WO2016010149A1 - Method of acclimating algae belonging to aurantiochytrium sp. to low-salt conditions - Google Patents
Method of acclimating algae belonging to aurantiochytrium sp. to low-salt conditions Download PDFInfo
- Publication number
- WO2016010149A1 WO2016010149A1 PCT/JP2015/070609 JP2015070609W WO2016010149A1 WO 2016010149 A1 WO2016010149 A1 WO 2016010149A1 JP 2015070609 W JP2015070609 W JP 2015070609W WO 2016010149 A1 WO2016010149 A1 WO 2016010149A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- algae
- culture
- medium
- salt
- strain
- Prior art date
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/12—Unicellular algae; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/01—Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
- C12P5/02—Preparation of hydrocarbons or halogenated hydrocarbons acyclic
Definitions
- 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.
- 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.
- 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.
- 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.
- 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)).
- 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).
- GTY medium D (+) glucose 20 g / L, tryptone 10 g / L, yeast extract 5 g / L, artificial seawater 8. 5 g / L
- Patent Document 2 Table 2012/0777799
- Patent Document 3 JP 2014-108101
- 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.
- 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.
- the culture plant 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.
- 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
- Both documents describe that an environmentally-adapted E. coli strain was obtained by applying stepwise environmental stresses over a period of several hundred days.
- the environmental adaptation process in E. coli cannot be discussed in the same way as eukaryotic microorganisms.
- 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).
- 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.
- the generational change of microalgae is remarkably slower than that of Escherichia coli.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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).
- 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.
- 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.
- the production method according to Item 10, wherein the hydrocarbon is squalene. 12 Item 12.
- the marine microalgae Aulanthiochytrium algae
- the marine microalgae 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.
- 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. 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
- 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.
- the microorganisms subjected to freshwater acclimation in the present invention may be any microorganisms belonging to the genus Aurantiochytrium.
- 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.
- 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).
- 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.
- Any known medium can be used as the medium.
- 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.
- the said culture medium can also contain vitamins suitably.
- the medium is prepared with artificial seawater with an appropriate salinity.
- 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)).
- 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%.
- 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.
- 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.
- 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.
- sodium chloride in an amount constituting an aqueous solution isotonic with seawater may be used instead of artificial seawater.
- natural seawater or seawater concentrate may be used instead of artificial seawater.
- 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.
- Acclimatization culture of alanthiochytrium algae is carried out by reducing the salinity of the culture medium used for the normal maintenance culture.
- 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.
- 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.
- 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.
- acclimatization culture such as 1/1000
- 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
- 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.
- 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.
- 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.
- the culture environment is appropriately adjusted so as to maintain the growth activity of the algae at an appropriate level.
- 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.
- 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.
- the acceptable level is 50% or more relative to the culture rate of the algae before acclimatization.
- the algal growth rate may be derived based on the rate at which the culture OD 660 increases.
- 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.
- auranthiochytrium algae can be acclimatized to conditions under which the amount of medium components other than salt is reduced.
- the Aulanthiochytrium algae are cultured in a medium in which any one or more components are reduced from the algal culture medium.
- the addition of one or more of glucose, tryptone and yeast extract is reduced.
- the addition amount of glucose, tryptone and yeast extract is reduced to 70% to 90% of the addition amount of normal culture medium.
- 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.
- 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.
- physiological activities such as growth rate and substance production ability.
- 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.
- 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.
- 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.
- 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.
- EMS Waako Pure Chemical Industries
- EMS Waako Pure Chemical Industries
- EMS Waako Pure Chemical Industries
- 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.
- 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. .
- 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).
- 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.
- Table 2 The procedure for the above-mentioned acclimation strain production experiment is shown in Table 2.
- eutrophic wastewater such as sewage and industrial wastewater for aurantiochytrium algae culture
- 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.
- an appropriate amount of salt is added.
- 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.
- 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.
- 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 ).
- the final turbidity was 2.8 times different. occured.
- 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.
- 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 ).
- 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.
- 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).
- 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.
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Microbiology (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Environmental & Geological Engineering (AREA)
- Biodiversity & Conservation Biology (AREA)
- Water Supply & Treatment (AREA)
- Hydrology & Water Resources (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Physics & Mathematics (AREA)
- General Chemical & Material Sciences (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicinal Chemistry (AREA)
- Botany (AREA)
- Cell Biology (AREA)
- Virology (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- Plant Pathology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Purification Treatments By Anaerobic Or Anaerobic And Aerobic Bacteria Or Animals (AREA)
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
本発明は、低塩分濃度条件下で増殖及び物質生産が可能なオーランチオキトリウム(Aurantiochytrium)属藻類を生産する方法、当該藻類を用いた炭化水素製造方法、及び当該藻類を利用した下水処理方法に関する。
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.
生物の同化産物から得られるバイオマス、特にバイオ燃料は、近年、地球温暖化又は埋蔵資源の枯渇等の問題から注目を集めている。このバイオマスの中でも、微生物が生産する炭化水素やトリアシルグリセロール等のオイルは、食料と競合せず、エネルギー生産効率が高く、生産規模の拡大が容易であることから、工業的利用の早期実現が望まれている。
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.
物質生産に利用される微生物の例として、ラビリンチュラ類(Labyrinthulomycetes)に属する藻類が挙げられる。ラビリンチュラ類藻類は様々な炭化水素や油脂を生産するものが報告されており、微生物を利用した物質生産技術の有望な材料として注目されている。例えば物質生産性ラビリンチュラ類藻類として、ドコサヘキサエン酸(DHA)やエイコサペンタエン酸(EPA)等の高度不飽和脂肪酸を多量に蓄積する性質を有するもの(SR21株、特許文献1:特許第2764572号)や、スクワレンを生産するものが知られている。(非特許文献1:G. Chen. et al. New Biotechnology 27, 382-289 (2010);非特許文献2:Q. Li et al., J. Agric. Food Chem. 57(10), 4267-4272 (2009);非特許文献3:K. W. Fan et al., World J. Microbiol. Biotechnol. 26, 1303-1309 (2010))。
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)).
ラビリンチュラ類藻類の中で、2007年に定義されたオーランチオキトリウム(Aurantiochytrium)属藻類は、汽水域に生息する従属栄養性藻類で、水中の栄養分を同化して脂質を生産し、細胞内に蓄積する特徴を有する。彼谷らは、スクワレンを生産するオーランチオキトリウム属藻類tsukuba-3株を同定し、その生産効率が、ボツリオコッカス・ブラウニー(Botryococcus braunii)等の従来から産業利用が研究されている炭化水素生産藻類よりも格段に優れていることを見出した(非特許文献4:BioScience, Biotechnology, and Biochemistry 75, 2246-2248)。
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).
オーランチオキトリウム属藻類の培養条件については鋭意検討が行われており、通常は、GTY培地(D(+)グルコース20g/L、トリプトン10g/L、酵母抽出物5g/L、人工海水8.5g/L)に種菌を播種し、常温で撹拌培養することにより行われる(特許文献2:再表2012/077799)。培養培地としてGTY培地に代えて透析排液や食品廃水、下水等の富栄養性の廃水を利用することにより、培地供給のコストを下げると共に廃水の浄化を促進する技術も報告されている(特許文献3:特開2014-108101)。
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).
ここで、オーランチオキトリウム属藻類は汽水域に生息する海洋性微細藻類であるため、通常培養には海水に近いレベルの塩分濃度を必要とする。従って、当該藻類は、低塩分濃度条件下では増殖速度や物質生産速度が著しく低下し、あるいは死滅する。そのような藻類の培養は、以下のような不都合を生じ得る。
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.
