JP2016140359A - Novel chlamydomonas and application of the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel chlamydomonas and application thereof.SOLUTION: Improved oil-productive chlamydomonas CPC1215 has characteristics such that it can be cultured by seawater, has high oil content, low adhesiveness and gravity settlement, and can be cultured outdoor. The present invention provides a method for synthesizing oil, includes a production method of biodiesel, and includes a food product and livestock feed of the above-described chlamydomonas.SELECTED DRAWING: Figure 10

Description

  The present invention relates to a technology that can be cultured using seawater, has high oil content, low tackiness, gravity subsidence, and can be cultured outdoors, and is applied to energy production. It relates to a novel Chlamydomonas orbular.

  As a next-generation biofuel, “microalgae” has been attracting attention around the world against issues such as rising oil prices, environmental pollution, and the greenhouse effect. Because microalgae are diverse and easy to cultivate, they have extremely high commercial availability, and are not limited to pharmaceuticals, health foods, agricultural fertilizers, fish farming feeds, cosmetics, biodiesel, etc. It is also used to produce biofuels and biochemical products.

As a result of scientists separating microalgae from the ocean and lakes, the structure of the oil and fat resembles general vegetable oil and fat, and C16 and C18 fatty acids are mainly used. The fatty acid content of higher plant seeds is only about 15% to 20% of the dry weight, and in microalgae, the fatty acid content reaches 70-80% of the dry weight of the cells when nitrogen is deficient.
The production volume of microalgae biodiesel per unit is about 10 to 20 times higher than that of other oil-producing plants, and is an excellent biodiesel raw material, which can be said to be a third generation biofuel. Research has shown that when biodiesel is produced using microalgae directly, 50% of the current transportation fuel can be substituted, and it is sufficient to use only about 3% of the current total agricultural land in the United States. is there. In the growth of microalgae, photosynthesis is performed using carbon dioxide as a carbon source, so that it is possible to simultaneously solve the increasingly severe greenhouse effect problem. Biodiesel produced using microalgae is non-toxic, has the advantages of being environmentally friendly and not occupying arable land, and can also reduce emissions of carbon dioxide and sulfate. it can.
The European Union (EU) has set a goal of 10% of all fuels as biofuels by 2020, and aviation fuel and diesel are also required to add 10% of biofuels. Therefore, it is expected that there will be a great demand for biodiesel in the near future, and therefore the search and development of biodiesel materials has become an urgent agenda in various countries around the world. A column titled “Algae bloom again” was published in Nature magazine in 2007, and the importance of bioenergy by algae was posted in the field of renewable energy, including environmental protection angle, energy angle, national economy, etc. It indirectly explains that algae plays an indispensable role in the national energy policy from any angle. How to achieve high economic effects, achieve both environmental protection, and increase the carbon separation efficiency and added value of different algal bodies is an issue that advanced countries have been focusing on research and development in recent years.

In addition, 1 kilogram (dry weight) of autotrophic microalgae absorbs about 1.8 kilograms of carbon dioxide and transforms it into biomass. Therefore, when microalgae grow, carbon dioxide, a greenhouse gas, is released into the atmosphere. Emissions are reduced. In addition, nitrogen and phosphorus necessary for culturing microalgae can be obtained from domestic wastewater or aquaculture wastewater, and since the wastewater is purified at the time of cultivation, the treatment effect of wastewater can also be achieved.
The largest bottleneck of biofuels from microalgae is too expensive to produce, much higher than fossil fuels, and the US Department of Energy's 2012 analysis of the sales price of microalgae algae oil is about 9 gallons. The price of diesel for fossil fuels was US $ 3.61 per gallon.
The United States Department of Energy announced in August 2013 that it will invest USD 220 million in the development of algae-based biofuels. In 2019, the cost of microalgae-based biofuels will be halved, resulting in large-scale commercialization. The sales price of algae oil from microalgae is expected to drop to US $ 2.27 per gallon, which will reduce costs. In other words, we improve the production efficiency of algae by genetic recombination method, improve the algae by artificially inducing mutation by the development of biochemical technology, increase the growth density of microalgae, increase the resistance to aquaculture environment, It aims to create super microalgae with extremely high commercial value.

