, the auranthiochytrium algae strain acclimatized to the low salinity obtained by the method of the present invention has a level of growth and inferiority compared to when cultured in seawater under low salinity conditions. Because it shows the rate of substance production, using the acclimatized strain as a material for hydrocarbon production technology avoids the various disadvantages expected when using a high salinity medium for marine algae culture. I can do it.

The low salinity-adapted algal strain obtained according to the method of the present invention may be evaluated for physiological activities such as growth rate and substance production ability. As the means for determining the physiological activity, those usually used in the technical field are used. For example, if it is a growth rate, absorbance measurement, cell counting, etc., and if it is a substance production capacity, it is assumed that the product is quantified by isolation and purification, chromatography, gel electrophoresis, etc.

The acclimatized strain has a reduced physiological activity compared to the culture rate of algae cultured in a medium having a normal salinity under low salinity conditions, but the degree of the decrease is at an acceptable level. To what extent the reduction in physiological activity is tolerated is specifically determined by a person skilled in the art attempting to produce a substance using a low salinity-adapted algal strain obtained according to the method of the present invention. Judgment is made by comparing and considering the final product yield reduction due to, and various disadvantages due to the requirement of adding salt to the medium.

The salinity that can be acclimatized in the method of the present invention is 1 / 100th of the seawater (salt concentration of 3.4% (w / v)) (salt concentration of 340 ppm) or less, preferably 1/1000 (salt concentration of 34 ppm). ) Or less, more preferably 1/600 (salt concentration 20.2 ppm) or less, still more preferably 1 / 200,000 (salt concentration 10.1 ppm) or less. Usually, if the salinity concentration is 100 ppm or less, most of various disadvantages caused by the addition of the salinity to the medium can be eliminated. That is, if auranthiochytrium algae can be cultured in a medium with a salinity of 100 ppm or less, there is no need to consider the cost of adding salt, so the location of the culture facility is not limited to the coastal area, and culture wastewater is Since it is not necessary to remove salt during treatment, the wastewater treatment process can be simplified and cost can be saved. There is no risk of damaging the culture equipment due to salt in the medium. Furthermore, when eutrophic wastewater is to be used for algae culture, the acclimatized strain can be cultured at a salt concentration of 100 ppm or less, which is present in the wastewater from the beginning, so that it is easy to prepare the medium and treat the culture wastewater. become.

Construction of a freshwater-adapted mutant of the Aulanthiochytrium algae An attempt was made to obtain a strain adapted to a low salt concentration environment by mutagenesis using ethyl methanesulfonate (EMS) as a mutagen. Aurantiochytrium tsukuba-3 strain was added to 20 ml of GTY medium (50% (v / v) artificial seawater equivalent to seawater (Osaka Yakken) 1.7% (w / v), D (+) glucose (Wako) 20 g / L, tryptone (GIBCO) 10 g / L, yeast extract (GIBCO) 5 g / L) for 16 to 100 hours. EMS (Wako Pure Chemical Industries) was added to the culture so that the final concentration was 30 to 50 mM, and further cultured at 25 ° C. for 7 hours. 2.0 ml of 10% (w / v) Na ₂ S ₂ O ₃ was added to the culture. This was centrifuged at 1000 xg for 5 minutes at room temperature, and the supernatant was removed. The step of adding 1 ml of GTY medium to the precipitated cell pellet, centrifuging at 1000 × g for 5 minutes at room temperature, and removing the supernatant was repeated twice. 1 ml of GTY medium was added to the obtained cell pellet, the number of cells in the suspension was counted, and this was spread on a GTY agar plate to which no artificial seawater was added so as to obtain 3000 to 120,000 cells / plate. . The experimental procedure is shown in Table 1.

The above experiment was repeated 10 times or more while changing the preculture time, the EMS concentration, the number of cells developed on the agar plate, the subsequent screening conditions, etc., but the target freshwater adaptive mutant could not be obtained. .