オーランチオキトリウム属藻類は光合成を行わない従属栄養性藻類であるため、大規模プラントで培養を行う際、光合成効率を考慮した広大な表面積の培養槽を要さず、光合成を行う独立栄養性藻類と比較してプラントの立地を比較的容易に確保することが出来るという利点がある。しかしながら、高い塩分濃度の培地が必要な当該藻類のプラント立地は、必然的に塩分供給源として海水の利用が容易な沿岸エリアに限定されてしまう。
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.
また、高い塩分濃度の培地を上記藻類の培養に用いた場合、培養の過程で高塩分濃度の廃液や残渣等の廃棄物が発生することとなるが、このような廃棄物の処分には専用設備と費用が必要となるため、事業化において負担となる。また、培地中に含まれる高濃度の塩分がプラント構造物を劣化させるため、当該培養プラントは塩耐性を備えるべく防錆などの表面処理が必要となり、プラントの建設コストや維持費用の支出が過大になるおそれもある。
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.
更に、上記先行技術に倣い様々な富栄養性廃水を藻類培養に利用しようとする場合、培養のため廃水に大量の塩分を添加して、培養後の廃水から費用を掛けて添加した塩分を処理しなければならず、工程が冗長となる。藻類培養への富栄養性廃水の利用は廃水浄化を兼ねているにもかかわらず、処理が必要な廃水が新たに生産されるのでは本末転倒である。
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.
従って、海洋性藻類であるオーランチオキトリウム属藻類を、上記弊害が生じないレベルの低塩分濃度で培養を可能とする技術は、当該藻類の培養による炭化水素生産技術の産業利用を見据えた発展に大いに貢献するものであり、当該技術分野における需要は極めて大きい。
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.
微生物を特定の環境に順応させる技術には、公知のものが存在する。例えば、大阪大学のグループは、大腸菌の高温順化株(非特許文献5:Kishimoto et al 2010, PLoS Genetics., 6(10), e1001164)やエタノール耐性株(非特許文献6:Horinouchi et al. 2010, BMC Genomics., 11(579), e579)の取得を報告している。両文献において、環境順応大腸菌株は、数百日に及ぶ期間に渡り段階的な環境ストレスを付与することにより取得されたことが記載されている。
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.
しかしながら、大腸菌における環境適応プロセスを、真核微生物と同列に議論することは出来ない。人工進化(研究室内進化)の研究では、出発微生物の世代時間が環境順応の成否に著しく影響する。即ち、微生物の環境耐性は数多の世代交代を繰り返すことにより獲得されるものであるため、世代交代の頻度が多い程(世代時間が短い程)環境順応の速度が増大する。ここで、上記大阪大学のグループの研究で用いられている大腸菌の世代時間は約20分、即ち一日の内に72回の世代交代が行われている。一方、微細藻類の世代交代は大腸菌と比較して著しく緩慢で、例えば本発明で用いられるオーランチオキトリウム属藻類の世代時間は2.86時間、即ち一日の世代交代回数は僅か8.4回であり、これは大腸菌に比べて8.6分の1の速度でしか環境順応が起こらないことを示している。故に、藻類が大腸菌と同等の世代交代回数を経て環境ストレスに対する耐性を獲得すると仮定した場合、環境順応株を取得するために数年~十年に及ぶ長大な期間の順化培養が必要となってしまう。
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.
従って、当業者は、仮に上記先行技術を参照してオーランチオキトリウム属藻類の環境適応を試みようとしても、所望の株を取得するために費やすことになる時間と労力が甚大となることを予想し、またいくら時間を掛けても目的の株が得られるか否かが不明であることを考慮し、計画を躊躇すると考えられる。
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.
彼谷らは、高いスクワレン生産能を有するオーランチオキトリウム・tsukuba-3株を樹立し、当該株を用いた様々な培養技術の研究開発を行っている。しかしながら、上述したように、海洋性藻類である当該株は培養において高濃度の塩分を要求するものであり、斯かる特性は、当該株を用いた炭化水素生産技術の産業上の利用を著しく阻害し、その価値を損なうおそれがある。
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.
そこで、発明者らは、通常の生育環境よりも低い塩分濃度で培養することが可能な、低塩分濃度条件に順化したオーランチオキトリウム属藻類の取得方法について鋭意研究した結果、驚くべきことに、当該藻類の培地中の塩分濃度を漸次減少させることにより、最終的に海水の数千分の1(~10ppm)の塩分濃度の培地中で持続的に増殖可能な順化株を取得出来ることが判明した。更に驚くべきことに、本発明における藻類の低塩分濃度順化プロセスは、約100日という、従来技術から予想されるよりも格段に短期間で達成されることが示された。
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.
なお、発明者らは、変異原を用いた突然変異誘導によるオーランチオキトリウム属藻類の低塩分濃度耐性株の取得を様々な条件で検討したが、結局所望の耐性株を取得することは出来なかった。
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.
当該順化培養において、当該藻類は120時間という比較的短い周期で継代が行われ、培養温度を至適温度条件の25℃よりも5℃低い20℃とすることにより、高い増殖力が長期間維持される。当該藻類の増殖活性を高いレベルに維持することが、低塩分濃度ストレスに対する耐性獲得を飛躍的に早めることに繋がった。
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.
従って、本願は、以下の発明を提供する。
1.低塩分濃度条件下で増殖及び物質生産が可能なオーランチオキトリウム(Aurantiochytrium)属藻類を生産する方法であって、以下の工程:
(i)海水の10%(v/v)以上に相当する塩分を添加した培養培地中で培養したオーランチオキトリウム属藻類を提供する工程;
(ii)上記藻類を、上記工程よりも塩分の添加量を減少させた培養培地中で培養し、当該藻類の増殖速度が基準値に達するまでこれを継代する工程;及び
(iii)上記工程(ii)を、更に低い塩分の添加量で繰り返して、所望の塩分濃度条件下で増殖及び物質生産が可能なオーランチオキトリウム属藻類を取得する工程;
を含む、生産方法。
2.前記オーランチオキトリウム属藻類が、オーランチオキトリウム・tsukuba-3株(受託番号FERM BP-11442)である、項1に記載の生産方法。
3.前記塩分の添加量の減少が、それまでの添加量の1/2~1/50に減少することである、項1又は2のいずれか1項に記載の生産方法。
4.前記所望の塩分濃度が100ppm以下である、項1~3のいずれか1項に記載の生産方法。
5.前記培養培地が、D(+)グルコース20g/L、トリプトン10g/L、酵母抽出物5g/Lを含む、項1~4のいずれか1項に記載の生産方法。
6.更に、以下の工程:
(iv)上記工程(iii)で取得した藻類を、塩分以外の1つ以上の成分の添加量を減少させた培養培地中で培養し、当該培養を当該藻類の増殖速度が基準値に達するまで続行する工程;及び
(v)上記工程(iv)を、当該成分の添加量を更に低下させて繰り返して、所望の栄養条件下で培養及び物質生産が可能なオーランチオキトリウム属藻類を取得する工程;
を含む、項1~5のいずれか1項に記載の生産方法。
7.前記培養培地の1つ以上の成分の添加量の減少が、それまでの当該成分の含有量の80%~90%に減少することである、項6に記載の生産方法。
8.前記1つ以上の成分が、D(+)グルコース、トリプトン、及び酵母抽出物の1つ以上である、項6又は7のいずれか1項に記載の生産方法。
9.前記所望の栄養条件が培養当初の栄養成分の70%以下である、項6~8のいずれか1項に記載の生産方法。
10.炭化水素を製造する方法であって、項1~9のいずれか1項に記載の方法により生産されたオーランチオキトリウム属藻類を低塩分濃度の培地中で培養すること、及び当該藻類細胞中に蓄積された炭化水素を分離精製することを含む、方法。
11.前記炭化水素がスクワレンである、項10に記載の製造方法。
12.培地が下水を含有する、項10又は11のいずれか1項に記載の製造方法。
13.前記下水が、10ppm以上の塩分を含有している、項12に記載の製造方法。
14.前記下水に、糖質、有機酸、無機酸、有機塩基、無機塩基、ビタミン、アミノ酸、ペプチド、タンパク質、ミネラルのいずれか1つ以上が添加されて成分調整が行われる、項12又は13のいずれか1項に記載の製造方法。
15.項12~14に記載の製造方法において下水を含有する培地を使用して当該下水中の栄養分を藻類に資化させることによる、下水を浄化する方法。 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 ofItems 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 toItem 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 ofItems 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 ofItems 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 toItem 10, wherein the hydrocarbon is squalene.