Inducing mutations artificially is an important mechanism in species evolution, and in the natural environment its probability of occurrence is only about 1 / 100,000 / 100,000, so artificial mutations Trigger. Examples of the method include radiation, ultraviolet rays, alkylating agents, nitroso compounds, and the like. Here, mutation by ultraviolet rays is taken as an example.
Irradiation with UV light causes pyrimidine dimers to form in DNA molecules, inhibits chromosomal genes from being placed in exact pairs, and causes mutations or death in microalgae genes. Normally, in this method, a myriad of mutant strains are obtained, followed by trait screening, obtaining microalgae with excellent oil production capacity, screening for the oil content of microalgae, and obtaining excellent oil production capacity. Select the microalgae you have on a large scale.

Chlamydomonas (Chlamydomonas) is a genus of Chlorophyta / Chlorophyceae, a unicellular organism with flagella, about 10 micrometers in diameter. Chlamydomonas and cyanobacteria are the most representative model organisms of the eukaryotic and prokaryotic algae of the microalgae world. Just as Drosophila has contributed to academic research in genetics, Chlamydomonas It has long been used as a research material for genetic engineering, and has been used to study the functions, morphological characteristics, functionality and regularity of biological molecules such as nucleic acids and proteins.
Chlamydomonas is an important research material in academic research, and Chlamydomonas reinhardtii 137C is the most commonly used algae (algae species). Chlamydomonas began research in biofuels in 1952 when Frenkel discovered that Chlamydomonas and other green algae generate hydrogen under certain circumstances.
However, the mechanism for generating hydrogen has not been elucidated, it is difficult to control the efficiency, and further research is expected to break through this bottleneck.

Chlamydomonas C.I. Reinhardtii 137C is a model algae for research on oil and fat production, and Chlamydomonas has already completed the decoding of three chromosomal genes of the cell nucleus, chloroplast, and mitochondria with genetic material, and an open database has been constructed and researchers Has been provided to. The above three strains of transformation tools have been further developed, and research on oil production has focused on the modification of model Chlamydomonas using genetic recombination technology. Reinhardtii 137C is artificially induced to mutate or remodel genes related to oil production, and research on the modification of oil production traits is progressing.
In previous studies, most of the found Chlamydomonas grows in freshwater environments, and its fat content is low (10-15%), compared to other microalgae that produce other fats (> 30%) It is not suitable for large-scale aquaculture. The algae that is most commonly used for oil production in commercial production is Chlorella vulgaris and has an oil production rate of about 50 to 100 mg / L / d. It grows in a freshwater environment. Many scientists have begun exploring algae that can be cultivated in seawater or saltwater, due to the need for large amounts of drinking water and the problem of competing for water resources. For this reason, microalgae that can be cultivated in seawater and have high fat content and high biomass production have become promising algae for producing biofuels.
Similarly, the novel Chlamydomonas that produces the fats and oils of the present invention can be cultured in seawater, has high oil content, low tackiness, gravity subsidence, outdoor culturing characteristics, etc., applied to energy production technology, and drinking water Has the advantage of not consuming.

  Reference: Chisti Y (2007) Biodiesel from microalgae. Biotechnol Adv 25: 294-306; Chisti Y (2008) Biodiesel microalgae beats bioethanol. Trends in biotechnology 26: 126-131; Fu Z, Yang F, An Y, Xue Y (2009) Characteristic of nitricate in situ and indirectivity. Bioresource Technology 100: 3015-3021; Grossman AR (2000) Chlamydomonas reinhardtii and photosynthesis: genetics to genomics. Current opinion in plant biology 3: 132-137; Haag AL (2007) Algae bloom against. Nature 447: 520-521; Rodolfi L, Chini Zittelli G, Bassi N, Padovani G, Biondi N, Bonini G, Tredici MR (2009) Microalgae for oil: strain selection, induction of lipid synthesis and outdoor mass cultivation in a low- cost photobioactor. Biotechnology and bioengineering 102: 100-112; Siaut M, Cuine S, Cagnon C, Fessler B, Nguyen M, Carrier P, Beilly A, Beisson F, Trison G, Tris. model green alga Chlamydomonas reinhardtii: charactorization, variant between common laboratories and relations with whip research. BMC biotechnology 11: 7; Spoleore P, Joannis-Cassan C, Duran E, Isambert A (2006) Commercial applications of microalgae. Journal of bioscience and bioengineering 101: 87-96; Wang B, Li Y, Wu N, Lan CQ (2008) CO (2) bio-mitigating using microalgae. Applied microbiology and biotechnology 79: 707-718.

  The present invention has been made in view of such conventional problems. In order to solve the above problems, the main object of the present invention is to provide an improved microalgae.

  Nitrogen-deficient conditions, high lipid content, cultivated in seawater and fresh water, gravity subsidence, low stickiness, etc., widely used in biodiesel production, carbon dioxide capture, food and feed production Applied.