Preparation of a freshwater acclimation strain of auranthiochytrium algae Auranthiochytrium tsukuba-3 strain was prepared by using 20 ml of GTY medium (50% (v / v) seawater equivalent artificial water (Osaka Yakuken) 1.7% (w / V), D (+) glucose (Wako) 20 g / L, tryptone (GIBCO) 10 g / L, yeast extract (GIBCO) 5 g / L) at 20 ° C. and 120 rpm for 72 hours. 1 ml of the medium was subcultured to a GTY medium prepared so that the salinity was 0.425% (w / v) (equivalent to 1.25% (v / v) seawater). Time swirling culture was performed. Further, the culture was subcultured in a GTY medium prepared so that the salinity was 20.2 ppm (equivalent to 0.06% (v / v) seawater) at a concentration of initial turbidity OD ₆₆₀ = 0.05. Then, swirl culture was performed at 20 ° C. and 120 rpm. At this time, multiple lines were prepared in consideration of the risk that the algae would be annihilated without being able to withstand the low salinity condition. In the middle, the passage was repeated to a new medium every 120 hours (initial turbidity OD ₆₆₀ = 0.05) to maintain each line. A line that was able to maintain physiological activity at an acceptable level through such a process was obtained as a 0.06% (v / v) seawater acclimated strain.

From the 0.06% (v / v) seawater acclimated strain, a strain that was viable under the condition that the salinity was further reduced was also obtained. The 0.06% (v / v) seawater acclimated strain culture obtained by performing acclimation culture in 0.06% (v / v) seawater for 1256 hours was further added to a salinity of 10.1 ppm (0 To a GTY medium prepared to be 0.03% (v / v seawater equivalent), and subcultured at a concentration of initial turbidity OD ₆₆₀ = 0.05, and subjected to swirling culture at 20 ° C. and 120 rpm. Subculture was repeated to a new medium every 120 hours (initial turbidity OD ₆₆₀ = 0.05), and the culture was carried out for 1200 hours. As a result, a 0.03% (v / v) seawater acclimated strain was obtained. .

From the 0.06% (v / v) seawater-acclimated strain, a strain that was viable under conditions in which the concentration of other medium components was further reduced was also obtained. The 0.06% (v / v) seawater acclimated strain culture obtained by performing acclimation culture in 0.06% (v / v) seawater for 334 hours was further added to D (+) glucose, tryptone, The initial turbidity OD ₆₆₀ = 0 in a GTY medium prepared such that the yeast extract content is reduced by 20% and the salinity is 20.2 ppm (equivalent to 0.06% (v / v) seawater). The cells were subcultured at a concentration of 0.05, and swirl culture was performed at 20 ° C. and 120 rpm. As a result of culturing for 451 hours, a 20% component weight loss / 0.06% (v / v) seawater acclimated strain was obtained.

Furthermore, the 20% component weight loss / 0.06% (v / v) seawater acclimated strain culture is reduced by 30% each in the content of D (+) glucose, tryptone, and yeast extract, and the salinity is reduced. The GTY medium prepared to 20.2 ppm (equivalent to 0.06% (v / v) seawater) was subcultured at a concentration of initial turbidity OD ₆₆₀ = 0.05 and swirled at 20 ° C. and 120 rpm. Cultured. As a result of carrying out the culture for 1627 hours, a 30% component weight loss / 0.06% (v / v) seawater-acclimated strain was obtained. The procedure for the above-mentioned acclimation strain production experiment is shown in Table 2.

Evaluation of acclimatized strain The success or failure of acclimatization under the above-mentioned conditions of low salinity or low salinity / component reduction is to pass OD ₆₆₀ = 0.05 and after culturing for 120 hours, reach OD ₆₆₀ ≧ 5.0. Judged as a guide.

Furthermore, it was also verified whether the acclimatized strain has squalene production capacity. 0.03% (v / v) seawater acclimated strain, 20% component weight loss / 0.06% (v / v) seawater acclimated strain, and 30% component weight loss / 0.06% (V / v) Seawater acclimated strains were collected by centrifugation, and the cell pellet was lyophilized. The lyophilized preparation was added to a mixed solution of chloroform and ethanol in a ratio of 2: 1 at a rate of 15 ml per 100 mg of dried cells, and this was incubated for 72 hours. 4 μl of a suspension formed by adding 1 ml of hexane to the obtained oil extract was developed in thin layer chromatography. The result of the experiment is shown in FIG. 0.03% (v / v) seawater acclimated strain, 20% component weight loss. 0.06% (v / v) seawater acclimated strain, and 30% component weight loss. 0.06% (v / v) seawater order In each of the chemical strains, a band corresponding to squalene was detected at the same position as the standard, and it was confirmed that all of the acclimated strains retained the squalene production capacity.