12Item 12. The method according to any one of Items 10 or 11, wherein the medium contains sewage.
13 Item 13. The method according toItem 12, wherein the sewage contains a salt content of 10 ppm or more.
14 Any ofparagraphs 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.
1.低塩分濃度条件下で増殖及び物質生産が可能なオーランチオキトリウム(Aurantiochytrium)属藻類を生産する方法であって、以下の工程:
(i)海水の10%(v/v)以上に相当する塩分を添加した培養培地中で培養したオーランチオキトリウム属藻類を提供する工程;
(ii)上記藻類を、上記工程よりも塩分の添加量を減少させた培養培地中で培養し、当該藻類の増殖速度が基準値に達するまでこれを継代する工程;及び
(iii)上記工程(ii)を、更に低い塩分の添加量で繰り返して、所望の塩分濃度条件下で増殖及び物質生産が可能なオーランチオキトリウム属藻類を取得する工程;
を含む、生産方法。
2.前記オーランチオキトリウム属藻類が、オーランチオキトリウム・tsukuba-3株(受託番号FERM BP-11442)である、項1に記載の生産方法。
3.前記塩分の添加量の減少が、それまでの添加量の1/2~1/50に減少することである、項1又は2のいずれか1項に記載の生産方法。
4.前記所望の塩分濃度が100ppm以下である、項1~3のいずれか1項に記載の生産方法。
5.前記培養培地が、D(+)グルコース20g/L、トリプトン10g/L、酵母抽出物5g/Lを含む、項1~4のいずれか1項に記載の生産方法。
6.更に、以下の工程:
(iv)上記工程(iii)で取得した藻類を、塩分以外の1つ以上の成分の添加量を減少させた培養培地中で培養し、当該培養を当該藻類の増殖速度が基準値に達するまで続行する工程;及び
(v)上記工程(iv)を、当該成分の添加量を更に低下させて繰り返して、所望の栄養条件下で培養及び物質生産が可能なオーランチオキトリウム属藻類を取得する工程;
を含む、項1~5のいずれか1項に記載の生産方法。
7.前記培養培地の1つ以上の成分の添加量の減少が、それまでの当該成分の含有量の80%~90%に減少することである、項6に記載の生産方法。
8.前記1つ以上の成分が、D(+)グルコース、トリプトン、及び酵母抽出物の1つ以上である、項6又は7のいずれか1項に記載の生産方法。
9.前記所望の栄養条件が培養当初の栄養成分の70%以下である、項6~8のいずれか1項に記載の生産方法。
10.炭化水素を製造する方法であって、項1~9のいずれか1項に記載の方法により生産されたオーランチオキトリウム属藻類を低塩分濃度の培地中で培養すること、及び当該藻類細胞中に蓄積された炭化水素を分離精製することを含む、方法。
11.前記炭化水素がスクワレンである、項10に記載の製造方法。
12.培地が下水を含有する、項10又は11のいずれか1項に記載の製造方法。
13.前記下水が、10ppm以上の塩分を含有している、項12に記載の製造方法。
14.前記下水に、糖質、有機酸、無機酸、有機塩基、無機塩基、ビタミン、アミノ酸、ペプチド、タンパク質、ミネラルのいずれか1つ以上が添加されて成分調整が行われる、項12又は13のいずれか1項に記載の製造方法。
15.項12~14に記載の製造方法において下水を含有する培地を使用して当該下水中の栄養分を藻類に資化させることによる、下水を浄化する方法。 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.
3. Item 3. The production method according to any one of
4).
5. Item 5. The production method according to any one of
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;
7). Item 7. The production method according to
8).
9. Item 9. The production method according to any one of
10. A method for producing a hydrocarbon, comprising culturing an auranthiochytrium algae produced by the method according to any one of
11. Item 11. The production method according to
12
13 Item 13. The method according to
14 Any of
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.
驚くべきことに、海洋性微細藻類であるオーランチオキトリウム属藻類は、対数増殖期を維持できる頻度で継代を行いながら培地の塩分濃度を漸次減少させることにより、50~100日程度の極めて短期間で低塩分濃度に順化出来ることが判明した。本発明の方法により取得された順化株は、海水中で培養した場合と比較して遜色のないレベルの増殖及び物質生産速度を示す。従って、本発明の方法により取得した順化株を炭化水素生産技術の材料として使用することにより、通常の当該藻類が高い塩分濃度を要求することにより想定される上記様々な不利益を回避することが出来る。
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.
1.オーランチオキトリウム属藻類
本発明において淡水順化が行われる微生物は、オーランチオキトリウム(Aurantiochytrium)属に属する微生物であればいずれのものでもよい。具体的には、例えば、上記オーランチオキトリウム・tsukuba-3株を用いることができるが、これに限らず、当該株と実質的に同一の菌学的性質を有する菌株であればいずれの菌株も使用することができる。 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.
本発明において淡水順化が行われる微生物は、オーランチオキトリウム(Aurantiochytrium)属に属する微生物であればいずれのものでもよい。具体的には、例えば、上記オーランチオキトリウム・tsukuba-3株を用いることができるが、これに限らず、当該株と実質的に同一の菌学的性質を有する菌株であればいずれの菌株も使用することができる。 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.
本発明において淡水順化が行われるオーランチオキトリウム属藻類は、ラビリンチュラ類(Labyrinthulomycetes)に属する微細藻類で、従属栄養性で、細胞内に多量の炭化水素を蓄積する。ラビリンチュラ類は、卵菌類に属するものであるが、系統的には他の菌類と異なり褐藻類や珪藻類等の不等毛植物と近縁の系統であり、不等毛植物とともにストラメノパイル系統を構成するものである。
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.
前記オーランチオキトリウム属藻類は、2007年に新たに定義された海洋性藻類で、脂質を生産して、細胞内に蓄積する特徴を有する。例えば、オーランチオキトリウムtsukuba-3株は、彼谷らが沖縄県の海岸に生息するマングローブの葉より採取及び分離したものである。当該藻類は、従来炭化水素を生産することが知られていたボツリオコッカス・ブラウニー等の他の微細藻類と比較して卓越して優れた炭化水素生産能力と増殖性を有することが見出され、藻類培養による炭化水素生産技術への利用が期待されている。オーランチオキトリウム・tsukuba-3株は、独立行政法人産業技術総合研究所(住所 日本国茨城県つくば市東1丁目1番地中央第6)に2010年12月9日付で寄託されている(受託番号FERM BP-11442)。
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.通常の藻類培養条件
上記オーランチオキトリウム属藻類の培養方法は、当該技術分野において確立されたものである。即ち、通常の維持培養は、成分調製した適当な培地に播種し、定法に従い行われる。培地としては、任意の公知のものを使用できる、例えば、炭素源としてはグルコース、フルクトース、サッカロース等の糖類がある。これらの炭素源を、例えば、培地1リットル当たり20~120gの濃度で使用する。窒素源としては、グルタミン酸ナトリウム、尿素等の有機窒素、又は酢酸アンモニウム、硫酸アンモニウム、塩化アンモニウム、硝酸ナトリウム、硝酸アンモニウム等の無機窒素、又は酵母抽出物、コーンスチープリカー、ポリペプトン、ペプトン、トリプトン等の生物由来消化物等がある。また、上記培地は適宜ビタミン類を含むこともできる。上記培地は、適切な塩分濃度の人工海水で調製される。好ましくは、当該培地は、最終的な塩分濃度が海水(塩分濃度3.4%(w/v))の約10%(v/v)以上、又は約10%(v/v)~約100%(v/v)、例えば塩分濃度が約1.0~3.0%(w/v)となるように調製される。より好ましくは、当該培地は、最終的な塩分濃度が海水の約20%(v/v)~80%(v/v)、約30%(v/v)~70%(v/v)、約40%(v/v)~60%(v/v)、又は約50%となるように調製される。 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%.