The present invention is a protozoal alga Chlamydomonas orbicularis CPC1 in which a large amount of fats and oils are accumulated, is an algae selected from a river estuary in central Taiwan. When cultured in a laboratory environment, the fat content is 43% of the dry weight. And when cultivated outdoors in a 50 liter photobioreactor, the average fat content is 20-30%.
When cultivating outdoors, due to uncontrollable factors such as light intensity is not constant, temperature is too high, contamination, etc., the cumulative amount of fat decreases, the growth rate decreases, and the amount of fat produced Diminished. For this reason, algae was improved by a method of inducing mutation by ultraviolet rays.
The present invention seeks mutant algae that generate mutations in CPC1 algae mainly by radiating ultraviolet rays and increase fat production by a large selection method. Mutations in the DNA of organisms by the radiation of ultraviolet rays, DNA breaks or thymine dimers are formed, and DNA is unable to maintain its normal function, altering genes, Affects the body's metabolic pathways and physiological properties. In order to analyze the fats and oils of algal bodies at high speed and accurately, the present invention analyzes fats and oils by the Nile red fluorescence technique. Nile red is a fluorescent staining reagent that detects lipid, and after binding to neutral lipid, it excites 530 nm light and emits 580 nm fluorescence.
Nile red is less accurate than gas chromatographic analysis of algal fats and oils, but it has the advantage that it can be operated quickly, is low cost, and has a short pretreatment time. Since the required sample volume is small, it is suitable for large-scale sorting and analysis.

  In order to solve the above-mentioned problems and achieve the object, the biological culture of the microorganism according to the present invention is Chlamydomonas orbicularis CPC1215, deposited with the Hsinchu Food Industry Development Research Institute in the Republic of China, and the accession number Is BCRC 980035.

  The above-mentioned Chlamydomonas CPC1215 has a similarity of 90% or more in the sequence of rbc L and the sequence provided in the present invention (SEQ ID NO: 6), a fat content of 30% or more, and an oil production rate of 100 mg / Points that reach L / day or higher.

  In addition, the method for synthesizing fats and oils of the present invention includes the fats and oils of the aforementioned culture, subjecting the biological culture to nitrogen starvation, and culturing the biological culture with a temperature range of 25 to 35 ° C. including.

  Fresh water or seawater with a salinity of 1 to 3% is disposed and used for the culture medium of the above-described biological culture to obtain a production amount of 1.5 g / L or more of biomass. For example, the culture is carried out in a 1 L photoreactor, Bold's Basal Medium (BBM) culture solution is employed, artificial sea salt is added, the culture is performed, aerated to 2% CO2, the light source is With a TL5 fluorescent lamp, the light intensity is 150 μmol / m 2 / s, the fat content reaches 51.8% at the maximum, and the fat production rate reaches 121.8 mg / L / day in 9 days. When induced at a high temperature of 35 ° C., the production of fats and oils reaches 140.8 mg / L / day. In addition, CPC1215 can be cultivated using fresh seawater or artificial seawater, has the characteristics of fast subsidence and low stickiness, can be cultivated in large quantities, and algae is cultivated to reduce carbon dioxide, Applied to production etc.

  Furthermore, the present invention provides a method for synthesizing biodiesel, including biodiesel of the aforementioned culture.

  In addition, this invention is a foodstuff containing the above-mentioned culture.

  Moreover, this invention is a feed containing the above-mentioned culture.

  The Chlamydomonas CPC1215 of the present invention is promising for commercialization because wild CPC1 can be cultivated in large quantities, can be easily collected, is naturally submerged, and has a high oil production rate.

It is a filtration process figure which shows the isolate (isolates) of this invention. It is an influence figure with respect to the biomass of CPC1 artificial aquaculture. It is a linear relationship figure which shows the density | concentration (OD680) of algae and Nile red fluorescence intensity. It is a comparison figure (250 ml photobioreactor culture) which shows Nile red fluorescence intensity of CPC1 and a mutant algae species. It is a comparison figure which shows the growth rate of CPC1215 mutant algae species and wild CPC1. It is a comparison figure which shows the fats and oils accumulation ability of CPC1215 mutant algae species and wild CPC1. It is a comparison figure in which CPC1215 mutant algae species shows the growth rate in high temperature culture. It is a comparison figure in which CPC1215 mutant algae species shows fats and oils accumulation ability in high temperature culture. It is an observation result with the staining microscope of CPC1215 mutant algae extra-species and Nile red. It is an observation result that the adhesive force of CPC1215 mutant algae species is low. It is an observation result that the settlement rate of CPC1215 mutant algae species wild CPC1 is fast.