Cultivation of acclimatized strains using sewage Utilizing eutrophic wastewater such as sewage and industrial wastewater for aurantiochytrium algae culture is beneficial both in terms of reducing culture costs and promoting wastewater treatment. However, normal eutrophic wastewater does not contain enough salt to cultivate auranthiochytrium algae, and in order to use this as the basis of the medium, an appropriate amount of salt is added. Must. In addition, since the wastewater with a high salinity concentration that needs further treatment is discharged in the course of the culture, the advantage of the use of promoting the wastewater treatment is lost.

On the other hand, if the low-salinity acclimated strain of the Aulanthiochytrium algae produced as described above can survive under the salinity conditions that ordinary eutrophic wastewater contains, the wastewater is the basis. Since there is no need to add salt to the prepared medium, the above disadvantages do not occur.

Therefore, a verification experiment was conducted to determine whether the low salinity acclimated strain prepared by the method of the present invention can actually be cultured in a medium based on eutrophic wastewater having a low salinity.

A GTY medium was prepared based on primary treated water produced at the Sendai City Nansei Purification Center instead of 50% (v / v) seawater. The growth rate in the medium was compared between the wild strain of Aurantiochytrium tsukuba-3 and the low salinity acclimated strain (0.03% (v / v) seawater acclimated strain). .

The acclimatized strain and the wild strain were cultured in a GTY medium based on sewage having a low salt content instead of artificial seawater. The sewage used as the foundation of the culture medium is the primary treated water produced at the Sendai City Nansei Purification Center. The salinity of the primary treated water is less than 100 ppm (99 to 65 ppm), and the salinity of the medium prepared based on this is the same.

Prior to the experiment, both the acclimatized strain and the wild strain were cultured in 50 mL of normal GTY medium prepared with 50% (v / v) artificial seawater for 72 hours (20 ° C., 120 rpm swirl culture). Then, in a GTY medium prepared by adding D (+) glucose (Wako) 20 g / L, tryptone (GIBCO) 10 g / L, and yeast extract (GIBCO) 5 g / L to the primary treated water. The cells were cultured for 96 hours (20 ° C., 120 rpm rotating culture), and OD ₆₆₀ was measured at five time points from the start of the culture. The result is shown in FIG.

The left and right graphs in FIG. 2 show the same result, but the right figure has a logarithmic scale on the Y axis. Squares indicate acclimatized strains and circles indicate wild-type cell concentrations (OD ₆₆₀ ). As shown in the left figure, at 96 hours after the start of culture, the acclimated strain had OD ₆₆₀ = 13.9, the wild strain had OD ₆₆₀ = 4.9, and the final turbidity was 2.8 times different. occured. Further, as shown in the right figure, the acclimatized strain was μd ⁻¹ = 2.12, and the wild strain was μd ⁻¹ = 1.45, which showed a difference of 1.46 times in specific growth rate. Therefore, the figure shows that the low salinity acclimated strain prepared by the method of the present invention is significantly superior to the wild strain in the GTY medium based on sewage with low salt content instead of artificial seawater. It demonstrates that it has a proliferative ability.

Furthermore, it was examined whether organic substances derived from sewage can be used as nutrients for algae culture. 6 mL of 72% H ₂ SO ₄ was added to 0.6 g of dry dewatered sludge produced by sewage treatment, and reacted at 30 ° C. for 1 hour. To the reaction product, 102 mL of the primary treated water was added and incubated at 120 ° C. for 1 hour. The salt concentration of the primary treated water is less than 100 ppm (99 to 65 ppm), and the salt concentration of the finally prepared medium is almost the same. The suspension was neutralized with CaCO ₃ and adjusted to pH 5.3. The suspension was filtered, and the filtrate was obtained as an acid saccharified solution (saccharide content 3.2 g / L). To the acid saccharified solution, tryptone 10 g / L and yeast extract 5 g / L were added and sterilized in an autoclave at 120 ° C. for 20 minutes to prepare an algal culture medium based on sewage.