上記オーランチオキトリウム属藻類の培養方法は、当該技術分野において確立されたものである。即ち、通常の維持培養は、成分調製した適当な培地に播種し、定法に従い行われる。培地としては、任意の公知のものを使用できる、例えば、炭素源としてはグルコース、フルクトース、サッカロース等の糖類がある。これらの炭素源を、例えば、培地1リットル当たり20~120gの濃度で使用する。窒素源としては、グルタミン酸ナトリウム、尿素等の有機窒素、又は酢酸アンモニウム、硫酸アンモニウム、塩化アンモニウム、硝酸ナトリウム、硝酸アンモニウム等の無機窒素、又は酵母抽出物、コーンスチープリカー、ポリペプトン、ペプトン、トリプトン等の生物由来消化物等がある。また、上記培地は適宜ビタミン類を含むこともできる。上記培地は、適切な塩分濃度の人工海水で調製される。好ましくは、当該培地は、最終的な塩分濃度が海水(塩分濃度3.4%(w/v))の約10%(v/v)以上、又は約10%(v/v)~約100%(v/v)、例えば塩分濃度が約1.0~3.0%(w/v)となるように調製される。より好ましくは、当該培地は、最終的な塩分濃度が海水の約20%(v/v)~80%(v/v)、約30%(v/v)~70%(v/v)、約40%(v/v)~60%(v/v)、又は約50%となるように調製される。 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%.
本明細書中、「塩分」とは、海水が含有する主要な塩類、即ち塩化ナトリウム、塩化マグネシウム、硫酸マグネシウム、硫酸カルシウム及び塩化カリウムの、海水と同一の構成比率の混合物を意味する。他の態様において、「塩分」とは、75%(w/w)以上の塩化ナトリウムと、塩化ナトリウム以外の1つ以上の上記海水が含有する主要な塩類との混合物、又は塩化ナトリウム単体を意味する。
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.
人工海水は、水に溶解させることで海水に近い各種イオン組成を構成することの出来る塩の混合物である。好ましくは、当該人工海水は、海水を模倣するように、適切な比率で塩化ナトリウム、塩化マグネシウム、硫酸マグネシウム、硫酸カルシウム、塩化カリウム、微量金属、有機物等を含有する。本明細書中、本発明の培地の成分として、人工海水に代えて、海水と等張の水溶液を構成する量の塩化ナトリウムを用いてもよい。あるいは、人工海水に代えて、天然の海水又は海水濃縮物を用いてもよい。
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.
好ましくは、上記培地は、50%(v/v)人工海水に、D(+)グルコース20g/L、トリプトン10g/L、酵母抽出物5g/Lを添加したもの(GTY培地)である。
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).
上記培地は、調製後、適当な酸又は塩基を加えることにより適宜pHを調整できる。培地のpHは、pH2.0~11.0、好ましくはpH3.0~10.0、より好ましくはpH4.0~9.0、より好ましくはpH4.5~9.0であり、最も好ましくはpH6.5を用いる。
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.
上記培地は、オーランチオキトリウム属藻類の播種前にオートクレーブにより殺菌されてもよい。
The above medium may be sterilized by an autoclave before seeding with the Aulanthiochytrium algae.
培養は、培養温度5~40℃、好ましくは10~35℃、より好ましくは10~30℃にて行われる。継代は、通常1~10日間、好ましくは3~7日間置きに行われる。培養は通気攪拌培養、振とう培養又は静置培養で行うことができるが、好ましくは通気攪拌培養又は振とう培養で培養する。
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.
通常、海洋性微細藻類であるオーランチオキトリウム属藻類の維持培養において、培養液に海水と同濃度~10%(v/v)濃度程度の塩分の添加は必須である。当該藻類の増殖活性は、維持培地中の塩分濃度の低下に依存して著しく低下し、塩分を添加せずに調製された培地中では、当該藻類は増殖を停止し、又は死滅する。
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.順化条件
オーランチオキトリウム属藻類の低塩分濃度順化培養は、上記通常の維持培養に用いられる培養培地の塩分濃度を減少させることにより行われる。好ましくは、低塩分濃度順化に用いる培地の組成は、塩分濃度を変化させる点を除いて、維持培養に用いる培地の組成と同一のものである。あるいは、本発明の方法に用いる具体的な藻類の種類、状態、実施環境に依存して、当該藻類の増殖活性を高いレベルに維持するために、維持培地の組成を適宜変更してもよい。 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.
オーランチオキトリウム属藻類の低塩分濃度順化培養は、上記通常の維持培養に用いられる培養培地の塩分濃度を減少させることにより行われる。好ましくは、低塩分濃度順化に用いる培地の組成は、塩分濃度を変化させる点を除いて、維持培養に用いる培地の組成と同一のものである。あるいは、本発明の方法に用いる具体的な藻類の種類、状態、実施環境に依存して、当該藻類の増殖活性を高いレベルに維持するために、維持培地の組成を適宜変更してもよい。 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.
上記低塩分濃度順化培養に用いる培地の塩分濃度の減少は、藻類の継代の際に用いる新鮮な培地の塩分濃度を減少させることにより行われる。当該塩分濃度の減少の幅は、2~100分の1であり、本発明の方法に用いる具体的な藻類の種類、状態、実施環境、所望される順化レベルに依存して選択される。減少の幅が大きくなる程順化の成功率は低下し、順化に掛かる時間は長くなる。一方、減少の幅を小さくすると、順化の成功率は増大し、順化に掛かる時間は短くなる。
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.
所望の順化レベルが1回の順化培養で達成出来ないものである場合(1000分の1等)、上記順化培養を二段階以上に分けることにより、即ち塩分濃度を漸次減少させることにより行う。例えば通常の塩分濃度の1000分の1の塩分濃度に順化した株を取得する場合、まず塩分濃度50分の1に順化した株を取得し、これを更に塩分濃度を20分の1に低下させて順化を行うことにより、所望の順化レベルの株を取得することが出来る。
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.
上記低塩分濃度順化培養における藻類の継代は、藻類培養物が定常期に入る前の時点で実施され、好ましくは対数増殖期の時点で実施される。藻類培養物の増殖段階は、OD660の吸光度測定等、当該技術分野で通常用いられる方法により容易に判定することが出来る。藻類培養物がいかなる増殖段階にあるかは、実際に藻類培養物をサンプリングしてOD660を測定することにより導き出してもよく、あるいは当該藻類における同一の条件下で作成した増殖曲線を参照して推定してもよい。例えば、本発明の実施例において、オーランチオキトリウム・tsukuba-3株は、OD660が0.05となるように新鮮な培地に播種した後、120時間の時点で対数増殖期にあると推定し、120時間周期で継代が行われた。
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.