  Preferred embodiments of the present invention will be described with reference to the accompanying drawings. The embodiments described below do not limit the contents of the present invention described in the claims. In addition, all of the configurations described below are not necessarily essential requirements of the present invention.

(First embodiment)
The present invention is a biological culture of microorganisms, the biological culture is Chlamydomonas orbicularis CPC1215, which is stored in the Food Industry Development Institute of the Republic of China, and the accession number is BCRC 980035. Preferably, Chlamydomonas CPC1215 refers to those in which the rbc L sequence and the sequence provided by the present invention (SEQ ID NO: 6) reach 90% or more. The aforementioned Chlamydomonas CPC1215 has a fat content of 30% or more and an oil production rate of 100 mg / L / day or more.
The present invention provides a method for synthesizing fats and oils, including producing fats and oils using Chlamydomonas orbicularis CPC1215. Preferably, the above-mentioned method is used in which Chlamydomonas is subjected to nitrogen starvation treatment, cultured at a temperature range of 25 to 35 ° C., and fresh water or seawater having a salinity of 1 to 3% is placed in the culture medium. And obtaining a production amount of 1.5 g / L or more of biomass.

  The present invention also provides a method for producing biodiesel, which is produced using Chlamydomonas orbicularis CPC1215.

  Furthermore, the present invention provides a food or feed comprising Chlamydomonas orbicularis CPC1215.

  In addition, the actual application scope of the present invention is proved by the specific embodiments described below, however, the scope of the present invention is not limited to any form.

The mainland microalga Chlamydomonas orbicularis CPC1 with high fat content is selected.
As shown in FIG. 1, the purification and separation process for algae obtained from the seawater environment of the present invention includes a sample collection process (S11), a pre-culture process (S12), and a single algal strain (algal strain). A screening step (S13), a liquid culture step (S14), a culture medium type and culture condition optimization step (S15), and an algal strain physiological condition analysis step (S16). . As a separation and purification method, a serial dilution method is adopted, and after algae strains are purified, algae are identified using 18S rRNA and rbc L gene sequences.
The forward direction of the PCR amplification primer of 18S DNA is CCA TGC ATG TCT AAG TAT AAA CTG CT (SEQ ID NO: 1), and the opposite direction is TAG GTG GGA GGG TTT AAT GAA CTT (SEQ ID NO: 2) The comparison result of the sequence alignment of the obtained sequence in GenBank is shown in SEQ ID NO: 5 and is the registration number KF383269.
The forward direction of the PCR amplification primer of rbc L is GTT CAA AGC CGG TGT AAA AGA C (SEQ ID NO: 3) and the opposite direction is CAT AGC TGA ATA CCA CCA GAA GCA (SEQ ID NO: 4) The result of comparing the sequence alignment in GenBank of SEQ ID NO: 6 is shown in SEQ ID NO: 6, and the two genes were combined with the sequence alignment of the GenBank database, and as a result, most similar to Chlamydomonas orbicularis SAG 11-19, and The outline matches Chlamydomonas and is named Chlamydomonas orbiralis CPC1.

CPC1 is cultured using fresh water and seawater.
In this study, fresh water and seawater (salinity of 3 to 3.5%) were first placed in the BBM culture medium, and 2% CO2 was aerated on CPC1 and cultured to accumulate the biomass of salinity and fats and oils. Investigate the impact on
As shown in FIG. 2, the growth days of CPC1 cultured in a seawater environment is 11 days, the biomass reaches a maximum of 1.42 g / L, and the growth days after being cultured in a freshwater environment is around 14 days. Biomass is better than seawater, reaching 2.32 g / L. Regarding the accumulation of fats and oils shown in Table 1, the oil content and oil production rate obtained by culturing CPC1 with seawater are both higher than those in freshwater culture, with a maximum of 43.39% and 64 mg / L / d. To reach. In addition, CPC1 has been successfully cultured in artificial seawater, natural seawater, and fresh seawater (storage time is shorter than one week), and the culture volume is 50 L.