Using the medium described above, the low salinity acclimated strain (0.03% (v / v) seawater acclimated strain) was cultured for 96 hours (20 ° C., 120 rpm swirl culture), and five time points from the start of the culture. The sugar concentration and OD ₆₆₀ in the medium were measured. The result is shown in FIG.

The circle connected by the dotted line indicates the organic substance concentration (g / L) in the medium, and the circle connected by the solid line indicates the cell concentration (OD ₆₆₀ ). Approximately 40 hours after the start of the culture, the cultured algae enters the logarithmic growth phase, and at the same time, the sugar concentration starts to decrease. Then, the sugar concentration continues to decrease in response to the increase in cell concentration. In addition, cells at 76 hours after the start of culture were stained with Nile red, and it was observed that remarkable oil droplets were formed in the cells under a fluorescence microscope (FIG. 4).

Therefore, according to the experiment, the low salinity acclimated strain was grown and oiled using nutrients derived from sewage in a culture medium prepared using primary treated water of sewage and acid saccharified product from dried dehydrated sludge. It was shown that production can be performed. This is because the auranthiochytrium tsukuba-3 low salinity acclimated strain obtained using the method of the present application can perform good growth and substance production in a sewage-based medium. This suggests that the acclimated strain can promote the purification of sewage by assimilating nutrients in the sewage through culturing.

Claims

A method for producing an aurantiochytrium algae capable of growth and substance production under low salinity conditions, comprising the following steps:
(I) a step of providing auranthiochytrium algae cultured in a culture medium to which a salt content corresponding to 10% (v / v) or more of seawater is added;
(Ii) culturing the algae in a culture medium in which the amount of salt added is less than that in the above step, and substituting the algae until the growth rate of the algae reaches a reference value; and (iii) the above step (Ii) is repeated at a lower salt addition amount to obtain an auranthiochytrium algae capable of growth and substance production under desired salt concentration conditions;
Including the production method.
The production method according to claim 1, wherein the auranthiochytrium algae is an auranthiochytrium tsukuba-3 strain (accession number FERM BP-11442).
The production method according to any one of claims 1 and 2, wherein the decrease in the amount of salt is reduced to 1/2 to 1/50 of the amount added so far.
The production method according to any one of claims 1 to 3, wherein the desired salt concentration is 100 ppm or less.
The production method according to any one of claims 1 to 4, wherein the culture medium contains 20 g / L of D (+) glucose, 10 g / L of tryptone, and 5 g / L of yeast extract.
In addition, the following steps:
(Iv) The algae obtained in the above step (iii) is cultured in a culture medium in which the addition amount of one or more components other than salt is reduced, and the culture is continued until the growth rate of the algae reaches a reference value. And (v) repeating the above step (iv) by further reducing the amount of the component added to obtain alanthiochytrium algae that can be cultured and produced under desired nutritional conditions. Process;
The production method according to any one of claims 1 to 5, comprising
The production method according to claim 6, wherein the decrease in the addition amount of one or more components of the culture medium is reduced to 80% to 90% of the content of the components until then.
The production method according to any one of claims 6 and 7, wherein the one or more components are one or more of D (+) glucose, tryptone, and yeast extract.
The production method according to any one of claims 6 to 8, wherein the desired nutrient condition is 70% or less of the nutrient component at the beginning of culture.
A method for producing a hydrocarbon, comprising culturing an auranthiochytrium algae produced by the method according to any one of claims 1 to 9 in a low salinity medium, and said algal cell Separating and purifying hydrocarbons accumulated therein.
The method according to claim 10, wherein the hydrocarbon is squalene.
The method according to any one of claims 10 and 11, wherein the medium contains sewage.
The manufacturing method according to claim 12, wherein the sewage contains a salt content of 10 ppm or more.
The component adjustment is performed by adding any one or more of carbohydrate, organic acid, inorganic acid, organic base, inorganic base, vitamin, amino acid, peptide, protein, and mineral to the sewage. The manufacturing method of any one of Claims.
15. A method for purifying sewage by using a culture medium containing sewage in the production method according to claim 12 to assimilate nutrients in the sewage to algae.