上記低塩分濃度順化培養において、温度、湿度、照明、撹拌速度、通気量、培養設備の構成等、諸々の培養環境は、藻類の増殖活性を高いレベルに維持するように適宜調整される。あるいは、増殖速度が過剰となりコストや労力の面で実施者の負担となる場合に、上記培養環境が当該藻類の増殖活性を適切なレベルに維持するように適宜調整される。例えば、本発明の実施例において、オーランチオキトリウム・tsukuba-3株は、最適な培養温度の25℃では増殖速度が過剰となり長期間の順化培養が困難となることが予想されたため、20℃で培養が行われた。
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.
一つの態様において、前記藻類がある塩分濃度に順化したか否かは、当該藻類の増殖速度が基準値に達したことによって判断される。即ち、塩分濃度を低下させる前(ある塩分濃度に順化した藻類を更に低い塩分濃度に順化させる場合、当該更なる順化を試みる前)の藻類の培養速度と比較して許容出来るレベルの増殖速度に達したことをもって、当該塩分濃度に順化したと判断する。好ましくは、当該許容出来るレベルとは、順化前の藻類の培養速度に対して50%以上である。当該判断において、藻類の増殖速度は、培養物のOD660が増大する速度に基づいて導き出されてもよい。他の態様において、前記藻類がある塩分濃度に順化したか否かは、増殖速度に代えて炭化水素等物質生産速度が基準値に達したことによって判断されてもよい。
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.
本発明において所望の低塩分濃度順化藻類株を取得するために要する時間は、本発明の方法に用いる具体的な藻類の種類、状態、実施環境、所望される順化レベル、塩分濃度減少の幅に依存して変動するが、数ヶ月で通常の培養培地の塩分濃度(50%(v/v)人工海水)の1000分の1以下のレベルまで順化させることが出来る。増殖速度で藻類を遥かに上回る大腸菌においてさえ環境ストレス耐性株を取得するのに数百日かかる(非特許文献5:Kishimoto et al 2010, PLoS Genetics., 6(10), e1001164;非特許文献6:Horinouchi et al. 2010, BMC Genomics., 11(579)ことを考慮すると、これは予想を上回る短期間といえる。
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.
低塩分濃度順化藻類株を取得するための本願発明の方法を応用して、オーランチオキトリウム属藻類を、塩分以外の培地構成成分の添加量を低下させた条件に順化させることも出来る。当該順化において、オーランチオキトリウム属藻類は、前記藻類培養培地から任意の1つ以上の成分を減少させた培地中で培養される。
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.
任意の成分を減少させた培地への順化を行う際に用いる諸条件は、減少させられる成分の種類に依存して当業者により検討されるべきであるが、上記低塩分濃度順化において用いられた条件を基礎としてもよい。
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.
例えば、藻類の培養にGTY培地(50%(v/v)人工海水に、D(+)グルコース20g/L、トリプトン10g/L、酵母抽出物5g/Lを添加したもの)を用いる場合、順化培地において、グルコース、トリプトン及び酵母抽出物の1つ以上の添加量が減少させられる。好ましくは、当該順化培地において、グルコース、トリプトン及び酵母抽出物の添加量が、通常の培養培地の添加量の70%~90%に減少させられる。
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.
オーランチオキトリウム属藻類は、培地中の栄養分の含有量に応じて増殖速度を柔軟に変動させるため、上記GTY培地の栄養分を減少させること自体は環境ストレスとはならない。しかしながら、当該培地中に比較的高濃度で存在する上記栄養成分を減少させることにより培地の浸透圧が著しく低下することとなり、これが藻類細胞に対するストレスとなる。実際に、GTY培地の上記栄養成分を減少させると藻類の増殖速度は低下するが、その培地で上記低塩分濃度順化の手順と同様に継代培養を続行すると、一定の期間を経て増殖速度が僅かに増大する。これは、GTY培地の栄養成分を減少させることにより浸透圧が低下した培地に藻類が順化したことを示唆する。
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.低塩分濃度順化藻類株の特性
オーランチオキトリウム属藻類は藻類培養による炭化水素生産技術において極めて有望な材料であるが、培養に高濃度の塩分が要求されるため、工業的規模の生産に用いることを考えた場合、プラント立地の制限やコストの増大等の不利益が懸念されていた。しかしながら、本発明の方法により取得された低塩分濃度に順化したオーランチオキトリウム属藻類株は、低塩分濃度条件下で、海水中で培養した場合と比較して遜色のないレベルの増殖及び物質生産速度を示すので、当該順化株を炭化水素生産技術の材料として使用することにより、海洋性藻類の培養に高塩分濃度の培地を使用する際に予想される上記様々な不利益を回避することが出来る。 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.
オーランチオキトリウム属藻類は藻類培養による炭化水素生産技術において極めて有望な材料であるが、培養に高濃度の塩分が要求されるため、工業的規模の生産に用いることを考えた場合、プラント立地の制限やコストの増大等の不利益が懸念されていた。しかしながら、本発明の方法により取得された低塩分濃度に順化したオーランチオキトリウム属藻類株は、低塩分濃度条件下で、海水中で培養した場合と比較して遜色のないレベルの増殖及び物質生産速度を示すので、当該順化株を炭化水素生産技術の材料として使用することにより、海洋性藻類の培養に高塩分濃度の培地を使用する際に予想される上記様々な不利益を回避することが出来る。 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.
本発明の方法に従い取得された低塩分濃度順化藻類株は、増殖速度や物質生産能力等の生理活性が評価されてもよい。当該生理活性の判定手段は、当該技術分野で通常用いられているものが用いられる。例えば増殖速度であれば吸光度測定や細胞計数等、また物質生産能力であれば生産物の単離精製、クロマトグラフィーやゲル電気泳動等による生産物の定量が想定される。
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.
当該順化株は、低塩分濃度条件下で、通常の塩分濃度の培地で培養した藻類の培養速度と比較して生理活性が低下するが、その低下の程度は、許容出来るレベルである。どの程度の生理活性の低下まで許容するかは、具体的に本発明の方法に従い取得された低塩分濃度順化藻類株を用いて物質生産を試みる当業者により、順化株の生理活性の低下による最終的な生産物の収量低下と、培地に塩分添加が要求されることによる様々な不利益とを比較考量して判断される。
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.
本発明の方法において順化が可能な塩分濃度は、海水(塩分濃度3.4%(w/v))の100分の1(塩分濃度340ppm)以下、好ましくは1000分の1(塩分濃度34ppm)以下、より好ましくは1600分の1(塩分濃度20.2ppm)以下、尚もより好ましくは3200分の1(塩分濃度10.1ppm)以下である。通常、塩分濃度が100ppm以下であれば、培地に塩分添加が要求されることによる様々な不利益の大半が解消され得る。即ち、オーランチオキトリウム属藻類を塩分濃度100ppm以下の培地で培養出来るのであれば、塩分添加のコストに配慮する必要が無いので、培養設備の立地が沿岸部に限定されず、また培養廃水を処理する際に塩分を除去する必要が無いので廃水処理工程の簡素化及びコスト節約が図れる。培地中の塩分により培養設備が損傷する恐れも無い。更に、富栄養性廃水を藻類培養に利用しようとする場合、当該順化株は廃水中に最初から存在する100ppm以下の塩分濃度で培養が可能であるため、培地調製や培養廃液の処理が容易になる。
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.
オーランチオキトリウム属藻類の淡水適応突然変異体の作製
変異原としてメタンスルホン酸エチル(EMS)を使用した突然変異誘導により、低塩濃度環境に適応した株の取得を試みた。オーランチオキトリウムtsukuba-3株を、20mlのGTY培地(50%(v/v)海水相当の人工海水(大阪薬研)1.7%(w/v)、D(+)グルコース(Wako)20g/L、トリプトン(GIBCO)10g/L、酵母抽出物(GIBCO)5g/L)中で16~100時間前培養した。当該培養物に、最終濃度が30~50mMとなるように、EMS(和光純薬)を添加し、更に25℃で7時間培養した。当該培養物に、10%(w/v)Na2S2O3を2.0ml添加した。これを室温下5分間1000xgで遠心分離し、上澄みを除去した。沈殿した細胞ペレットに、更にGTY培地1mlを添加し、室温下5分間1000xgで遠心分離し、上澄みを除去する工程を2回繰り返した。得られた細胞ペレットにGTY培地を1ml添加し、懸濁物の細胞数を計測し、これを3000~120000細胞/プレートとなるように、人工海水を添加していないGTY寒天平板上に展開した。当該実験の手順を、表1に示す。
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.