Mutation technology by ultraviolet rays of CPC1.
CPC1 passes through BG-11 solid culture medium, selects algae, replaces with BBM culture medium, performs preculture, and after exponential phase, transfers 5 mL of algae liquid to a sterile culture container and Make a mutation.
In this patent, a 6 W short wavelength (254 nm) ultraviolet lamp is used, the distance between the light source and the algal fluid is 12.5 centimeters, and the irradiation time is 2 minutes. After the irradiation with ultraviolet light is completed, light on the alga liquid is blocked for 24 hours, and then the algal liquid is diluted 1000 times and applied to the solid culture medium of BG-11.

Oils and fats of algal bodies are detected by Nile red fluorescent staining technology.
CPC1 algae solution is cultured for 7 to 12 days, and after waiting for oil and fat to accumulate, it is diluted with 20% ethanol and fixed at a concentration of OD680. After the total volume reaches 1 mL, 1 μL of Nile red staining Add reagent (0.1 mg / mL in acetone) and mix evenly for 10 minutes at 100 rpm at room temperature. In the staining process, the sample is covered with an aluminum foil in the entire process so that the staining reagent does not lose its activity when irradiated with light. After completion of the staining, the sample is moved to a quartz tube dedicated for the fluorescence spectrometer, and the excitation wavelength (excitation wavelength: 530 nm) is detected by the fluorescence spectrometer.
In FIG. 3, after the CPC1 algae liquid is diluted to different concentrations, the fluorescence results detected by the Nile red staining reagent show a linear relationship between the alga body concentration and the fluorescence intensity, and the R square value (R square) is 0. 9998, indicating a high linear relationship. Nile red fluorescence is a highly sensitive detection technology. In the present invention, when OD680 is higher than 0.2, the cell concentration is too high, the fluorescence value becomes unstable, and it is impossible to measure accurately. Then, Nile red fluorescence measurement is performed in the range of OD680 = 0.06 to 0.1.

Analysis method of fatty acid composition component, oil content and oil production rate of microalgae.
To analyze the fatty acid composition of microalgae, first obtain dried algae powder, add 0.5N potassium hydroxide (KOH) in methanol, and remove the algal cells using an ultrasonic crusher. The saponification reaction (saponification) is performed on the crushed algal liquid after crushing, and then the esterification reaction is performed by adding methanol. The total amount and composition are analyzed, the fat content of the algal bodies is estimated, and the oil content is displayed as a percentage by weight.
Due to the synergy between the oil content (%) and the biomass production (g / L / day), the calculated oil production rate becomes the weight of fats and oils produced in the culture solution per liter every day. The calibration curve is set to n-hexane for methyl fatty acid standards (including C14: 0, C16: 0, C16: 1, C18: 0, C18: 1, C18: 2, C20: 5, C22: 6, etc.). The composition of the algal oil is identified by mixing and analyzing the residence time of each fatty acid methyl by gas chromatography.

Selection flow chart of CPC1215 mutant algae and measurement of oil production rate.
Algae mutated by ultraviolet rays are first cultured in 5 mL of BBM in a glass test tube, rotated at 60 rpm at room temperature, and cultured for 10-14 days. Detection is performed, 16 mutant algae having higher fluorescence intensity than the native species are selected, and the measurement proceeds to the second step.
Culturing in a 250 mL optical bioreactor, adding 2% artificial sea salt to BBM to make a culture solution, culturing for 7 days, and then analyzing the fat content by gas chromatography, the oil content is wild CPC1 After being confirmed to be higher, the measurement was performed by expanding to a 1 L photobioreactor, and the culture solution was also BBM with 2% sea salt added.

After culturing in a 250 mL photobioreactor and selecting 5 mutant mutants with high oil content using the Nile red staining reagent (see FIG. 4), the FAME content is detected by GC. Three strains of algae (CPC1215 / 1218/1266) with the highest content are selected and grown in a 1 L photobioreactor, and the fat accumulation test results are as follows.
FIG. 5 illustrates the comparison result of the growth speed. The growth rate of CPC1215 is the fastest and after entering the log phase, the biomass is clearly higher than the wild (CPC1) and other mutant algae. The same situation occurs in the stationary phase, reaching 2.44 g / L on the 14th day and increasing CPC1 to 8.9% at 2.24 g / L. In addition, CPC1218 and CPC1266 also have higher stationary biomass than wild (CPC1), and on day 14 are 2.41 and 2.38 g / L, respectively. The portion of CPC 1215 that has a preferred fat accumulation capacity is that the fat accumulation rate and total amount are both higher than wild CPC1 (see FIG. 6). The difference on day 9 is most noticeable, with the oil content of CPC1215 reaching 46.5%, 20.8% higher than the oil content of wild CPC1 of 38.5%. The oil content on day 14 exceeds 50% and reaches 51.8%. As can be seen from the above results, the growth rate and fat content of CPC1215 are both improved as compared to wild CPC1.