変異原としてメタンスルホン酸エチル(EMS)を使用した突然変異誘導により、低塩濃度環境に適応した株の取得を試みた。オーランチオキトリウムtsukuba-3株を、20mlのGTY培地(50%(v/v)海水相当の人工海水(大阪薬研)1.7%(w/v)、D(+)グルコース(Wako)20g/L、トリプトン(GIBCO)10g/L、酵母抽出物(GIBCO)5g/L)中で16~100時間前培養した。当該培養物に、最終濃度が30~50mMとなるように、EMS(和光純薬)を添加し、更に25℃で7時間培養した。当該培養物に、10%(w/v)Na2S2O3を2.0ml添加した。これを室温下5分間1000xgで遠心分離し、上澄みを除去した。沈殿した細胞ペレットに、更にGTY培地1mlを添加し、室温下5分間1000xgで遠心分離し、上澄みを除去する工程を2回繰り返した。得られた細胞ペレットにGTY培地を1ml添加し、懸濁物の細胞数を計測し、これを3000~120000細胞/プレートとなるように、人工海水を添加していないGTY寒天平板上に展開した。当該実験の手順を、表1に示す。
前培養時間、EMS濃度、寒天平板上に展開する細胞数、その後のスクリーニング条件等を変更しつつ上記実験を10回以上繰り返したが、目的の淡水適応突然変異体を取得することは出来なかった。
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. .
オーランチオキトリウム属藻類の淡水順化株の作製
オーランチオキトリウムtsukuba-3株を、20mlのGTY培地(50%(v/v)海水相当の人工海水(大阪薬研)1.7%(w/v)、D(+)グルコース(Wako)20g/L、トリプトン(GIBCO)10g/L、酵母抽出物(GIBCO)5g/L)中、20℃、120rpmで、72時間旋回培養した。当該培地1mlを、塩分濃度が0.425%(w/v)(1.25%(v/v)海水相当)となるように調製したGTY培地に継代し、20℃、120rpmで、120時間旋回培養した。更に当該培養物を、塩分濃度が20.2ppm(0.06%(v/v)海水相当)となるように調製したGTY培地に、初期濁度OD660=0.05となる濃度で継代し、20℃、120rpmで、旋回培養した。この時、低塩分濃度条件に耐えられず藻類が全滅するリスクを考慮し、複数系統を用意した。途中、120時間毎に新規培地に継代を繰り返して(初期濁度OD660=0.05)、各系統を維持した。斯かるプロセスを経て許容出来るレベルで生理活性を維持することが出来た系統を、0.06%(v/v)海水順化株として取得した。 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.
オーランチオキトリウムtsukuba-3株を、20mlのGTY培地(50%(v/v)海水相当の人工海水(大阪薬研)1.7%(w/v)、D(+)グルコース(Wako)20g/L、トリプトン(GIBCO)10g/L、酵母抽出物(GIBCO)5g/L)中、20℃、120rpmで、72時間旋回培養した。当該培地1mlを、塩分濃度が0.425%(w/v)(1.25%(v/v)海水相当)となるように調製したGTY培地に継代し、20℃、120rpmで、120時間旋回培養した。更に当該培養物を、塩分濃度が20.2ppm(0.06%(v/v)海水相当)となるように調製したGTY培地に、初期濁度OD660=0.05となる濃度で継代し、20℃、120rpmで、旋回培養した。この時、低塩分濃度条件に耐えられず藻類が全滅するリスクを考慮し、複数系統を用意した。途中、120時間毎に新規培地に継代を繰り返して(初期濁度OD660=0.05)、各系統を維持した。斯かるプロセスを経て許容出来るレベルで生理活性を維持することが出来た系統を、0.06%(v/v)海水順化株として取得した。 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.
上記0.06%(v/v)海水順化株から、更に塩分濃度を減少させた条件下で生存可能な株の取得も実施された。上記0.06%(v/v)海水への順化培養を1256時間実施して取得した0.06%(v/v)海水順化株培養物を、更に塩分濃度が10.1ppm(0.03%(v/v)海水相当)となるように調製したGTY培地に、初期濁度OD660=0.05となる濃度で継代し、20℃、120rpmで、旋回培養した。120時間毎に新規培地に継代を繰り返して(初期濁度OD660=0.05)、当該培養を1200時間実施した結果、0.03%(v/v)海水順化株が取得された。
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. .
上記0.06%(v/v)海水順化株から、更に他の培地成分の濃度を減少させた条件下で生存可能な株の取得も実施された。上記0.06%(v/v)海水への順化培養を334時間実施して取得した0.06%(v/v)海水順化株培養物を、更にD(+)グルコース、トリプトン、酵母抽出物の含有量をそれぞれ20%減少させ、かつ塩分濃度が20.2ppm(0.06%(v/v)海水相当)となるように調製したGTY培地に、初期濁度OD660=0.05となる濃度で継代し、20℃、120rpmで、旋回培養した。当該培養を451時間実施した結果、20%成分減量・0.06%(v/v)海水順化株が取得された。
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.
更に、当該20%成分減量・0.06%(v/v)海水順化株培養物を、D(+)グルコース、トリプトン、酵母抽出物の含有量をそれぞれ30%減少させ、かつ塩分濃度が20.2ppm(0.06%(v/v)海水相当)となるように調製したGTY培地に、初期濁度OD660=0.05となる濃度で継代し、20℃、120rpmで、旋回培養した。当該培養を1627時間実施した結果、30%成分減量・0.06%(v/v)海水順化株が取得された。以上の順化株作製実験の手順を、表2に示す。
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.
順化株の評価
上記低塩分又は低塩分・成分減量条件における順化の成否は、OD660=0.05となる濃度で継代し120時間培養後、OD660≧5.0に達することを目安として判断した。 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.
上記低塩分又は低塩分・成分減量条件における順化の成否は、OD660=0.05となる濃度で継代し120時間培養後、OD660≧5.0に達することを目安として判断した。 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.
更に、当該順化株が、スクワレン生産能力を保持しているか否かも検証された。上記のようにして取得した0.03%(v/v)海水順化株、20%成分減量・0.06%(v/v)海水順化株、及び30%成分減量・0.06%(v/v)海水順化株を遠心分離にて回収し、細胞ペレットを凍結乾燥した。当該凍結乾燥調製物を、クロロホルムとエタノールを2:1の割合で混合した溶液に、乾燥細胞100mgあたり15mlの割合で添加し、これを72時間インキュベーションした。取得されたオイル抽出物にヘキサン1mlを添加して形成した懸濁物4μlを、薄層クロマトグラフィーに展開した。当該実験の結果を、図1に示す。0.03%(v/v)海水順化株、20%成分減量・0.06%(v/v)海水順化株、及び30%成分減量・0.06%(v/v)海水順化株のいずれにおいてもスタンダードと同一の位置にスクワレンに相当するバンドが検出されており、いずれの順化株もスクワレン生産能力を保持していることが確認された。
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.
下水を利用した順化株の培養
下水や産業廃水等の富栄養性廃水をオーランチオキトリウム属藻類培養に利用することは、培養コストの削減及び廃水処理の促進の両面において有益である。しかしながら、通常の富栄養性廃水はオーランチオキトリウム属藻類を培養するのに充分な濃度の塩分を含有しておらず、これを培地の基礎とするためには、適切な量の塩分を添加しなければならない。また、当該培養の過程で、更に処理が必要な高塩分濃度の廃水が排出されるので、廃水処理の促進という当該利用の利点が没却してしまう。 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.
下水や産業廃水等の富栄養性廃水をオーランチオキトリウム属藻類培養に利用することは、培養コストの削減及び廃水処理の促進の両面において有益である。しかしながら、通常の富栄養性廃水はオーランチオキトリウム属藻類を培養するのに充分な濃度の塩分を含有しておらず、これを培地の基礎とするためには、適切な量の塩分を添加しなければならない。また、当該培養の過程で、更に処理が必要な高塩分濃度の廃水が排出されるので、廃水処理の促進という当該利用の利点が没却してしまう。 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.
一方、上記のように作製したオーランチオキトリウム属藻類の低塩分濃度順化株が、通常の富栄養性廃水が含有する程度の塩分濃度条件下で生存が可能であれば、廃水を基礎とした培地に塩分を添加する必要が無いので、上記のような不利益が発生しない。
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.
そこで、本発明の方法により作製した低塩分濃度順化株が、塩分濃度の低い富栄養性廃水を基礎とした培地中で実際に培養可能であるかについて、検証実験を行った。
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.
50%(v/v)海水に代えて、仙台市南蒲生浄化センターで生産された一次処理水を基礎として、GTY培地を作製した。オーランチオキトリウムtsukuba-3株の野生株と、上記低塩分濃度順化株(0.03%(v/v)海水順化株)との間で、当該培地中での増殖速度を比較した。
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). .
人工海水に代えて塩分含有量の低い下水を基礎としたGTY培地中で、上記順化株と野生株を培養した。培地の基礎として利用した下水は、仙台市南蒲生浄化センターで生産された一次処理水である。当該一次処理水の塩分濃度は100ppm未満(99~65ppm)であり、これを基礎として調製した培地の塩分濃度も同様である。
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.
実験に先立って、順化株、野生株共に、50%(v/v)人工海水で調製した通常のGTY培地50mL中で、72時間培養した(20℃、120rpm旋回培養)。そして、これらを、上記一次処理水に、D(+)グルコース(Wako)20g/L、トリプトン(GIBCO)10g/L、酵母抽出物(GIBCO)5g/Lを添加して調製したGTY培地中で96時間培養し(20℃、120rpm旋回培養)、培養開始から5箇所のタイムポイントでOD660を測定した。その結果を図2に示す。
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.
図2の左右のグラフは同一の結果を示すものであるが、右図はY軸が対数目盛になっている。正方形が順化株、円が野生株の細胞濃度(OD660)を示す。左図が示すように、培養開始後96時間の時点で、順化株はOD660=13.9、野生株はOD660=4.9となり、最終到達濁度に2.8倍の差が生じた。また、右図が示すように、順化株はμd-1=2.12、野生株はμd-1=1.45となり、比増殖速度に1.46倍の差が生じた。従って、当該図は、人工海水に代えて塩分含有量の低い下水を基礎としたGTY培地中で、本発明の方法により作製した低塩分濃度順化株が、野生株と比較して格段に優れた増殖能力を有することを実証するものである。
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.
更に、下水由来の有機物を藻類培養の栄養分として利用することが可能かを検討した。下水処理で生産された乾燥脱水汚泥0.6gに、6mLの72%H2SO4を添加し、30℃で1時間反応させた。当該反応物に、前記一次処理水102mLを添加し、120℃で1時間インキュベートした。当該一次処理水の塩分濃度は100ppm未満(99~65ppm)であり、最終的に調製される培地の塩分濃度も、概ね同様である。当該懸濁物を、CaCO3で中和して、pH5.3に調整した。当該懸濁物を濾過して、濾液を酸糖化液として取得した(糖類含有量3.2g/L)。当該酸糖化液に、トリプトン10g/L、酵母抽出物5g/Lを添加し、これをオートクレーブで120℃、20分間滅菌して、下水を基礎とした藻類培養培地を調製した。
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.
上記培地を用いて、上記低塩分濃度順化株(0.03%(v/v)海水順化株)を96時間培養し(20℃、120rpm旋回培養)、培養開始から5箇所のタイムポイントで、培地中の糖濃度及びOD660を測定した。その結果を図3に示す。
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.
点線で結んだ円が培地中の有機物濃度(g/L)、実線で結んだ円が細胞濃度(OD660)を示す。培養開始後約40時間で培養藻類が対数増殖期に入り、同時に糖濃度が低下し始める。そして、細胞濃度の増大に対応して、糖濃度が低下し続ける。また、培養開始後76時間時点の細胞をNile redで染色し、蛍光顕微鏡下で細胞内に顕著な油滴が形成されることを観察した(図4)。
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).
従って、当該実験により、上記低塩分濃度順化株が、下水の一次処理水及び乾燥脱水汚泥由来の酸糖化物を利用して調製した培養培地中で、下水由来の栄養分を用いて増殖及びオイル生産を行うことが出来ることが示された。これは、本願方法を用いて取得されたオーランチオキトリウムtsukuba-3低塩分濃度順化株が、下水を基礎とする培地中で良好な増殖及び物質生産を行うことが可能であると共に、当該培養を通じて、当該順化株が下水中の栄養分を同化することにより、下水の浄化を促進することも可能であることを示唆する。
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 (15)
- 低塩分濃度条件下で増殖及び物質生産が可能なオーランチオキトリウム(Aurantiochytrium)属藻類を生産する方法であって、以下の工程:
(i)海水の10%(v/v)以上に相当する塩分を添加した培養培地中で培養したオーランチオキトリウム属藻類を提供する工程;
(ii)上記藻類を、上記工程よりも塩分の添加量を減少させた培養培地中で培養し、当該藻類の増殖速度が基準値に達するまでこれを継代する工程;及び
(iii)上記工程(ii)を、更に低い塩分の添加量で繰り返して、所望の塩分濃度条件下で増殖及び物質生産が可能なオーランチオキトリウム属藻類を取得する工程;
を含む、生産方法。 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. - 前記オーランチオキトリウム属藻類が、オーランチオキトリウム・tsukuba-3株(受託番号FERM BP-11442)である、請求項1に記載の生産方法。 The production method according to claim 1, wherein the auranthiochytrium algae is an auranthiochytrium tsukuba-3 strain (accession number FERM BP-11442).
- 前記塩分の添加量の減少が、それまでの添加量の1/2~1/50に減少することである、請求項1又は2のいずれか1項に記載の生産方法。 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.
- 前記所望の塩分濃度が100ppm以下である、請求項1~3のいずれか1項に記載の生産方法。 The production method according to any one of claims 1 to 3, wherein the desired salt concentration is 100 ppm or less.
- 前記培養培地が、D(+)グルコース20g/L、トリプトン10g/L、酵母抽出物5g/Lを含む、請求項1~4のいずれか1項に記載の生産方法。 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.
- 更に、以下の工程:
(iv)上記工程(iii)で取得した藻類を、塩分以外の1つ以上の成分の添加量を減少させた培養培地中で培養し、当該培養を当該藻類の増殖速度が基準値に達するまで続行する工程;及び
(v)上記工程(iv)を、当該成分の添加量を更に低下させて繰り返して、所望の栄養条件下で培養及び物質生産が可能なオーランチオキトリウム属藻類を取得する工程;
を含む、請求項1~5のいずれか1項に記載の生産方法。 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 - 前記培養培地の1つ以上の成分の添加量の減少が、それまでの当該成分の含有量の80%~90%に減少することである、請求項6に記載の生産方法。 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.