Table 2 shows the calculation results of the oil production rate of CPC1215. The oil production rate of CPC1215 is clearly higher than that of the wild (CPC1) and other mutant strains (CPC1218 / 1266), and the oil production rate on the 7th day of culture. Becomes 118.8 mg / L / day, 35.3% higher than wild CPC1. On the 9th day, the highest oil production rate is achieved, reaching 121.8 mg / L / day, 30.8% higher than 93.2 mg / L / day for wild CPC1.
The CPC 1215 is stored in the Food Industry Development Laboratory, and the accession number is BCRC980035. Both CPC1215 used in the present invention are the same as the above-mentioned stored algae.

CPC1215 is cultured at high temperature to increase the accumulation of fats and oils.
The culture temperature is increased from the original 25 ° C. to 35 ° C. and the Bold's Basal Medium (BBM) culture medium is used. The culture volume is 1 L, 1.0% seawater salinity, 2% carbon dioxide is used as the carbon source, and the starting algae amount is 0.305 g / L. As shown in FIG. 7, the growth rate of CPC1215 mutant algae is not suppressed by high temperature, and when cultured at room temperature and high temperature for 11 days of operation, the concentration of algal bodies is 2.46 g / L and 2 respectively. Reaching 51 g / L.
According to FIG. 8, the CPC1215 cultured at 35 ° C. has a high fat accumulation rate and fat content, and the difference on day 7 is most noticeable, and the fat and oil accumulation capacity was cultured at a high temperature. No. The oil content of 215 is 42.5%, 10.7% higher than that cultured at room temperature (38.4%) and 20.7% higher than 35.2% of the native species . The oil content on the 11th day reached 51.0%. As shown in Table 3, the production rate of fats and oils cultured at high temperature is clearly high, and the oil production rate becomes 140.8 mg / L / day when cultured for 7 days.

CPC1215's low tack and fast settlement characteristics.
CPC1215 shows low adhesion in the culture process. For example, when the outer shape is observed with a microscope (see FIG. 9), the cell shape of the mutant CPC1215 is round compared to wild CPC1 and stained with Nile red. The stained oil droplets of mutant strain CPC1215 are clear and large in quantity. When the algae was removed, the algal bodies remained on the culture container of the mutant strain CPC1215 (see FIG. 10), and as a result of three or more experiments, the adhesiveness of the mutant strain CPC1215 to the container was low. I understood that.
As shown in FIG. 11, CPC1215 was more likely to sink than in the wild, and after standing at room temperature for 5 minutes, CPC1215 had a faster settlement rate than in the wild. When the above observations and tests are combined, CPC1215 can be cultivated in large quantities compared to wild CPC1, easily collected, subsided naturally, and has a high oil production rate. Chlamydomonas orbicularis CPC1215 has been commercialized. Promising.

  The embodiments disclosed herein are for the purpose of explaining, not limiting the present invention, and the spirit and scope of the present invention are not limited by such embodiments. The scope of the present invention should be construed according to the claims, and all technologies within the equivalent scope should be construed as being included in the scope of the present invention.

Claims (10)

Chlamydomonas orbicularis CPC1215,
The Chlamydomonas CPC1215 is deposited with the Hsinchu Food Industry Research Institute in the Republic of China as the accession number BCRC980035,
A biological culture of microorganisms.   The microbial biological culture according to claim 1, wherein the Chlamydomonas CPC1215 has a rbc L sequence (SEQ ID NO: 6) similarity of 90% or more.   The microbial biological culture according to claim 2, wherein the Chlamydomonas CPC1215 has a fat content of 30% or more and an oil production rate of 100 mg / L / day or more.   A method for synthesizing fats and oils, comprising producing fats and oils using the biological culture of microorganisms according to claim 1.   The method for synthesizing fats and oils according to claim 4, comprising a nitrogen starvation treatment of the biological culture.   The method for synthesizing fats and oils according to claim 4, wherein the biological culture is cultured in a temperature range of 25 to 35 ° C.   The fresh culture medium or seawater with a salinity of 1 to 3% is used for the culture medium of the biological culture to obtain a production amount of 1.5 g / L or more of biomass. Of oil and fat.   A method for producing biodiesel, comprising producing biodiesel using the microbial biological culture of claim 1.   A food comprising the microorganism-based biological culture of claim 1.   A feed comprising the microorganism-based culture of claim 1.