- 前記1つ以上の成分が、D(+)グルコース、トリプトン、及び酵母抽出物の1つ以上である、請求項6又は7のいずれか1項に記載の生産方法。 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.
- 前記所望の栄養条件が培養当初の栄養成分の70%以下である、請求項6~8のいずれか1項に記載の生産方法。 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.
- 炭化水素を製造する方法であって、請求項1~9のいずれか1項に記載の方法により生産されたオーランチオキトリウム属藻類を低塩分濃度の培地中で培養すること、及び当該藻類細胞中に蓄積された炭化水素を分離精製することを含む、方法。 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.
- 前記炭化水素がスクワレンである、請求項10に記載の製造方法。 The method according to claim 10, wherein the hydrocarbon is squalene.
- 培地が下水を含有する、請求項10又は11のいずれか1項に記載の製造方法。 The method according to any one of claims 10 and 11, wherein the medium contains sewage.
- 前記下水が、10ppm以上の塩分を含有している、請求項12に記載の製造方法。 The manufacturing method according to claim 12, wherein the sewage contains a salt content of 10 ppm or more.
- 前記下水に、糖質、有機酸、無機酸、有機塩基、無機塩基、ビタミン、アミノ酸、ペプチド、タンパク質、ミネラルのいずれか1つ以上が添加されて成分調整が行われる、請求項12又は13のいずれか1項に記載の製造方法。 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.
- 請求項12~14に記載の製造方法において下水を含有する培地を使用して当該下水中の栄養分を藻類に資化させることによる、下水を浄化する方法。 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.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2016534508A JP6611715B2 (en) | 2014-07-18 | 2015-07-17 | Method for acclimatizing low salinity conditions of Aurantiochytrium algae |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2014147476 | 2014-07-18 | ||
JP2014-147476 | 2014-07-18 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2016010149A1 true WO2016010149A1 (en) | 2016-01-21 |
Family
ID=55078634
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP2015/070609 WO2016010149A1 (en) | 2014-07-18 | 2015-07-17 | Method of acclimating algae belonging to aurantiochytrium sp. to low-salt conditions |
Country Status (2)
Country | Link |
---|---|
JP (1) | JP6611715B2 (en) |
WO (1) | WO2016010149A1 (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2017205045A (en) * | 2016-05-17 | 2017-11-24 | 株式会社 ヒガシマル | Method using marine heterotrophic algae, amino acid composition suitable for feed, as protein component of feed |
CN113774092A (en) * | 2021-09-27 | 2021-12-10 | 哈尔滨工业大学(深圳) | Method for synthesizing isoprene from environmental wastewater |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2012077799A1 (en) * | 2010-12-09 | 2012-06-14 | 国立大学法人筑波大学 | Novel microorganism having high squalene-producing ability, and method for producing squalene by means of same |
JP2013126404A (en) * | 2011-12-19 | 2013-06-27 | Sumitomo Heavy Ind Ltd | Fat production method and fat production apparatus |
JP2014030383A (en) * | 2012-08-03 | 2014-02-20 | Fuji Electric Co Ltd | Method and device for producing oil |
JP2014108101A (en) * | 2012-12-04 | 2014-06-12 | Univ Of Tsukuba | Method for culturing algae using peritoneal dialysis wastewater as culture medium |
-
2015
- 2015-07-17 JP JP2016534508A patent/JP6611715B2/en active Active
- 2015-07-17 WO PCT/JP2015/070609 patent/WO2016010149A1/en active Application Filing
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2012077799A1 (en) * | 2010-12-09 | 2012-06-14 | 国立大学法人筑波大学 | Novel microorganism having high squalene-producing ability, and method for producing squalene by means of same |
JP2013126404A (en) * | 2011-12-19 | 2013-06-27 | Sumitomo Heavy Ind Ltd | Fat production method and fat production apparatus |
JP2014030383A (en) * | 2012-08-03 | 2014-02-20 | Fuji Electric Co Ltd | Method and device for producing oil |
JP2014108101A (en) * | 2012-12-04 | 2014-06-12 | Univ Of Tsukuba | Method for culturing algae using peritoneal dialysis wastewater as culture medium |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2017205045A (en) * | 2016-05-17 | 2017-11-24 | 株式会社 ヒガシマル | Method using marine heterotrophic algae, amino acid composition suitable for feed, as protein component of feed |
CN113774092A (en) * | 2021-09-27 | 2021-12-10 | 哈尔滨工业大学(深圳) | Method for synthesizing isoprene from environmental wastewater |
Also Published As
Publication number | Publication date |
---|---|
JPWO2016010149A1 (en) | 2017-04-27 |
JP6611715B2 (en) | 2019-11-27 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Shen et al. | Effect of nitrogen and extraction method on algae lipid yield | |
JP5466120B2 (en) | Method for producing high levels of DHA in microalgae using modified amounts of chloride and potassium | |
Talukder et al. | Immobilization of microalgae on exogenous fungal mycelium: a promising separation method to harvest both marine and freshwater microalgae | |
JP5942197B2 (en) | Novel microorganism having high squalene production ability and method for producing squalene using the same | |
US9102552B2 (en) | Production of cyanobacterial or algal biomass using chitin as a nitrogen source | |
Oliveira et al. | Bioflocculation of cyanobacteria with pellets of Aspergillus niger: Effects of carbon supplementation, pellet diameter, and other factors in biomass densification | |
CN107164238B (en) | Schizochytrium limacinum strain and mutagenesis method and application thereof | |
JPWO2020071444A1 (en) | How to cultivate freshwater microalgae | |
US10173913B2 (en) | Process of treating buchu mercaptan production wastewater using microalgae and chitin as a nitrogen source | |
JP6611715B2 (en) | Method for acclimatizing low salinity conditions of Aurantiochytrium algae | |
AU2012284645B2 (en) | Method for obtaining an open phototrophic culture with improved storage compound production capacity | |
KR101521274B1 (en) | Novel microalgae aurantiochytrium sp. la3 (kctc12685bp) and method for preparing bio-oil using thereof | |
CN114729297B (en) | Method for producing astaxanthin by heterotrophic culture of haematococcus pluvialis | |
RU2639557C1 (en) | Xanthomonas campestris bacteria strain - xanthan producer | |
TW202016289A (en) | Novel microalgal strains of thraustochytrium genus, and producing polyunsaturated fatty acids using the same | |
KR101631591B1 (en) | Treatment method of swine wastewater by microalgae, Chlorella sorokiniana | |
KR101654219B1 (en) | Thraustochytriidae sp. strain containing high content of polyunsaturated fatty acid and uses thereof | |
WO2018064036A1 (en) | Methods of culturing aurantiochytrium using acetate as an organic carbon source | |
Shetty et al. | Variations among antioxidant profiles in lipid and phenolic extracts of microalgae from different growth medium | |
Manheim | Improved microalgal biomass harvesting using optimized environmental conditions and bacterial bioflocculants | |
Fábregas et al. | Use of agricultural surpluses for production of biomass by marine microalgae | |
Farhadian et al. | Density, growth and biochemical composition of Tetraselmis tetrathele cultured in molasse and beer wastewater as media | |
Doukani et al. | Development of experimental production of algal biomass: case of Dunaliela salina of Arzew’s Salines (Western Algeria) | |
RU2575804C1 (en) | STRAIN OF YEAST Candida parapsilosis - PRODUCER OF LIPASE | |
PL231760B1 (en) | Method for obtaining citric acid |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 15822544 Country of ref document: EP Kind code of ref document: A1 |
|
ENP | Entry into the national phase |
Ref document number: 2016534508 Country of ref document: JP Kind code of ref document: A |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
122 | Ep: pct application non-entry in european phase |
Ref document number: 15822544 Country of ref document: EP Kind code of ref document: A1 |